RNA Extraction from Clinical Samples, Cell Culture Lysates, … · CDC protocols for the molecular epidemiology of measles virus and rubella virus; version of 05/19/2012 2 Controls

CDC protocols for the molecular epidemiology of measles virus and rubella virus; version of 05/19/2012

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RNA Extraction from Clinical Samples, Cell Culture Lysates, Plasma and Serum Using the Qiagen Viral RNA Mini Kit Purpose The following protocol is to be used for the extraction of RNA for use in RT-PCR reactions. This kit is designed for the purification of viral RNA from plasma, serum, and clinical samples including urine and throat swabs, and cell culture lysates. The Qiagen Viral RNA kit handbook recommends using only cell-free samples, but the laboratory at CDC has successfully extracted RNA from cell-containing lysates. Important

1. This is a condensed protocol derived from the kit manual. Please read the manufacturer’s manual before first use.

2. Extraction of RNA from FTA disks requires a modified protocol (see below). Reagents and materials needed

• Gloves

• Lab coat

• RNaseZap (Sigma, # R2020-250ML)

• Nuclease-free (autoclaved) 1.5 ml microcentrifuge tubes

• Qiagen Viral RNA mini kit (Qiagen, #52904)

• 95-100% Ethanol

• 70% Ethanol

• PBS, RNase-free Equipment needed

• -20oC freezer

• Micropipettors • Bucket with ice

• Class II BSC with UV light

• Microcentrifuge

• Micropipettors and sterile pipette tips with aerosol-resistant filters

• Vortex

• Waterbath at 37 °C Recommendations for working with RNA

• Use dedicated equipment, rooms and biosafety cabinets for all pre-PCR procedures. Post amplification analysis and processing should be performed in separate rooms using dedicated equipment. Do not share equipment (including lab coats) between pre-PCR and post-PCR procedures. Wear gloves throughout experiments to prevent contamination from RNases found on human hands.

• Change gloves after touching skin (e.g. your face), door knobs, and common surfaces.

• Have a dedicated set of pipettors that are used solely for RNA work.

• Use filter tips and tubes that are tested and guaranteed to be RNase-free.

• Use RNase-free chemicals and reagents.

• Reduce RNase contamination by cleaning tube racks, micropipettors, and the work surface with 70% ethanol and with RNaseZap wipes.

• Reduce DNA contamination with UV light exposure for 15 minutes.


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Controls It is highly recommended to include a negative sample (uninfected cells or cell culture medium) as an extraction control to monitor for contamination introduced during the extraction procedure. Preparing Kit for First Use

• Add 310 µl of the AVE buffer to the tube containing lyophilized carrier RNA. Vortex thoroughly.

• Divide carrier RNA solution into aliquots, store at -20°C. Do not freeze-thaw more than 3 times.

• Add 25 ml of 95-100% Ethanol to AW1 buffer (as indicated on the bottle)

• Add 30 ml of 95-100% Ethanol to AW2 buffer (as indicated on the bottle) Assay Protocol

1. In a BSC designated for working with RNA, heat AVL buffer in 37°C water bath until all crystals have been dissolved. Vortex thoroughly.

2. Mix buffer AVL and carrier RNA solution at a ratio of 100 parts AVL plus 1 part carrier RNA, e.g. for 10 samples, mix 5.6 ml AVL with 56 µl carrier RNA. Remember to include an extraction control. It is recommended to make more AVL with carrier RNA than needed to allow for pipetting losses.

3. Add 560 µl of AVL buffer to nuclease-free 1.5 ml tube. 4. Add 140 µl of sample (clinical sample or cell lysate), pulse vortex for 15 seconds and

incubate at room temperature for 10 minutes. 5. Briefly spin tube in a microcentrifuge to remove drops from inside the lid. 6. Add 560 µl of 95-100% Ethanol to each tube and vortex for 15 seconds. Spin briefly. 7. For each sample, label a Mini spin column and place in a 2 ml collection tube (both

provided in kit). Add 630 µl of the mixture to column and centrifuge for 1 minute at 8,000 rpm

8. Empty the collection tube and repeat step 7. 9. Remove the column from the collection tube and place in a clean collection tube. Add

500 µl of buffer AW1. Centrifuge for 1 minute at 8,000 rpm. 10. Remove the column from the collection tube and place in a clean collection tube. Add

500 µl of AW2. Centrifuge for 3 minutes at 14,000 rpm. 11. Remove the column from the collection tube and place in a nuclease-free, labeled 1.5 ml

tube. 12. Add 60 µl of AVE buffer, incubate 1 minute at room temperature and centrifuge for 1

minute at 8,000 rpm 13. Remove and discard column; place 1.5 ml tube containing RNA on ice for immediate use

or store in -20oC or -70oC freezer for future use. Modified Protocol for Extraction of RNA from FTA disks Use forceps to handle FTA disks. Forceps must be cleaned to prevent cross-contamination. See protocol for use of FTA disks.

1. Heat AVL buffer in 37°C water bath until all crystals have been dissolved. 2. Mix buffer AVL and carrier RNA solution at a ratio of 100 parts AVL plus 1 part carrier

RNA, e.g. for 10 samples, mix 6.0 ml AVL with 60 µl carrier RNA. Remember to include an extraction control. It is recommended to make more AVL with carrier RNA than needed to allow for pipetting losses.

3. Pipet 600 µl of prepared buffer AVL containing carrier RNA into a 1.5 ml micro centrifuge containing an FTA disk.

4. Add 150 µl 1x PBS. Mix by pulse-vortexing for 15 seconds.


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5. Incubate at room temperature for 10 minutes. 6. Centrifuge at 13000 rpm for 2 minutes. 7. Remove 700 µl of the liquid to a new 1.5 ml tube, avoiding the filter paper or any fibers. 8. Add 560 µl of ethanol. Mix by pulse vortexing for 15 seconds. 9. Spin briefly to collect liquid from the lid. 10. Next, follow the standard Qiagen kit protocol for RNA extraction, step 7, above.


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Real-time RT-PCR Assays for the Detection of Measles Virus (MeV) N Gene RNA and Human RNase P mRNA (a cellular reference gene) using the ABI 7500 Real-Time Thermocycler Purpose The following protocol is to be used to detect RNA of measles virus (MeV) for diagnostic purposes. Primers and control RNA are supplied by CDC as a kit. For these procedures, RNA is extracted from a clinical sample or from cell culture (See RNA Extraction protocol). If samples give a positive result in the real-time RT-PCR assays, the RNA may be tested in a measles genotyping RT-PCR assay to amplify the target for sequence analysis and genotyping (See Measles Genotyping RT-PCR protocol). Important: This is a general protocol for use with the measles real-time RT-PCR kit supplied by CDC. Please check the package insert of the kit for updates to the protocol. Reagents and materials needed

• 70% ethanol

• Aluminum foil

• Gloves

• Lab coat

• MicroAmp optical 96-well reaction plate with barcode (Applied Biosystems #4306737)

• Nuclease-free water (Ambion #9937)

• Optical adhesive cover (Applied Biosystems #4311971)

• RNase inhibitor 2000 units (Applied Biosystems N-808-0119)


• Sterile 1.5 ml microcentrifuge tubes

• SuperScript III Platinum OneStep qRT- PCR Kit (Invitrogen, #11732-020) Equipment needed

• -70oC and -20oC freezers

• ABI 7500 Real-time PCR System

• Bucket with ice

• Centrifuge with holder for 96 well plates

• Class II BSC with UV light designated for PCR set up

• Microcentrifuge


• Vortex

• Water bath Recommendations for working with RNA

• Use dedicated equipment, rooms and biosafety cabinets for all pre-PCR procedures. Post amplification analysis and processing should be performed in separate rooms using dedicated equipment. Do not share equipment (including lab coats) between pre-PCR and post-PCR procedures. Wear gloves throughout experiments to prevent contamination from RNases found on human hands.






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• Reduce RNase contamination by cleaning tube racks, pipettors, and the work surface of the PCR hood with 70% ethanol and with RNaseZap wipes.

• Reduce DNA contamination with UV light exposure for 15 minutes. Kit contents The MeV real-time RT-PCR kit contains

• MeV primer/probe mix: One tube with 100 µl of a mix of primers and probe for MeV real-time reactions. Content needs to be diluted before use (see below).

• RNase P primer/probe mix: One tube with 50 µl of a mix of primers and probe for RNase P real-time reactions. Content needs to be diluted before use (see below).

• MeV control RNA (high concentration control). Contains synthetic MeV RNA (MeV-N3in) and total human RNA. Content is dried and needs to be rehydrated and diluted before use (see below). This control can be used for both MeV and RNase P reactions.

• MeV control RNA (low concentration control). Contains synthetic MeV RNA (MeV-N3in) and total human RNA. Content is dried and needs to be rehydrated and diluted before use (see below). This tube contains less synthetic MeV RNA than the high control, but the same amount of total human RNA. This control can be used for both MeV and RNase P reactions.

• 2 ml TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) for rehydration of controls. Information about primers and probes

• Primer and probe stock solutions are stored at –20oC. Keep probes protected from light.

• Probes are labeled at the 5’ terminus with a fluorescent reporter dye, 6-carboxy-fluorescein (FAM), and at the 3’ terminus with a non-fluorescent quencher, black hole quencher-1 (BHQ1).

• Final concentration in the reaction mix: o MeV primers and RNase P primers: 300 nM o MeV probe: 250 nM o RNase P probe: 100 nM

• MeV primer sequences o Forward Primer (MVN1139-F): 5’ TGG CAT CTG AAC TCG GTA TCA C 3’ o Reverse Primer (MVN1213-R): 5’ TGT CCT CAG TAG TAT GCA TTG CAA 3’ o Probe (MVNP1163-P): 5’ FAM CCG AGG ATG CAA GGC TTG TTT CAG A BHQ

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• RNase P primer sequences o Forward Primer (HURNASE-P-F): 5’ AGA TTT GGA CCT GCG AGC G 3’ o Reverse Primer (HURNASE-P-R): 5’ GAG CGG CTG TCT CCA CAA GT 3’ o Probe (BHQ1 HURNASE-P): 5’ FAM TTC TGA CCT GAA GGC TCT GCG CG

BHQ1 3’ Preparation of working solutions of primer/probe mixes Primer/probe mixes are supplied as 10x (10 fold) concentrated stocks. It is necessary to prepare a working solution of each mix before setting up a real-time RT-PCR reaction. Store diluted primers and probes at -20 oC. Wrap tubes in aluminum foil to protect from light. Aliquots should be thawed no more than three times.

• MeV primer/probe mix: Mix 10 µl stock solution with 90 µl nuclease-free water. Vortex.

• RNase P primer/probe mix: Mix 10 µl stock solution with 90 µl nuclease-free water. Vortex.


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Preparation of control RNA stocks Control RNAs are supplied as dried RNA. It is necessary to rehydrate these controls before the first use of the kit. Always work with RNA on ice. Do not work with control RNAs in the same PCR hood where master mix preparation is carried out.

1. To each tube (high and low control RNA) add 100 µl nuclease-free TE (supplied in kit) and vortex for 15 seconds.

2. Heat tubes to 50oC for 15 minutes in water bath then vortex for 15 seconds. Spin briefly to collect.

3. Prepare 10 aliquots of 10 µl each and store at -70oC. Each aliquot should be thawed only once to make a working solution (see below).

Preparation of working solutions of control RNAs 1. Thaw one tube with 10 µl high control RNA stock and one tube with 10 µl low control

RNA stock. 2. To each tube, add 90 µl nuclease-free TE (supplied in kit). Vortex. Spin briefly to collect. 3. Prepare 10 aliquots of 10 µl each and store at -70oC. 4. Use one aliquot for each real-time RT-PCR assay. Discard leftover working solution.

Sample Preparation RNA samples (extracted from a clinical sample or from cell culture) are stored at -70oC. For the assay, 2.5 µl of sample are used per reaction. Addition of different volumes requires adjustment of added nuclease-free water to result in a final volume of 25 µl. Assay Controls

• Every RNA sample should be tested in parallel with the MeV primer/probe and the RNase P primer/probe. The purpose of the RNase P control reaction is to monitor the integrity of the RNA.

• The following standards and controls should be run on each plate as indicated on the Measles Real-time Plate Layout. They must be included in master mix calculations for each primer/probe set:

o Negative controls � NTC: add 2.5 µl/well nuclease-free water instead of RNA to the wells

labeled MeV NTC and RNP neg as indicated on Measles Real-time Plate Layout.

� Extraction control: mock-extracted RNA obtained by extraction of water or medium using the Qiagen method. In the calculations for the number of reactions (see below) the extraction control is tested as an additional sample.

o Two control RNAs (high control and low control) � Rehydration and dilutions should be done separately from set up of

master mix. � Either one of the controls can also be used as RNase P controls. � Add 2.5 µl/well of each control RNA as indicated on Measles Real-time

Plate Layout.


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Preparations for assay set up

• Thaw kit reagents: 2X SS buffer, ROX, and primer/probe mix and briefly vortex.

• Spin down all reagents (including enzymes) in microcentrifuge and keep on ice until ready to dispense.

• Thaw RNA samples and keep on ice during assay set up.

• Record open date of reagents on Measles Real-time Master Mix worksheet. Assay Protocol

1. Determine the number of reactions (n) based on the number of RNA samples to be tested and the format of the Measles Real-time Plate Layout. Each clinical sample and the extraction control should be tested in triplicate with the MeV primer set, and in a single well for the RNase P primer set. Controls for the MeV primer/probe set (MeV high control, MeV low control, MeV-NTC) are tested in duplicate. Controls for the RNase P primer/probe set (RNase P positive control, RNase P-NTC) are tested in a single well. Either the MeV high control or the MeV low control tube can be used as an RNase P control. Prepare excess reaction volumes (n + 1) for each primer/probe set to allow for pipetting errors. Calculating the number of reactions: For the MeV primer set, the number of reactions is 3 times the number of samples (which includes the extraction control) plus 6 for the real-time controls plus 1 to allow for pipetting losses. For the RNase P primer set, the number of reactions is the number of samples (which includes the extraction control) plus 2 for the real-time controls plus 1 to allow for pipetting losses.

Example: If there are 4 specimens and 1 extraction control: make a master mix for 22 reactions with the MeV primer set:

• 5 samples (specimens and extraction control) measured in triplicate=15 reactions

• 2 NTCs

• 2 reactions high control RNA

• 2 reactions low control RNA

• 1 extra to allow for pipetting losses

Make a master mix for 8 reactions with RNase P primers:

• 5 samples (specimens and extraction control)

• 1 NTC

• 1 reaction with either high or low control RNA


2. Enter the ID# of the sample(s) on the Measles Real-time Plate Layout. 3. Enter the number of samples in the Real-time Master Mix worksheet to determine

volumes of each reagent to be added. There are separate calculations for the measles primers and the RNase P primers.

4. In the BSC dedicated for master mix preparation, for the MeV primer/probe set, add the first 4 reagents (nuclease-free water through ROX reference) to a pre-chilled 1.5 ml microcentrifuge tube. Invert, briefly centrifuge, and keep on ice.

5. Add RNase inhibitor and enzyme mix to master mix tube. Vortex and chill briefly on ice. 6. In another 1.5 ml microcentrifuge tube, repeat steps 4 and 5 for the RNase P master

mix. 7. Proceed to a separate BSC designated for template addition. Dispense 1 reaction

volume of master mix into appropriate wells according to the Measles Real-time Plate


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Layout, using a new tip for each master mix. Reaction volume is 22.5 µl/well for MeV and RNase P.

8. Add nuclease-free water (NTC), extraction control, sample RNA or positive control standards as indicated on the Measles Real-time Plate Layout, using a new tip for each well. Sample volume is always 2.5 µl/well. The total volume in each well should now be 25 µl.

9. Seal plate with optical adhesive cover. 10. Centrifuge the sealed plate at 1500 rpm for 1 minute at room temperature.

Assay Run

1. Launch software by double-clicking the 7500 software icon on the desktop. The following instructions are for v2.0.4 of the software.

2. Turn on the 7500 thermocycler – connection is usually automatic. 3. Select Advanced Set-Up. 4. The Experiment Properties screen will open. Name the experiment (e.g. date and

initials). The settings should be 7500 (96 wells), Quantitation-Standard Curve, TaqMan reagents, Standard Ramp Speed.

5. Go to Plate Setup: Define Targets and Samples. Enter name of each sample to be tested by clicking on Add New Sample. Include extraction controls. Each sample must be listed twice: once for measles (e.g. sample 1) and once for RNase P (e.g. sample 1-R).

6. Go to Define Targets. There should be one target (FAM) and one quencher (NFQ-MGB, Blackhole Quencher). The target is the fluorescent dye that the instrument will detect. The probes for measles and RNase P are labeled with the same fluorescent dye (FAM), so all wells will be assigned to the same target. It is not necessary to name the target.

7. Go to Assign Targets and Samples. Using the Plate Layout as a guide, highlight all the wells that will contain the test samples, the extraction control, and the RNase P controls. Do not highlight empty wells.

8. Check the box under ‘assign’. All the marked wells will display ‘U’ for ‘unknown’. 9. To define standards, highlight the wells with the high control. Under ‘Assign Targets’,

click on ‘S’ for standard. Under ‘quantity’, fill in 100000 (105). Repeat for the low control with 1000 (103) as quantity.

10. To define the NTC, highlight the wells with the measles water control. Under ‘Assign Targets’, click on ‘N’ for NTC.

11. To assign samples, highlight the 3 wells that contain the MeV master mix for the first test sample (e.g. sample 1). Under ‘Assign samples to the Selected Wells’ check the box for sample 1. Repeat for the well that contains sample 1 and RNase P master mix, check the box for sample 1-R. Repeat this process for each sample. Each well in use should now contain a target, a task (NTC, standard or unknown) and a sample name.

12. Make sure ROX is listed as the passive dye. 13. Go to Run Method. Set the Sample Volume to 25 µl. Set the step parameters

(time/temp) on the thermal profile to the following:

• RT Step: 48 oC /30 minutes

• Activation: 95 oC /5 minutes

• PCR (40 cycles): 95 oC /15 seconds, 60 oC /1 minute 14. Select File > Save As and save file in user folder using a standard format: (e.g.

Date_initials.eds) 15. Open the door of the 7500 by pushing in the indentation on the front. 16. Place a plate into the instrument tray. Orient the A1 well on the plate with the A1 position

on the instrument tray.


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17. Select Start Run. Under Run, select Amplification Plot to monitor the run, which should be complete in approximately 2 hours. Time left in run will be given.

18. At the end of the assay, the green Analyze button will appear. 19. Click on Save to save your data.

Assay Quality Control

1. After the run has finished, click on Analyze. The program will calculate an automatic threshold. Do not change the threshold for the analysis of the controls.

2. The results will appear. 3. Checklist for quality control: A real-time RT-PCR assay is valid if:

• The NTCs for MeV and RNase P and the extraction controls are undetermined (negative).

• The automatic threshold is located in the exponential phase of the standard amplification curves.

• The Ct values for the duplicate samples of the MeV high control are between 24.6-29.3 (see picture below).

• The Ct values for the duplicate samples of the MeV low control are between 31.7-37.2 (see picture below).

• The Ct value for the single sample of the RNase P control is between 24.0-29.5. If any of these criteria are not fulfilled, the assay must be repeated. If the extraction control is positive, the RNA extraction must be repeated.

4. Monitoring real-time RT-PCR assay over time: Ct values for positive controls should be recorded for subsequent runs. After several assays, a typical range of Ct values for each positive control should be determined and used for the analysis of subsequent runs. Changes in these values may indicate problems.

5. Important: the range of Ct values for the positive controls may vary between different lots of the Measles Real-time RT-PCR kit. Please check the package insert for updates.

Figure 1: Amplification plot with high and low control curves.


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Sample Data Analysis

1. Check the Multicomponent Data Pane icon on all samples with Ct values < 40 to confirm true amplification as indicated by a rise in FAM fluorescence.

2. Some wells may be highlighted by yellow flags. Click on the QC Summary Pane to see the reason why the wells were flagged. For example, if a sample well did not produce a signal, the program will flag this with No amplification. However, if the sample is negative (for example the extraction control), a lack of amplification is the expected result and not a quality control issue.

3. Adjusting the threshold (if necessary): Highlight the wells containing the samples and the NTC and examine the amplification curves. If a sample has an exponential curve that does not cross the threshold, it is possible to lower the threshold for the analysis of patient samples. Only lower the threshold after the Ct values for the standards have been determined as described in Assay Quality Control.

• Click on Analysis Settings. Click on Edit Default Settings. Remove the check from Auto Threshold. Click on Save Change and Apply Analysis Settings. Close.

• In the amplification plot, click on the threshold and drag it to the desired position. Every time the threshold is moved, the Ct values for the samples will be automatically recalculated.

• Important: The signal for the NTC must remain below the threshold setting. The threshold must be located in the lower third of the exponential phase of the graph. Figure 2 below shows the 2 positive controls and a typical positive test sample.

• If the threshold has to be moved, write down the value for threshold, because the program does not save this information.

Figure 2: Amplification plot with standards and typical sample


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Interpretation of results for MeV, RNase P

Viral gene RNase P gene Specimen Result

< 40 < 40 positive < 40 undetermined positive undetermined < 40 negative undetermined undetermined indeterminate

• If the PCR with the MeV primers/probe produced a Ct below 40, the specimen is positive for measles. It does not matter whether the RNase P reaction is positive or negative.

• If the result of the MeV PCR is undetermined but the RNase P PCR produced a Ct below 40, the specimen is negative for measles. The positive RNase P result indicates that sufficient RNA was extracted from the specimen to allow amplification to occur.

• If the result of the MeV PCR and the RNase P PCR are undetermined, the specimen is judged to be indeterminate. It is not possible determine a result, because there was not enough RNA to allow amplification to occur.

• Clinical samples with 2 or 3 positive reactions (out of the 3 replicates run on the plate) will be reported positive.

Exporting Data To save the data to an Excel file: Click on Export, then

1. Under: select data to export, choose Results 2. Under: Select one file or separate files, choose One file 3. Choose a file name and browse to the location where the file should be saved. 4. Click Open, then click Start Export. 5. Close the Export Tool.


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Worksheet for Measles Real-time RT-PCR for Measles RNA Detection (page 1 of 2) Below is an example of a worksheet for Measles Real-time Master-mix. An Excel spreadsheet with the worksheet is provided in a separate file.

Technician:

File name:

Test date:

Kit: Superscript III One-Step qRT-PCR (Invitrogen #11730-020)

Date opened:

Primer mix lot number:

Number of specimens (including extraction control): 0 Please insert number of

Number of reactions for measles reaction mix: 7 specimens in blue field

(number of specimens multiplied by 3 plus 7 )

Number of reactions for Rnase P reaction mix:

(number of specimens plus 3)

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Master mix worksheet for measles primer/probe set

Component Vol/rxn (µl) Final conc. # of rxns Total vol (µl)

Nuclease-free water 7.2 7 50.4

2x SS buffer 12.5 1x 7 87.5

MeV primer/probe mix 2 7 14

ROX reference 0.05 0.25 U/µl 7 0.35

RNase inhibitor 0.25 0.4 U/µl 7 1.75

SS III/Taq mix (5U/µl) 0.5 0.025 U/µl 7 3.5

RNA 2.5 xxxx xxxx

Total 25

Master mix worksheet for RNaseP primer/probe set


Nuclease-free water 7.2 3 21.6

2x SS buffer 12.5 1x 3 37.5

RNP primer/probe mix 2 3 6




RNA 2.5 xxxx xxxx

Total 25

Dispense master mix: 22.5 µl/well for both master mixes. Add 2.5 µl template


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Worksheet for Measles Real-time RT-PCR for Measles RNA Detection (page 2 of 2) Below is an example of a plate layout with two samples. An Excel spreadsheet with the Measles Real-time plate layout is provided in a separate file. Plate layout for real-time RT-PCR assay Sample 1 (S1) tested in triplicate with MeV primers 1 2 3 4 5 6 7 8 9 10 11 12 A S1 S1 S1 S1-R MeV high MeV high B S2 S2 S2 S2-R MeV low MeV low C - - D MeV NTC MeV NTC E - - F RNP pos RNP neg G - - H - - Sample 1 (S1) tested with primers for RNaseP MeV high: MeV control RNA (high) MeV low: MeV control RNA (low) NTC: negative control for MeV (water) RNP pos: positive control for RNase P (MeV control RNA high or low) RNP neg: negative control for RNase P (water) White field: MeV master mix Grey field: RNase P master mix


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RT-PCR Protocol for Measles Genotyping (Version 2) Purpose The following protocol is to be used for the amplification of a fragment of the measles virus (MeV) nucleoprotein (N) gene. This DNA fragment may be used for genetic characterization of measles virus. The protocol can also be used for detection of MeV RNA; however, this reaction is less sensitive than real-time RT-PCR. For these procedures, RNA is extracted from a clinical sample or from cell culture. This is a one-tube reaction, so there is a minimum of specimen handling. Primers and control RNA are supplied by CDC as a kit. Important: This is a general protocol for use with the measles genotyping kit supplied by CDC. Please check the package insert of the kit for updates to the protocol. What to do with the PCR products Analyze PCR products on agarose gels (See Agarose Gel Electrophoresis protocol). Laboratories with sequencing capacity should purify and sequence the PCR products (See protocols for Clean-up of PCR Products, Sequencing (Measles) and Sequencing (Rubella). Laboratories without sequencing capacity should ship the PCR product to the regional reference laboratory for sequencing. PCR products can be shipped without drying or purification. Transfer the PCR reaction into a 1.5 ml tube, close the lid, seal with parafilm and ship at ambient temperature. PCR products are stable at room temperature for at least one month. Reagents and materials needed

• 70% ethanol

• Lab coat

• Gloves

• RNase inhibitor 2000 units (Applied Biosystems N-808-0119)



• Nuclease-free, deionized water (NF water)

• Qiagen One-step RT-PCR kit (Qiagen CAT# 210210 or 210212)

• Autoclaved PCR tubes (0.2 ml, thin-walled)

• Measles Genotyping RT-PCR Kit, Version 2.0 (primers and control RNA)

Equipment needed


• Bucket with ice

• Class II biological safety cabinets with UV light (BSC)


• Vortex mixer

• Metal cooling rack for 0.2 ml tubes

• Waterbath at 50 °C

• Thermocycler (AB GeneAmp PCR System 9700)

• Microcentrifuge, refrigerated to 4 °C with rotor for 1.5 ml tubes and 0.2 ml tubes


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Recommendations for working with RNA

• Use dedicated equipment, rooms and biosafety cabinets for all pre-PCR procedures. Post amplification analysis and processing should be performed in separate rooms using dedicated equipment. Do not share equipment (including lab coats) between pre-PCR and post-PCR procedures.

• Wear gloves throughout experiments to prevent contamination from RNases found on human hands.





• Reduce RNase contamination by cleaning tube racks, micropipettors, and the work surface of the PCR hood with 70% ethanol and with RNaseZap wipes.

• Reduce DNA contamination with UV light exposure for 15 minutes. Kit contents The MeV genotyping kit contains

• 25 µl 10x measles virus forward primer MeV216. Content needs to be diluted before use (see below).

• 25 µl 10x measles virus reverse primer MeV214. Content needs to be diluted before use (see below).

• Dried measles RNA control. Content is dried and needs to be rehydrated and diluted before use (see below).

• 1 ml TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) for rehydration of controls. Information about primers

• Previously used primers MV60 and MV63.3 have been replaced by primers MeV214 and MeV216.

• MeV primer sequences o Forward Primer (MeV216): 5’ TGG AGC TAT GCC ATG GGA GT 3’ o Reverse Primer (MeV214): 5’ TAA CAA TGA TGG AGG GTA GG 3’

• The location of the primer binding sites is described in a separate file (Primers and reference sequences).

• Primer stock solutions are stored at -20oC.

• Final concentration of primers in the reaction mix is 600 nM. Information about the positive control The measles RNA control is an RNA transcript of a measles nucleoprotein (N) gene with a 220 nucleotide insert in the 3’ variable region. Amplified products will thus be 220 nucleotides larger than RT-PCR products from patient samples. Using this control simplifies the identification of contamination. Important: The new synthetic RNA, MeV-N3in, will work with both the previously recommended primer set (MV60, MV63.3) and the new primer set (MeV214, MeV216). But the synthetic RNA MVN216 which was used with the previously recommended primer set will not work with the new primers. Preparation of working solutions of primers Primers are supplied as 10X (10 fold) concentrated stocks. The concentration in the stocks is 200 µM. It is necessary to prepare a working solution of each primer prior to setting up a RT-


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PCR reaction. The concentration of the working dilution is 20 µM. Store diluted primers at -20 oC. Do not freeze-thaw more than 5 times.

• MeV214: Mix 10 µl stock solution with 90 µl nuclease-free water. Vortex.

• MeV216: Mix 10 µl stock solution with 90 µl nuclease-free water. Vortex. Preparation of control RNA stocks The measles control RNA is supplied as dried RNA. It is necessary to rehydrate it before the first use of the kit. Always work with RNA on ice. Do not work with control RNA in the same PCR hood where master mix preparation is carried out.

1. Add 100 µl nuclease-free TE (supplied in kit) and vortex for 15 seconds. 2. Heat tube at 50oC for 15 minutes in waterbath then vortex for 15 seconds. Spin briefly to

collect. 3. Prepare 10 µl aliquots and store at -70oC.

Preparation of working solutions of control RNA

1. Thaw one tube with 10 µl control RNA stock. 2. Add 90 µl nuclease-free TE (supplied in kit). Vortex. Spin briefly to collect. 3. Prepare 10 µl aliquots and store at -70oC for long-term storage and -20oC for short-term

storage. 4. Use 1 µl per RT-PCR reaction plus 4 µl nuclease-free water.

Sample Preparation RNA samples (extracted from a clinical sample or from cell culture, see RNA extraction protocol) are stored at -70oC. Usually, RNA is extracted from one 25 cm2 flask of infected cells or from 100-200 µl of clinical material. Most of the extraction protocols yield 40-50 µl of RNA. For the assay, 5 µl of sample are used per reaction. The volume of RNA can be increased, but this will not significantly improve the sensitivity. Addition of different volumes requires adjustment of added nuclease-free water to result in a final volume of 50 µl. . Assay Controls

• The following controls must be run in each assay. They must be included in master mix calculations:

o Negative controls � water control: add 5 µl nuclease-free nuclease-free water instead of RNA � Extraction control: mock-extracted RNA obtained by extraction of water or

cell culture medium. In the calculations for the number of reactions (see below) the extraction control is tested as an addtional sample.

o Positive control (MeV control RNA) � Rehydration and dilutions should be done separately from set up of

master mix. � Add 1 µl control RNA plus 4 µl of nuclease-free water.

Preparations for Assay Set-up

• Thaw kit reagents: 5X buffer, dNTP mix, and primers and briefly vortex.

• Spin down all reagents (including enzymes) in microcentrifuge and keep on ice until ready to dispense. Enzymes must be kept on ice at all times.

• Thaw RNA samples and keep on ice during assay set up.

• Record open date of reagents on worksheet for measles genotyping RT-PCR.


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Assay Protocol 1. Determine the number of reactions (n) based on the number of RNA samples to be

tested. Prepare excess reaction volumes (n + 1) to allow for pipetting errors. Calculating the number of reactions: the number of reactions is the number of samples (which includes the extraction control) plus 2 for the nuclease-free water control and the positive control plus 1 to allow for pipetting losses.

Example: If there are 4 specimens and 1 extraction control: make a master mix for 8 reactions:

• 5 samples (specimens and extraction control)

• 1 nuclease-free water control

• 1 MeV control RNA


2. Enter the number of samples in the Excel Measles genotyping RT-PCR master mix worksheet to determine volumes of each reagent to be added.

3. Turn on the thermocycler. 4. In the BSC designated for master mix preparation: Label appropriate number of 0.2 ml

thin-walled reaction tubes and place in pre-chilled metal cooling rack. Keep cooling rack on ice for entire protocol.

5. Add appropriate volumes of the first 5 reagents (nuclease-free water through reverse primer) to a pre-chilled 1.5 ml microcentrifuge tube. Vortex and keep tube on ice.

6. Add RNase inhibitor and enzyme mix to master mix tube. Vortex, spin briefly and chill on ice.

7. Dispense 45 µl master mix (see worksheet) to each reaction tube. Keep reaction tubes on ice in the metal cooling rack after the master mix is dispensed.

8. Proceed to the separate BSC designated for template addition. Using a new, clean pipette tip for each transfer, add RNA to each tube and close the cap. The final volume is 50 µl.

9. Add negative controls (nuclease-free water and extraction control), then the positive control (1 µl control RNA and 4 µl nuclease-free water). Mix.

10. Spin the tubes briefly (10,000 rpm for 1 minute) in a chilled microcentrifuge and immediately return the tube to the metal cooling rack.

11. Start the appropriate program in the thermocycler (see cycling conditions below). Place the samples in the block and start the run.

Cycling parameters for Qiagen kit-Measles 50°C for 30 minutes 95°C for 15 minutes 40 cycles of: 95°C for 30 seconds 55°C for 30 seconds 72°C for 30 seconds Followed by 72°C for 10 minutes Final: hold at 4°C


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Worksheet for Measles Genotyping RT-PCR (Qiagen One-step kit) Below is an example of a worksheet. An Excel spreadsheet with the worksheet is provided in a separate file. Technician: Test date: Kit: Qiagen One-step RT-PCR kit (Qiagen CAT# 210210 or 210212) Date opened: Primer lot number: Number of specimens (including extraction control): Number of additional reactions: 3

Component Vol/rxn Final conc. # of rxns Total vol. 1. NF water 27.5 µl 2. 5x Qiagen One-Step RT-PCR buffer 10 µl 1x 3. dNTP mix 2 µl 0.4 mM 4. Forward primer MeV216 (20 µM) 1.5 µl 0.6 µM 5. Reverse primer MeV 214 (20 µM) 1.5 µl 0.6 µM 6. RNase inhibitor 40 U/µl 0.5 µl 0.4 U/µl 7. Qiagen One-Step Enzyme Mix 2 µl 8. RNA 5 µl xxxxx xxxxx For positive control add 1 µl control RNA and 4 µl NF water

Forward Primer:_______________ Reverse Primer:_______________ Total reaction volume: 50 µl Positive control:____________ Negative control:____________


1

Sequencing protocol for measles genotyping Purpose The following protocol is to be used derive the sequence of the 450 nucleotide region of the measles N gene that is required to determine the genotype. The DNA fragment is a PCR product generated in the measles genotyping RT-PCR reaction (See Measles Genotyping RT-PCR protocol). The primers supplied in the measles genotyping kit are also used for sequencing. This protocol describes the set-up of sequencing reactions, clean-up of sequencing reactions, and quality control for chromatograms. For analysis of chromatograms for genotyping, please see the Sequence Analysis for Measles Genotyping protocol. Important note This protocol was developed for the ABI Prism 3100 Genetic Analyzer. Use of other sequencing instruments may require significant changes to the protocol. Please contact the person responsible for sequencing at your institution to discuss details of the procedure. Reagents and materials needed

• Gloves

• Lab coat


• 0.2 ml thin walled autoclaved PCR tubes

• BigDye Terminator v1.1 (ABI part no. 4336774)

• Sequencing primers MeV214 and MeV216

• Nuclease-free water

• 5X Sequence reaction buffer provided in the BigDye version 1.1 kit

• 96 well plate for thermocycling (AB MicroAmp optical 96 well reaction plate, catalog number N-801-0561)

• 96 well Septa (AB 4319533) Equipment needed


• ABI Prism 3100 Genetic Analyzer

• Bucket with ice

• 96 Well cold plate

• Microcentrifuge with rotor for 1.5 ml tubes and 0.2 ml tubes.


• Vortex

• Thermocycler (ABI GeneAmp PCR system 9700)

Preparation of working solutions for primers Primers are supplied as 200 µM concentrated stocks in the Measles Genotyping Kit Version 2.0. Additional information about the primers can be found in the kit insert. It is necessary to prepare a working solution of each primer prior to setting up a sequencing reaction. Sequencing primers are used at a concentration of 3.2 µM. Do not use the 20 µM working solution for sequencing reactions. Store diluted primers at -20oC.

• MeV214: Mix 1.6 µl stock solution with 98.4 µl nuclease-free water. Vortex.

• MeV216: Mix 1.6 µl stock solution with 98.4 µl nuclease-free water. Vortex


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Template concentrations Generally, use 1 µl of the purified PCR product. A template gel should be run after purification to verify recovery of the PCR product after cleanup (See Clean-up of PCR Products protocol). If the band on the template gel is difficult to see, the amount of template in the sequencing reaction can be increased to 5 µl. The total volume of the sequencing reaction is 20 µl. Addition of different template volumes requires adjustment of added nuclease-free water to result in a final volume of 20 µl. Preparations for assay set up

• Thaw 5X Sequence Reaction Buffer and primers and briefly vortex. Thaw Big Dye Terminator on ice, mix by inverting tube.

• Spin down all reagents in microcentrifuge at 4 °C and keep on ice until ready to dispense.

• Thaw templates and keep on ice during assay set up.

Assay Protocol 1. Determine the number of reactions (n) based on the number of templates to be

sequenced using the Excel spreadsheet for sequencing master mix. Each template is sequenced with the forward and the reverse primer. Sequences are set up in duplicate, resulting in 4 sequencing reactions for each template. Prepare excess reaction volumes (n + 1) for each master mix to allow for pipetting errors. Example: If there are 4 templates: make a master mix for 9 reactions with each MeV primer.

2. Enter the number of samples in the Excel Sequencing Master Mix worksheet to determine volumes of each reagent to be added.

3. Label 0.2 ml thin walled autoclaved PCR tubes and place in 96 well cold plate on ice. 4 tubes are needed for each template.

4. Add all the reagents for the MeV214 master mix to a pre-chilled 1.5 ml microcentrifuge tube. Vortex, briefly centrifuge, and keep on ice.

5. In another 1.5 ml microcentrifuge tube, repeat step 4 for the MeV216 master mix. 6. Dispense 19 µl of master mix into appropriate tubes. Keep reaction tubes on ice in the

metal cooling rack after the master mix is dispensed. 7. Add 1 µl of template to each tube. 8. Close tubes, mix, spin briefly. 9. Start the appropriate program in the thermocycler (see cycling conditions below) and let

the temperature of the block get to 90°C. Pause the run. 10. Add tubes to the thermocycler and continue the run. 11. After the program is completed, proceed to clean-up. Alternatively, reactions can be

stored at 4°C in the dark. Cycling parameters 25 cycles of: 96°C for 30 seconds 50°C for 15 seconds 60°C for 4 minutes Final: hold at 4°C


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Clean-up of sequencing reactions The sequencing reaction products must be purified before loading on the ABI Prism 3100 Genetic Analyzer. Purification removes excess nucleotides, primers and enzyme. Please contact the person responsible for sequencing at your institution to discuss options for clean-up. Many different kits for clean-up of sequencing products are commercially available. It is recommended to use a commercially available kit rather than methods such as ethanol precipitation, because the concentration of recovered DNA with kits is higher and more reproducible. Good results have been obtained with the following kits: CleanSeq kit (Agencourt, catalog # A29150) CentriSep Dye Terminator Removal (Princeton Separations, catalog # CS-900) Other kits are acceptable if they produce purified sequencing reaction products suitable for analysis. Please refer to the kit manual for instructions. Important: In order to place the samples in the ABI Prism 3100 Genetic analyzer, the purified reaction products must be loaded in a MicroAmp optical 96 well reaction plate and covered with a 96 well Septa lid. It is possible to perform the sequencing reaction in the thermocycler in the MicroAmp optical 96 well reaction plate. Alternatively, samples can be transferred before clean-up or afterwards, depending on the clean-up kit used. Operating the sequencing machine Please contact the person responsible for sequencing at your institution to discuss details of the procedure. Saving Data Please save backups of chromatograms. Quality Control To examine the quality of the chromatograms, the chromatograms can be imported into the Sequencing Analysis program which is part of the ABI program package. This program displays the quality of each individual base call in form of a colored bar above the nucleotide sequence (see Figure 1 below). Blue bars indicate that the base call is of acceptable quality while yellow or red bars indicate that the base call is not of acceptable quality. There are four sequences for each template (two forward and two reverse sequences). At least one sequence must have a base call of acceptable quality for each nucleotide position in the sequencing window. Please read the help file of the Sequencing Analysis Program for further details.


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Figure 1. Screenshot of a quality analysis of an ABI sequence file using the Sequence Analysis program.


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Worksheet for Sequencing Master Mix (measles) Below is an example of a worksheet. An Excel spreadsheet with the worksheet is provided in a separate file.

Note: Excel will automatically calculate the volume of the reaction components after

the number of templates is inserted into the blue field.

Technician:

Test date:

Kit: BigDye Terminator v1.1 (ABI part no. 4336774)

Date opened:

Primer lot number:

Number of specimens: 0 Please insert number of

Number of reactions for MeV214 master mix: 1 specimens in blue field


Number of reactions for MeV216 master mix:

(number of specimens multiplied by 2 plus 1 ) 1

Master mix worksheet for MeV214


Nuclease-free water 12 1 12

5x sequence reaction buffer

2 1x 1 2

big dye v1.1 4 1 4

primer (3.2 µM) 1 0.16 µM 1 1

template 1 xxxxxx

Total 20

Master mix worksheet for MeV216


Nuclease-free water 12 1 12

5x sequence reaction buffer

2 1x 1 2

big dye v1.1 4 1 4

primer (3.2 µM) 1 0.16 µM 1 1

template 1 xxxxx

Total 20


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Sequence analysis for measles genotyping Purpose The following protocol is to be used for the analysis of chromatograms to determine the genotype based on the sequence obtained from the sample. Chromatograms are generated by sequencing of PCR products (See Sequencing Protocol for Measles Genotyping). Important: There are many methods available to generate sequence alignments and phylogenetic trees. This protocol described here has been used in inter-country training courses for LabNet and is based on free software (Mega vs. 5.05) which can be downloaded from the internet. Other sequence analysis software may be available to some of the laboratories and this software can be used if it produces results that are consistent with those described below. Equipment needed

• Computer • Mega version 5.05 software. The full manual for Mega vs. 5.05 can be downloaded from:

http://www.megasoftware.net/. This free program can be used to view and edit chromatogram files, create a final sequence, create an alignment with reference sequences, and determine a genotype by phylogenetic analysis.

• Microsoft Office PowerPoint • Text file with measles reference sequences. Please contact Dr. Paul Rota

([email protected]) to obtain this file. • Internet access (for BLAST searches)

File organization

• Before beginning the sequence analysis, copy the chromatogram files (.abi) into a separate folder on the computer and include the WHO reference strains in this folder.

• Write the output files into this folder so that they are easily identified

• Edit one template at a time. Importation of chromatogram files (.abi files from the sequencer)

1. Open MEGA program. 2. Click on Align, then choose Edit/Build alignment. 3. Choose create a new alignment, then click OK. 4. Click on DNA for Are you building a DNA or Protein sequence alignment? An

alignment window will open.

5. Go to Edit, choose Insert Sequence from File (or click the “insert sequence” icon). 6. Browse to the folder containing the .abi sequences and click on the forward primer.abi

file for one template. Click file name to select the sequence. 7. Using this procedure insert the files for the 2 forward sequencing reactions and 2

reverse sequencing reactions into the alignment window. Editing sequences The sequences derived from the reverse primer (MeV 214) must be reverse complemented to align with the forward sequences. Both ends of the sequences must be cropped to obtain the 450 nucleotide sequencing window. The goal of the alignment is to have a sequence that covers the 450 nucleotide window and in which all of the nucleotides of the 2 forward and reverse sequences are aligned in vertical columns.


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1. Change the order of the sequences in the window by clicking on the sequence name on the left and dragging it up or down. Group sequences such that the two forward sequences are on top.

2. Reverse complement the 2 reverse sequencing reactions (primer 214). Click on the sequence name on the left to choose a sequence, then go to Data>Reverse Complement.

3. To align the 4 sequences, select them by clicking on the sequence names while holding the shift key. Go to Alignment>Align by Clustal W. A window will open. Click OK.

4. To find the beginning of the measles genotyping sequence go to Search>Find Motif (or

icon). Type in AAGGTCA (GTC is the starting trinucleotide of the 450-nt window in the measles N gene). Click OK.

5. The query sequence will be highlighted in yellow and this sequence is typically near position 100 in the forward sequences and position 120-140 in the reverse sequences. Use the mouse to select the nucleotides from the beginning of the sequence to the G of the GTC (alternatively, highlight the first nucleotide; hold the shift key then click on the nucleotide before the GTC. This will allow you to highlight the region to be cut). Click on the X to delete the selected nucleotides.

6. The first four nucleotides should now be GTCA. Repeat for the other three sequences. 7. The sequence for this fragment should be about 450 nucleotides long. Look at the

bottom left of the window where it says “Site #” and type “450” then press the Enter key. Nucleotide 450 will be highlighted in white in the middle of the window. Click on the grey bar above the nucleotide to verify the nucleotide number.

8. The 3 nucleotides following immediately after the sequencing window are TAG (stop codon of the N protein). They are not part of the window. Click on the T of the TAG and drag the mouse to the end of the sequence (alternatively, highlight the first nucleotide then scroll down to the end of the sequence; hold the shift key then click on the last nucleotide. This will allow you to highlight the region to be cut). Delete the selected nucleotides.

9. Repeat for the other three sequences. 10. The alignment window should now show 4 sequences, 450 nucleotides in length. If there

are no errors or artifacts, all of the sequences should be identical. Use the slider bar at the bottom of the screen to scroll though the sequences noting positions where all 4 nucleotides are not identical. Look at the grey bars above the nucleotides. There will be an asterisk at every residue where there is agreement between the nucleotides of the 4 sequences at the same position.

11. Check that all sequences stop and start at the same nucleotide. Resolving mismatches If there are any mismatches, look at the chromatograms to resolve the problem:

1. To open the chromatograms, go to Sequencer>Edit Sequence File. Browse to the folder with the .abi files, select and open the appropriate .abi file.

2. The peak height can be adjusted with the slider bars on the right of the chromatogram. 3. To view a chromatogram from the reverse primers, reverse complement the

chromatogram. Choose EDIT>Reverse Complement for each reverse primer. 4. Warning: The nucleotide numbers in the chromatogram do not correspond to the

numbers in the alignment window because the sequences in the alignment window have been cropped. To locate the mismatch in the chromatogram, choose the alignment window, highlight 8-10 nucleotides before the mismatch and right-click to copy the

sequence. Switch to the chromatogram window, click on and right click to paste the


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copied sequence. Compare the sequence preceding the highlighted area to make sure the correct position is highlighted.

5. Compare the mismatch position for all four sequences. If at least one sequence has a high quality peak, the other sequences can be edited to match. Do not change the chromatogram. If changes need to be made, go back to the sequence in the alignment window to make the corrections.

6. Under Edit make sure allow base editing is checked. Click after a nucleotide and backspace to delete, then type in the correct nucleotide.

7. In some cases, especially with the reverse primers, the last 15-20 nucleotides of the 450 nucleotide sequencing window may be difficult to read because these are close to the primer binding site. This proximity to the binding site often leads to high background and “noise” for the first bases. If this is the case, delete these bases.

Saving one sequence for phylogenetic analysis

1. After all nucleotides match, select one of the full length sequences to be used for further analysis and delete the remaining three sequences using Edit->Cut. This leaves a single sequence for the sample.

2. Go to Data>Export Alignment and save the sequence as a .fas (FASTA) file (for example “virus name.fas”). This fragment should be exactly 450 nucleotides in length. The .fas file can be opened with the Microsoft Text Editor if you need to check it.

3. If more than one PCR product was sequenced, repeat the alignment and editing process for all the sequences and generate individual FASTA files for each sequence.

Alignment of sample sequence with reference sequences and phylogenetic analysis

1. If the sample sequence is not in the alignment window, locate the file and open it. (See Importation of chromatogram files above). If more than one PCR product was sequenced, all edited sequences may be imported to be analyzed in one tree. Alternatively, each sample can be analyzed in a separate tree.

2. Go to Insert Sequence from File and browse to find the FASTA file containing the WHO reference sequences. Highlight and import. Check the alignment carefully to make sure that the new sequence aligns with the reference sequences. Check carefully for misalignments in all of the vertical columns. If there is a problem with the alignment, check the chromatograms again.

3. Go to Data>Export Alignment and choose Mega Format. Save the file in mega format (virus name.meg). When prompted to input title of the data, choose a title (e.g. the virus name or outbreak name). When asked whether the sequence is protein-coding nucleotide data, click on Yes.

4. Go to Data>Phylogenetic Analysis. When asked whether the sequence is protein-coding nucleotide data, click on Yes.

5. Minimize the Alignment window. 6. Click on the MEGA 5.05 window. Three squares will be shown in the window, one

labeled TA, one labeled Close data, and one a series of lines (represents the alignment file). Clicking on TA shows the data explorer, which displays variable nucleotide positions. Minimize this window.

7. Proceed to Phylogenetic analysis. Choose: Phylogeny>Construct/Test Maximum Parsimony Tree(s). When asked Would you like to use the currently active data? Choose Yes. Use default setting to draw a tree. Click on Compute. When the tree appears, click on the tree topology button (see below) to turn it off. This will generate a tree with the correct topology.


4

Screen shot of phylogenetic tree before and after the tree topology button was inactivated:

Saving a phylogenetic tree in PowerPoint In the tree window, choose Image, then copy to clipboard then paste into PowerPoint. In PowerPoint, select the tree and then use the Ungroup command twice to generate a tree that can be edited. The labels of the different taxa can now be edited. In the tree window, click on Caption. A window with a figure legend and references for publication will open. You may use this information for publications and your records. After importing the tree into PowerPoint, close the MEGA tree window. Click OK to discard the results. Interpretation of a phylogenetic tree The horizontal lines depict nucleotide differences between strains as indicated by the scale at the bottom of the tree. The differences between two isolates can be calculated by adding the length of the horizontal bars that connect them. Vertical lines are added by the program to facilitate visualization but they do not indicate nucleotide differences. The nodes show where different genotypes branch off. If an unknown sequence branches off the same node as a reference strain, it belongs to the same genotype. Because several of the reference strains were isolated many years ago, more recent isolates of the same genotype can display considerable sequence variation. Always check that the overall topology of the tree is intact. For example, all clade D genotypes (D3, D4, D5 etc.) should branch off the same central node. Optional: BLAST Search using the NCBI website The BLAST search can be used to find sequences in GenBank that are closely related to the sample sequences. Submission of sequences to GenBank is optional, so sequences that are present in MeaNS may not be present in GenBank. The BLAST search can be performed as an additional, optional control for the phylogenetic analysis. For example, if the new sequence is genotype D4, then the most closely related sequences identified in the BLAST search should also be D4. Also, the closely related sequences identified in the BLAST search can be integrated into a phylogenetic tree to show the relationship of the sample sequence to more


5

recent virus isolates. In order to carry out a BLAST search, your computer must be able to access the internet.

1. Open an internet browser and type the address www.ncbi.nlm.nih.gov/BLAST. 2. Select Nucleotide Blast 3. Open the .fas file of the new sequence and copy the sequence using the Microsoft text

editor, highlight the sequence only and copy. 4. On the BLAST webpage, paste the sequence into the field Enter accession numbers. 5. Under database click on ‘others’ and choose the Nucleotide collection (nr/nt), which

includes measles sequences. 6. At the bottom of the page, click on ‘BLAST’. After a brief wait, the results page will be

displayed (see below).

7. Scroll down to the table. Look at the columns query coverage and max identity. Query

coverage should be 100%, indicating that the retrieved sequences cover the entire 450 nucleotide sequencing window. Max identity should be as close to 100% as possible.

8. Look at the column description. Not all descriptions will include a genotype, but those that do should list the same genotype that was identified in the phylogenetic analysis. The most closely related sequences will be listed at the top.

9. Below the table are sequence comparisons of the query sequence with the sequences

listed in the table. Check for the number of nucleotide differences between the query sequence and the retrieved sequences. Click on the square button at the top of each


6

comparison to choose the retrieved sequence for further analysis. Select 4-6 sequences.

10. Click on get selected sequences at the top of the first sequence comparison. The

sequences will then be listed. 11. Click on display settings at the top left and select FASTA (txt) and click Apply. The

FASTA file will be displayed. 12. On the desktop, open the Microsoft text editor. Copy the FASTA file from the BLAST

web site and paste it into the open text file. Save the file with the extension .fas.

13. This file can be edited to add more sequences or change the sequence names. The file

can also be imported into the alignment editor in MEGA to generate a phylogenetic tree as described above.

Reporting

• Mandatory: To fulfill WHO accreditation requirements, LabNet laboratories must submit genotype information to the WHO database within 2 months of completion of the sequence. The Measles-Rubella Genotype Database at WHO/HQ (SharePoint) is a limited access database. The genotype and additional epidemiologic and virologic information must be reported. To get access, contact Dr. Featherstone ([email protected])


7

• Highly recommended: Submission to MeaNS. The MeaNS database, which now contains over 8000 measles sequences, serves as the Global Measles Sequence Database for the WHO LabNet. Genotype and sequence information should be submitted to the MeaNS database at the Health Protection Agency in London. Sequences are automatically submitted by MeaNS to the genotype database at WHO Headquarters and to GenBank (submission to GenBank is optional, but strongly encouraged).

• An account on MeaNS can be requested at: http://www.who-measles.org. Parts of the website are open to the public; however, the ability to submit and analyze sequence data is restricted to members of the WHO LabNet. All contributing users must agree to adhere to the terms and conditions of use which are clearly stated in the database.

• MeaNS will verify the genotype assignment of the submitted sequence. In addition, users can search the MeaNS database to find sequences that most closely match the sequences that they are submitting.

• At present, MeaNS accepts sequences in ASCII format. The FAST files generated above can be cut and pasted into MeaNS. All sequences must have a WHO name and an onset date to be reported to MeaNS. MeaNS will generate a WHO report using information supplied by the submitter. For more information see WER 2012, vol 87, p73-80.


1

Real-time (TaqMan®) RT-PCR Assays for the Detection of Rubella Virus (RuV) E1 Gene RNA and Human RNase P mRNA (a cellular reference gene) using the ABI 7500 Real-Time Thermocycler Purpose The following protocol is to be used to detect RNA of rubella virus (RuV) for diagnostic purposes. For these procedures, RNA is extracted from a clinical sample or from cell culture. If samples give a positive result in the real-time RT-PCR assays, the RNA may be tested in a rubella genotyping RT-PCR assay to amplify the target for sequence analysis and genotyping. Important: This is a general protocol for use with the rubella real-time RT-PCR kit supplied by CDC. Please check the package insert of the kit for updates to the protocol. Reagents and materials needed

70% ethanol

Aluminum foil

Gloves

Lab coat

MicroAmp optical 96-well reaction plate with barcode (Applied Biosystems #4306737)

Nuclease-free water (Ambion #9937)

Optical adhesive cover (Applied Biosystems #4311971)

RNase inhibitor 2000 units (Applied Biosystems N-808-0119)

RNaseZap (Sigma, # R2020-250ML)

Sterile 1.5 ml microcentrifuge tubes

SuperScript Platinum OneStep qRT- PCR Kit (Invitrogen, #11732-020) Equipment needed

-70oC and -20oC freezers

ABI Prism 7500 Sequence Detection System

Bucket with ice

Centrifuge with holder for 96 well plates

Class II BSC with UV light designated for PCR set up

Microcentrifuge

Micropipettors and sterile pipette tips with aerosol-resistant filters

Vortex

Waterbath Recommendations for working with RNA

Wear gloves throughout experiments to prevent contamination from RNases found on human hands.

Change gloves after touching skin (e.g. your face), door knobs, and common surfaces.

Have a dedicated set of pipettors that are used solely for RNA work.

Use filter tips and tubes that are tested and guaranteed to be RNase-free.

Use RNase-free chemicals and reagents.

Reduce RNase contamination by cleaning tube racks, micropipettors, and the work surface of the PCR hood with 70% ethanol and with RNaseZap.

Reduce DNA contamination in the BSC with UV light exposure for 15 minutes.


2

Kit contents The RuV real-time RT-PCR kit contains

RuV primer/probe mix: One tube with 100 μl of a mix of primers and probe for RuV real-time reactions. Content needs to be diluted before use (see below).

RNase P primer/probe mix: One tube with 50 μl of a mix of primers and probe for RNase P real-time reactions. Content needs to be diluted before use (see below).

RuV control RNA (high control). Contains synthetic RuV RNA and total human RNA. Content is dried and needs to be rehydrated and diluted before use (see below). This control can be used for both RuV and RNase P reactions.

RuV control RNA (low control). Contains synthetic RuV RNA and total human RNA. Content is dried and needs to be rehydrated and diluted before use (see below). This tube contains less synthetic RuV RNA than the high control, but the same amount of total human RNA. This control can be used for both RuV and RNase P reactions.

One tube with 2ml TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) for rehydration of controls.

Information about primers and probes

Primer and probe stock solutions are stored at –20oC. Keep probes protected from light.

Probes are labeled at the 5’ terminus with a fluorescent reporter dye, 6-carboxy-fluorescein (FAM), and at the 3’ terminus with a non-fluorescent quencher, black hole quencher-1 (BHQ1).

Final concentration in the reaction mix: o RuV primers: 400 nM o RNase P primers: 300 nM o RuV probe: 200 nM o RNase P probe: 100 nM

RuV primer sequences

o Forward primer (RV11): 5’ CAA CAC GCC GCA CGG ACA AC 3’

o Reverse primer (RV12): 5’ CAA CAC GCC GCA CGG ACA AC 3’

o Reverse primer (RV12-2): 5’ CCA CGA GCC GCG AAC AGT CG 3’ o Probe(RuV): 5’ FAM AG GTC CAG GTC CCG CCC GAC BHQ 3’

RNase P primer sequences o Forward Primer (HURNASE-P-F): 5’ AGA TTT GGA CCT GCG AGC G 3’ o Reverse Primer (HURNASE-P-R): 5’ GAG CGG CTG TCT CCA CAA GT 3’ o Probe (BHQ1 HURNASE-P): 5’ FAM TTC TGA CCT GAA GGC TCT GCG CG

BHQ1 3’ Preparation of working solutions of primer/probe mixes Primer/probe mixes are supplied as 10x (10 fold) concentrated stocks. It is necessary to prepare a working solution of each mix prior to setting up a real-time RT-PCR reaction. Store diluted primers and probes at -20oC. Wrap tubes in aluminum foil to protect from light.

RuV primer/probe mix: Mix 10 μl stock solution with 90 μl nuclease-free water. Vortex.

RNase P primer/probe mix: Mix 10 μl stock solution with 90 μl nuclease-free water. Vortex.


3

Preparation of control RNA stocks Control RNAs are supplied as dried RNA. It is necessary to rehydrate these controls before the first use of the kit. Always work with RNA on ice. Do not work with control RNAs in the same PCR hood where master mix preparation is carried out.

1. To each tube (high and low control RNA) add 100 µl nuclease-free TE (supplied in kit) and vortex for 15 seconds.

2. Heat tubes to 50oC for 10 minutes in water bath then vortex for 15 seconds. Spin briefly to collect.

3. Prepare 10 aliquots of 10 μl each and store at -70oC. Each aliquot should be thawed only once to make a working solution (see below).

Preparation of working solutions of control RNAs 1. Thaw one tube with 10 μl high control RNA stock and one tube with 10 μl low control

RNA stock. 2. To each tube, add 90 μl nuclease-free TE (supplied in kit). Vortex. Spin briefly to collect. 3. Prepare 10 aliquots of 10 μl each and store at -70oC. 4. Use one aliquot for each real-time RT-PCR assay. Discard leftover working solution.

Sample Preparation RNA samples (extracted from a clinical sample or from cell culture) are stored at -70oC. For the assay, 2.5 µl of sample are used per reaction. Addition of different volumes requires adjustment of added water to result in final volume of 25 µl. Assay Controls

Every RNA sample should be tested in parallel with the RuV primer/probe and the RNase P primer/probe. The purpose of the RNase P control reaction is to monitor the integrity of the RNA.

The following standards and controls should be run on each plate as indicated on the real-time 7500 control sheet. They must be included in master mix calculations for each primer/probe set:

o Negative controls Non-template control (NTC): add 2.5 µl/well nuclease-free water instead

of RNA for each primer/probe set evaluated as indicated on real-time 7500 control sheet.

Extraction control: mock-extracted RNA obtained by extraction of water or media using the Qiagen method. In the calculations for the number of reactions (see below) the extraction control is tested as an additional sample

o Two control RNAs (high control and low control) Rehydration and dilutions should be done separately from set up of

master mix. Either control can be used as the RNase P positive control. Add 2.5 µl/well of each control RNA as indicated on real-time control

sheet. Preparations for assay set up

Thaw kit reagents: 2X SS buffer, ROX, and primer/probe mix and briefly vortex.

Spin down all reagents (including enzymes) in microcentrifuge and keep on ice until ready to dispense.


4

Thaw RNA samples and keep on ice during assay set up.

Record open date of reagents on real-time 7500 control sheet. Assay Protocol

1. Determine the number of reactions (n) based on the number of RNA samples to be tested and the format of the real-time 7500 control sheet. Each clinical sample and the extraction control should be tested in triplicate with the RuV primer set, and in a single well for the RNase P primer set. Controls for the RuV primer/probe set (RuV high control, RuV low control, RuV-NTC) are tested in duplicate. Controls for the RNase P primer/probe set (RNase P positive control, RNase P NTC) are tested in a single well. Either the RuV high control or the RuV low control tube can be used as an RNase P positive control. Prepare excess reaction volumes (n + 1) for each primer/probe set to allow for pipetting errors. Calculating the number of reactions: For the RuV primer set, the number of reactions is 3 times the number of samples (which includes the extraction control) plus 6 for the real-time controls plus 1 to allow for pipetting losses. For the RNase P primer set, the number of reactions is the number of samples (which includes the extraction control) plus 2 for the controls plus 1 to allow for pipetting losses.

Example: If there are 4 specimens and 1 extraction control: make a master mix for 22 reactions with the RuV primer set:

5 samples (specimens and extraction control) measured in triplicate=15 reactions

2 NTCs

2 high control RNA

2 low control RNA

1 extra to allow for pipetting losses

Make a master mix for 8 reactions with RNase P primers:

5 samples (specimens and extraction control)

1 NTC

1 with either high or low control RNA


2. Enter the ID# of the sample(s) on the control sheet in the Plate Layout section. 3. Enter the number of samples and controls in the Master Mix worksheet to determine

volumes of each reagent to be added. There are separate worksheets for the rubella primers and the RNase P primers.

4. In the BSC dedicated for master mix preparation, for the RuV primer/probe set, add reagents in a 1.5 ml microcentrifuge tube in the order indicated on the worksheet except for the RNA. Invert, briefly centrifuge, and keep on ice.

5. In another 1.5 ml microcentrifuge tube, add reagents for the RNase P master mix. 6. Proceed to a separate BSC designated for template addition. Dispense 1 reaction

volume of master mix into appropriate wells according to the real-time 7500 control sheet, using a new tip for each master mix. Reaction volume is 22.5 µl/well for RuV and RNase P.

7. Add water (NTC), extraction control, sample RNA or positive control standards as indicated on the real-time control sheet, using a new tip for each well. Sample volume is always 2.5 µl/well. The total volume in each well should now be 25 µl.

8. Seal plate with optical adhesive cover.


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9. Centrifuge the sealed plate at 1500 rpm for 1 minute at room temperature. Assay Run

1. Launch software by double-clicking the 7500 software icon on the desktop. The following instructions are for v2.0.4 of the software.

2. Turn on the 7500 thermocycler – connection is usually automatic. 3. Select Advanced Set-Up. 4. The Experiment Properties screen will open. Name the experiment (usually date and

initials). The settings should be 7500 (96 wells), Quantitation-Standard Curve, TaqMan reagents, Standard Ramp Speed.

5. Go to Plate Setup: Define Targets and Samples. Enter name of each test sample and the extraction control to be tested by clicking on Add New Sample. Each test sample and the extraction control must be listed twice: once for rubella (e.g. sample 1) and once for RNase P (e.g. sample 1-R). Also, add new samples named NTC, High Standard, Low Standard, RNAseP positive, and RNAseP negative.

6. Go to Define Targets. There should be one target (FAM) and one quencher (NFQ-MGB, Blackhole Quencher). This target should be used for every reaction of the plate.

7. Go to Assign Targets and Samples. Using the Plate Layout and screen shot of layout (below) as a guide, highlight all the wells that will contain the test samples, the extraction control, and the RNAse P positive control.

8. Check the box under ‘assign’. All the marked wells will display ‘U’ for ‘unknown’. 9. To define standards, highlight the wells with the high control. Under ‘Assign Targets’,

click on ‘S’ for standard. Under ‘quantity’, fill in 100000 (105). Repeat for the low control with 1000 (103) as quantity.

10. To define the NTC and the RNAseP negative control, highlight the wells for these controls. Under ‘Assign Targets’, click on ‘N’ for NTC.

11. To assign samples, highlight the 3 wells that contain the RuV master mix for the first test sample (e.g. sample 1). Under ‘Assign samples to the Selected Wells’ check the box for sample 1. Repeat for the well that contains sample 1 and RNAseP master mix, check the box for sample 1-R. Repeat this process for each sample. Each well in use should now contain a target, a task (NTC, standard or unknown) and a sample name.

12. Make sure ROX is listed as the passive dye.


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Screen shot from ABI 7500 of the plate layout for rubella real-time RT-PCR: example with 2 rubella test samples. The target is named FAM_NFQ-MGB (the 2 fluorescent dyes used in the assay).

13. Go to Run Method. Set the Sample Volume to 25 μl. Set the step parameters (time/temp) on the thermal profile to the following: 14. RT Step: 48 oC /30 minutes 15. Activation: 95 oC /5 minutes 16. PCR (40 cycles): 95 oC /15 seconds, 60 oC /1 minute 17. Select File > Save As and save file in user folder using a standard format: (e.g.

Date_initials.eds) 18. Open the door of the 7500 by pushing in the indentation on the front. 19. Place a plate into the instrument tray. Orient the A1 well on the plate with the A1 position

on the instrument tray and push the door closed. 20. Select Start Run. Under Run, select Amplification Plot to monitor the run, which should

be complete in approximately 2 hours. Time left in run will be given. 21. At the end of the assay, the green Analyze button will appear.


7

22. Click on Save to save your data.

Assay Quality Control

1. After the run has finished, click on Analyze. The program will calculate an automatic threshold. Do no change the threshold for the analysis of controls.

2. The results will appear. 3. Checklist for quality control: A real-time RT-PCR assay is valid if:

The NTCs for RuV and RNase P and the extraction controls are undetermined (negative).

The automatic threshold is located in the exponential phase of the standard amplification curves.

The Ct values for the duplicate samples of the RuV high control are between 22 and 27 (see picture below).

The Ct values for the duplicate samples of the RuV low control are between 29 and 34 (see picture below).

The Ct value for the single sample of the RNase P control is between 26 and 30.

If any of these criteria are not fulfilled, the assay must be repeated. If the extraction control is positive, the RNA extraction must be repeated.

4. Monitoring real-time RT-PCR assay over time: Ct values for positive controls should be recorded for subsequent runs. After several assays, a typical range of Ct values for each positive control should be determined and used for the analysis of subsequent runs. Changes in these values may indicate problems.

5. Important: the range of Ct values for the positive controls may vary between different lots of the rubella real-time RT-PCR kit. Please check the package insert for updates.

(Picture 1) Sample Data Analysis

1. Check the Multicomponent Data Pane icon on all samples with Ct values < 40 to confirm true amplification as indicated by a rise in FAM fluorescence.

2. Some wells may be highlighted by yellow flags. Click on the QC Summary Pane to see the reason why the wells were flagged. For example, if a sample well did not produce a signal, the program will flag this with No amplification. However, if the sample is negative


8

(for example the extraction control), a lack of amplification is the expected result and not a quality control issue.

3. Adjusting the threshold (if necessary): Highlight the wells containing the samples and the NTC and examine the amplification curves. If a sample has an exponential curve that does not cross the threshold, it is possible to lower the threshold for the analysis of patient samples. Only lower the threshold after the Ct values for the standards have been determined as described in Assay Quality Control.

Click on Analysis Settings. Click on Edit Default Settings. Remove the check from Auto Threshold. Click on Save Change and Apply Analysis Settings. Close.

In the amplification plot, click on the threshold and drag it to the desired position. Every time the threshold is moved, the ct values for the samples will be automatically recalculated.

Important: The signal for the NTC must remain below the threshold setting. The threshold must be located in the lower third of the exponential phase of the graph. See picture 2 (shows the 2 positive controls and a typical positive test sample).

(Picture 2)

If the threshold has to be moved, write down the value for threshold, because the program does not save this information.

Interpretation of results for RuV, RNase P

Viral gene RNase P gene Specimen Result

< 40 < 40 positive

< 40 undetermined positive

undetermined < 40 negative

undetermined undetermined indeterminate

If the PCR with the RuV primers/probe produced a Ct below 40, the specimen is positive for rubella. It does not matter whether the RNase P reaction is positive or negative.


9

If the result of the RuV PCR is undetermined but the RNase P PCR produced a Ct below 40, the specimen is negative for rubella. The positive RNase P result indicates that sufficient RNA was extracted from the specimen to allow amplification to occur.

If the result of the RuV PCR and the RNase P PCR are undetermined, the specimen is judged to be indeterminate. It is not possible determine a result because there was not enough RNA to allow amplification to occur.

Clinical samples with 2 or 3 positive reactions (out of the 3 replicates run on the plate) will be reported positive.

Exporting Data To save the data to an Excel file: Click on Export, then

1. Under: select data to export, choose Results 2. Under: Select one file or separate files, choose One file 3. Choose a file name and browse to the location where the file should be saved. 4. Click Open, then click Start Export. 5. Close the Export Tool.


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Worksheet for Rubella Real-time RT-PCR for Rubella RNA Detection (page 1 of 2) Below is an example of a worksheet. An Excel spreadsheet with the worksheet is provided in a separate file. Technician: File name: Test date: Kit: Superscript III One-Step qRT-PCR (Invitrogen #11730-020) Date opened: Primer mix lot number: Number of specimens (including extraction control): Number of additional reactions for Rubella primer set: 7 Number of additional reactions for RNase P primer set: 3 Master Mix Worksheet for rubella virus primer/probe set


RNase-free water 7.2

2x SS buffer 12.5 1x

RuV primer/probe mix 2.0

SS III/Taq mix (5U/µl) 0.5 0.025 U/µl

ROX reference 0.05 0.25 U/µl

RNase inhibitor 0.25 0.4 U/µl

RNA 2.5 xxxx xxxx

Total 25

Master Mix Worksheet for human RNase P primer/probe set


RNase-free water 7.2

2x SS buffer 12.5 1x

RnaseP primer/probe mix 2.0

SS III/Taq mix (5U/µl) 0.5 0.025 U/µl

ROX reference 0.05 0.25 U/µl

RNase inhibitor 0.25 0.4 U/µl

RNA 2.5 xxxx xxxx

Total 25

Dispense master mix: 22.5 µl/well for both master mixes. Add 2.5 µl template.


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Worksheet for Rubella Real-time RT-PCR for Rubella RNA Detection (page 2 of 2) Below is an example of a worksheet with two samples. An Excel spreadsheet with the worksheet is provided in a separate file. Plate layout for real-time RT-PCR assay Sample 1 (S1) tested in triplicate with RuV primers

1 2 3 4 5 6 7 8 9 10 11 12

A S1 S1 S1 S1-R RuV high RuV high

B S2 S2 S2 S2-R RuV low RuV low

C - -

D RuV NTC RuV NTC

E - -

F RNP pos RNP neg

G - -

H - -

Sample 1 (S1) tested with primers for RNaseP RuV high: RuV control RNA (high) RuV low: RuV control RNA (low) NTC: negative control for RuV (water) RNP pos: positive control for RNase P (RuV control RNA high or low) RNP neg: negative control for RNase P (water) White field: RuV master mix Grey field: RNase P master mix

Worksheet for Rubella Real-time Master Mix

Note: Excel will automatically calculate the volume of the reaction components after

the number of specimens is inserted into the blue field.

Technician:

File name:

Test date:

Kit: Superscript III One-Step qRT-PCR (Invitrogen #11732-020)

Date opened:

Primer mix lot number:

Number of specimens (including extraction control): 0 Please insert number of

Number of reactions for rubella reaction mix: 7 specimens in blue field


Number of reactions for Rnase P reaction mix:

(number of specimens plus 3) 3

Master mix worksheet for rubella primer/probe set


RNase-free water 7.2 7 50.4

2x SS buffer 12.5 1x 7 87.5

RuV primer/probe mix 2 7 14




RNA 2.5 xxxx xxxx

Total 25

Master mix worksheet for Rnase P primer/probe set


RNase-free water 7.2 3 21.6

2x SS buffer 12.5 1x 3 37.5

RNP primer/probe mix 2 3 6




RNA 2.5 xxxx xxxx

Total 25

Plate Layout for Rubella Real-time

Technician:

File name:

Test date:

1 2 3 4 5 6 7 8 9 10 11 12

A RuV high RuV high

B RuV low RuV low

C - -

D RuV NTC RuV NTC

E - -

F RNP pos RNP neg

G - -

H - -

RuV master mix

RNase P master mix

RuV high: RuV control RNA (high)

RuV low: RuV control RNA (low)

NTC: negative control for RuV primers (water)

RNP pos: positive control for RNase P primers (RuV control RNA high or low)

RNP neg: negative control for RNase P primers (water)


1

RT-PCR protocol for rubella genotyping (2 Fragment System) Purpose The following protocol is to be used for the amplification of 2 fragments from the rubella virus (RuV) envelope protein 1 (E1) coding region. Sequences derived from the 2 overlapping DNA fragments can be combined to generate the recommended 739nt sequence for rubella virus genotype determination. This protocol should not be used for detection of RuV RNA; the Rubella Real-time RT-PCR protocol or the Rubella Detection RT-PCR protocol are more sensitive and should be used instead. For these procedures, RNA is extracted from a clinical sample or from cell culture. This is a one-tube reaction, so there is a minimum of specimen handling. Primers and control RNA are supplied by CDC as a kit. Important: This is a general protocol for use with the rubella genotyping kit supplied by CDC. Please check the package insert of the kit for updates to the protocol. What to do with the PCR products Two PCR products should be produced: the expected product size for the 5’ fragment (Fragment 1) is 480 nts and the product size for the 3’ fragment (Fragment 2) is 633 nts. Analyze PCR products on agarose gels (See Agarose Gel Electrophoresis protocol) Laboratories with sequencing capacity should purify and sequence the PCR products (See Clean-up of PCR Products and Rubella Sequencing Reactions protocol). Laboratories without sequencing capacity should ship the PCR product to the regional reference laboratory for sequencing. PCR products can be shipped without drying or purification. Transfer the PCR reaction into a 1.5 ml tube, seal with parafilm and ship at ambient temperature. PCR products are stable at room temperature for at least one month. Reagents and materials needed

70% ethanol

Lab coats

Disposable gloves

RNase inhibitor 2000 units (Applied Biosystems N-808-0119)

RNaseZap (Sigma, # R2020-250ML)

Sterile 1.5 ml microcentrifuge tubes

Nuclease-free, deionized water (NF water)

Qiagen One-step RT-PCR kit (Qiagen CAT# 210210 or 210212)

Autoclaved PCR tubes (0.2 ml, thin-walled)

Rubella Genotyping RT-PCR Kit, (primers and control RNA)

Equipment needed

-70oC and -20oC freezers

Bucket with ice

Class II biological safety cabinet with UV light (BSC)

Micropipettors and sterile pipette tips with aerosol-resistant filters

Vortex mixer

Metal cooling rack for 0.2 ml tubes

Thermocycler (AB GeneAmp PCR System 9700)

Microcentrifuge, refrigerated to 4 °C with rotor for 1.5 ml tubes and 0.2 ml tubes


2

Recommendations for working with RNA

Use dedicated equipment, rooms and biosafety cabinets for all pre-PCR procedures. Post amplification analysis and processing should be performed in separate rooms using dedicated equipment. Do not share equipment (including lab coats) between pre-PCR and post-PCR procedures.

Wear gloves throughout experiments to prevent contamination from RNases found on human hands.

Change gloves after touching skin (e.g. your face), door knobs, and common surfaces.

Have a dedicated set of pipettors that are used solely for RNA work.

Use filter tips and tubes that are tested and guaranteed to be RNase-free.

Use RNase-free chemicals and reagents.

Reduce RNase contamination by cleaning tube racks, micropipettors, and the work surface of the PCR hood with 70% ethanol and RNaseZap.

Reduce DNA contamination in the BSC with UV light exposure for 15 minutes. Kit contents The RuV genotyping kit contains

50 ul 10x rubella virus forward primers RV8633 and RV8945. Content needs to be diluted before use (see below).

50 μl 10x rubella virus reverse primers RV9112 and RV9577. Content needs to be diluted before use (see below).

Dried rubella RNA control. Content is dried and needs to be rehydrated and diluted before use (see below).

Information about primers

RuV primer sequences o Forward Primer 1 (RV8633): 5’ AGC GAC GCG GCC TGC TGG GG 3’ o Forward Primer 2 (RV8945): 5’ TGG GCC TCC CCG GTT TG 3’ o Reverse Primer 1 (RV9112): 5’ GCG CGC CTG AGA GCC TAT GAC 3’ o Reverse Primer 2 (RV9577): 5’ CGC CCA GGT CTG CCG GGT CTC 3’

The location of the primer binding sites is described in a separate file (Primers and Reference sequences).

Primer stock solutions are stored at -20oC.

Final concentration of primers in the reaction mix is 600 nM. Information about the positive control The rubella RNA control is an RNA transcript of the rubella E1 coding region which has been modified so that amplified products for both fragments will be approximately 80 nt smaller than RT-PCR products from patient samples. Using this control simplifies the identification of contamination. Preparation of working solutions of primers Primers are supplied as 10x (10 fold) concentrated stocks. The concentration in the stocks is 200 μM. It is necessary to prepare a working solution of each primer prior to setting up a RT-PCR reaction. The concentration of the working dilution is 20 μM. Store diluted primers at -20oC. Do not freeze-thaw more than 5 times.

RV8633: Mix 10 μl stock solution with 90 μl NF water. Vortex.


3




Preparation of control RNA stocks The rubella control RNA is supplied as dried RNA. It is necessary to rehydrate it before the first use of the kit. Always work with RNA on ice. Do not work with control RNA in the same PCR hood where master mix preparation is carried out.

1. Add 100 µl nuclease-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) and vortex for 15 seconds.

2. Incubate the tube at room temperature for 10-15 minutes then vortex for 15 seconds. Spin briefly to collect.

3. Prepare 10 μl aliquots and store at -70oC. Preparation of working solutions of control RNA

1. Thaw one tube with 10 μl control RNA stock. 2. Add 90 μl nuclease-free water or TE buffer. Vortex. Spin briefly to collect. 3. Prepare 10 μl aliquots and store at -70oC for long-term storage and -20oC for short-term

storage. 4. Use 5 µl per RT-PCR reaction.

Sample Preparation RNA samples (extracted from a clinical sample or from cell culture) are stored at -70oC. Usually, RNA is extracted from one 25 cm2 flask of infected cells or from 100-200 µl of clinical material. Most of the extraction protocols yield 50-60 µl of RNA. For the assay, 5 µl of sample are used per reaction. The volume of RNA can be increased, but this will not significantly improve the sensitivity. Addition of different volumes to a reaction requires adjustment of water to result in a final volume of 50 µl. . Assay Controls

The following controls must be run in each assay. They must be included in master mix calculations:

o Negative controls Water control: add 5 µl nuclease-free water instead of RNA Extraction control: mock-extracted RNA obtained by extraction of water or

media. In the calculations for the number of reactions (see below) the extraction control is tested as an addtional sample.

o Positive control (RuV control RNA) Rehydration and dilutions should be done separately from set up of

master mix. Add 5 µl..

Preparations for Assay Set-up Note: rubella virus has a high GC content and amplification will be enhanced by the addition of a GC melt reagent such as the Qiagen Q solution included in the kit. Always add this reagent for rubella genotyping reactions.

Thaw kit reagents: 5X buffer, Q solution, dNTP mix, and primers and briefly vortex.

Spin down all reagents (including enzymes) in microcentrifuge and keep on ice until ready to dispense. Enzymes must be kept on ice at all times.

Thaw RNA samples and keep on ice during assay set up.


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Record open date of reagents on worksheet for rubella genotyping RT-PCR. Assay Protocol

1. For rubella genotyping 2 master mixes need to be set up – one for each primer set. For each primer set determine the number of reactions (n) based on the number of RNA samples to be tested. Prepare excess reaction volumes (n + 1) to allow for pipetting errors. Calculating the number of reactions: for each master mix the number of reactions is the number of samples (which includes the extraction control) plus 2 for the water control and the positive control plus 1 to allow for pipetting losses.

Example: If there are 4 specimens and 1 extraction control: make master mix 1 for 8 reactions, then repeat for master mix 2:

5 samples (specimens and extraction control)

1 water control

1 RuV control RNA


2. Enter the number of samples in the Excel Genotyping Master Mix worksheet to determine volumes of each reagent to be added.

3. In the BSC designated for master mix preparation: Label appropriate number of 0.2 ml thin-walled reaction tubes and place in pre-chilled metal cooling rack. Keep cooling rack on ice for entire protocol.

4. For each master mix, add appropriate volumes (see Excel Worksheet for Rubella Genotyping RT-PCR) of the first 6 reagents (NF water through reverse primer) to pre-chilled 1.5 ml microcentrifuge tubes. For master mix 1 use primers RV8633/RV9112 and for master mix 2 use primers RV8945/RV9577. Vortex and keep tubes on ice.

5. Add RNase inhibitor and enzyme mix to master mix tubes. Vortex and chill briefly on ice. 6. Briefly spin down the master mix tubes in a microcentrifuge. Dispense 45 µl master mix

(see worksheet) to each reaction tube. Keep reaction tubes on ice in the metal cooling rack after the master mix is dispensed.

7. Proceed to the separate BSC designated for template addition. Using a new, clean pipette tip for each transfer, add test sample RNA(s) to the appropriate tube(s) and close the cap. The final volume is 50 µl.

8. Add negative controls (NF water and extraction control), then the positive control (5 µl control RNA). Mix.

9. Spin the tubes briefly (10,000 rpm for 1 minute) in a chilled microcentrifuge and immediately return the tube to the metal cooling rack.

10. Start the appropriate program in the thermocycler (see cycling conditions below). Place the samples in the block and start the run.

Cycling parameters for Qiagen-Rubella 50°C for 30 minutes 95°C for 15 minutes 40 cycles of: 95°C for 30 seconds 60°C for 30 seconds 72°C for 1minute Followed by 72°C for 10 minutes and final hold at 4°C

user

螢光標示

We have only one BSC in the room for template addition (tentatively in Room 907). It is the same room as RNA extraction. Template will be added on open bench. Same for preparation of positive RNA control.

user

螢光標示

We don't have enough pre-chilled metal cooling rack. We can provide trays with ice for the participants to put their racks on.

user

螢光標示

We don't have this equipment.


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Worksheet for Rubella Genotyping RT-PCR (Qiagen One-step kit) Below is an example of a worksheet. An Excel spreadsheet with the worksheet is provided in a separate file. Technician: Test date: Kit: Qiagen One-step RT-PCR kit (Qiagen CAT# 210210 or 210212) Date opened: Primer lot number: Number of specimens (including extraction control): Number of additional reactions: 3

Component: Master Mix Fragment 1 Vol/rxn Final conc. # of rxns Total vol.

1. NF water 17.5 µl

2. 5x Qiagen One-Step RT-PCR buffer 10 µl 1x

3.Q Solution 10 µl

4. dNTP mix 2 µl 0.4 mM

5. Forward primer RV8633 (20 µM) 1.5 µl 0.6 µM

6. Reverse primer RV9112 (20 µM) 1.5 µl 0.6 µM

7. RNase inhibitor 40 U/µl 0.5 µl 0.4 U/µl

8. Qiagen One-Step Enzyme Mix 2 µl

9. RNA 5 µl xxxxx xxxxx

Total reaction volume: 50 µl Positive control:____________ Negative control:____________

Component: Master Mix Fragment 2 Vol/rxn Final conc. # of rxns Total vol.

1. NF water 17.5 µl

2. 5x Qiagen One-Step RT-PCR buffer 10 µl 1x

3.Q Solution 10 µl

4. dNTP mix 2 µl 0.4 mM

5. Forward primer RV8945 (20 µM) 1.5 µl 0.6 µM

6. Reverse primer RV9577 (20 µM) 1.5 µl 0.6 µM

7. RNase inhibitor 40 U/µl 0.5 µl 0.4 U/µl

8. Qiagen One-Step Enzyme Mix 2 µl

9. RNA 5 µl xxxxx xxxxx

Total reaction volume: 50 µl Positive control:____________ Negative control:____________


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Sequence analysis for rubella genotyping Purpose The following protocol is to be used for the analysis of chromatograms to determine the genotype of the sequenced specimen. Chromatograms are generated by sequencing of PCR products (protocol Sequencing for rubella genotyping). Important: This protocol is based on free software which can be downloaded from the internet.. Equipment needed

Computer Mega version 5.05 software. The full manual for Mega v5.05 can be downloaded from:

http://www.megasoftware.net/. This free program can be used to view and edit chromatogram files, create a final sequence, create an alignment with reference sequences, and determine a genotype by phylogenetic analysis.

Microsoft Office PowerPoint Text file with rubella reference sequences. Please contact Emily Abernathy

([email protected]) to obtain this file. File organization

Before beginning the sequence analysis, make a folder on your desktop. Copy the chromatogram files (.abi) into the folder and also copy the file with the WHO reference strains.

As you proceed with the analysis, write the output files into this folder so that you will be able to find everything easily.

Edit one template at a time. Importation of chromatogram files (abi files from the sequencer)

1. Open MEGA program. 2. Click on Align. Next, choose Edit/Build alignment. 3. Choose create a new alignment, then click OK. 4. Click on DNA for Are you building a DNA or Protein sequence alignment? An

alignment window will open.

5. Go to Edit, choose Insert Sequence from File (or click the “insert sequence” icon). 6. Browse to the folder containing the .abi sequences and click on the Fragment 1 forward

primer.abi file. Click file name to select the sequence. 7. Using this procedure insert the files for Fragment 1 (primers 8633 and 9112).

Editing of sequences for Fragment 1 The sequence derived from the reverse primer must be reverse complemented to align with the forward sequence. The forward and reverse sequences must be aligned. Both ends of the sequences must be cropped to obtain a 350 nt sequence. The goal of the alignment is to have a sequence that covers the 350 nucleotide window in which all of the nucleotides match.

1. Reverse complement the primer RV9112 reverse sequence. Click on the sequence name on the left to choose a sequence, then go to Data>Reverse Complement.

2. To align the 2 sequences, select them by clicking on the sequence name of both primers while holding the shift key. Go to Alignment>Align by Clustal W. A window will open. Click OK.

http://www.megasoftware.net/

mailto:[email protected]


2

3. To find the beginning of the rubella fragment 1 sequence go to Search>Find Motif (or

icon). Type in GTTYCAYAC (GTT is the starting trinucleotide of the 739-nt genotyping window and Y can be a T or a C). Click OK.

4. The query sequence will be highlighted in yellow. Use the mouse to select the nucleotides from the beginning of the sequence for both sequences to the G of the GTT (alternatively, highlight the first nucleotide; hold the shift key then click on the nucleotide before the GTT. This will allow you to highlight the region to be cut). Click on the X to delete the selected nucleotides.

5. The first 3 nucleotides should now be GTT for both sequences. 6. The sequence for this fragment should be 350 nt long. Look at the bottom left of the

window where it says “Site#” and type “350” then press the Enter key. Nucleotide 350 will be highlighted in white in the middle of the window. Click on the grey bar above the nucleotide to verify the nucleotide #.

7. To crop the sequences to the proper length, click on nt 351 for both sequences. Scroll to the end. Holding down the shift key, click on the last nt. The nts to be cut for both sequences should now be blue. Click on the X to delete the selected nucleotides. The final 6 nts of Fragment 1 should now be AGGTGT.

8. The alignment window should now show 2 sequences, 350 nt in length. If there are no errors or artifacts, the sequences should be identical. Use the slider bar at the bottom of the screen to scroll though the sequences noting positions where the 2 nucleotides are not identical. Look at the grey bars above the nucleotides. There will be an asterisk at every residue where there is agreement between the nucleotides of the 2 sequences.

9. Check that both sequences stop and start at the correct nucleotides (errors such as insertions or deletions in one of the sequences may affect the length of the fragment).

Resolving mismatches If there are any mismatches, look at the chromatograms to resolve the problem:

1. To open the chromatograms, go to Sequencer>Edit Sequence File. Browse to the folder with the .abi files, select and open the appropriate .abi file.

2. The peak height can be adjusted with the slider bars on the right of the chromatogram. 3. If you are viewing a chromatogram from one of the reverse primers, you need to reverse

complement the chromatogram. Choose EDIT>Reverse Complement for each reverse primer.

4. Warning: The nucleotide numbers in the chromatogram do not correspond to the numbers in the alignment window because the sequences in the alignment window have been cropped. To locate the mismatch in the chromatogram, choose the alignment window. Highlight 8-10 nucleotides before the mismatch and right-click to copy the

sequence. Switch to the chromatogram, click on and right click to paste the copied sequence. Compare the sequence preceding the highlighted area to make sure that you have found the correct position.

5. Compare the mismatch position for the 2 sequences. If at least one sequence has a high quality peak, you can edit the other sequence to match. Do not change the chromatogram. If you find that changes need to be made, go to back to the sequence in the alignment window and make the correction.

6. Under Edit make sure allow base editing is checked. You can click after a nucleotide and backspace to delete, then type in the correct nucleotide.

7. After all nucleotides match, select one of the sequences to be used for further analysis and delete the remaining three sequences using Edit->Cut. This leaves a single sequence for the sample.

GW

螢光標示

Should be "Sequencer"

user

註解

“Unmarked”的設定者是“user”

GW

螢光標示

Should be "sequence"


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8. Go to Data>Export Alignment and save the sequence as a .fas (FASTA) file (for example “virus name_frag1.fas”). This fragment should be exactly 350 nt in length. The .fas file can be opened with the Microsoft Text Editor if you need to check it.

Editing of sequences for Fragment 2 The above process needs to be repeated with Fragment 2 to complete the 739 nt window.

1. Open a new Alignment window, then browse to select the Fragment 2 primers RV8945 and RV9577.

2. Reverse complement the primer RV9577 reverse sequence. Click on the sequence name on the left to choose a sequence, then go to Data>Reverse Complement.

3. To align the 2 sequences, select them by clicking on the sequence name of both primers while holding the shift key. Go to Alignment>Align by Clustal W. A window will open. Click OK.

4. To find the beginning of the rubella fragment 1 sequence go to Search>Find Motif (or

icon). Type in GGGTSACSCC where S can be a C or G. This will be the beginning of fragment 2. Click OK.

5. The query sequence will be highlighted in yellow. Use the mouse to select the nucleotides from the beginning of the sequence for both sequences to the G of the GGG (alternatively, highlight the first nucleotide; hold the shift key then click on the nucleotide before the GGG. This will allow you to highlight the region to be cut). Click on the X to delete the selected nucleotides.

6. The first 3 nucleotides should now be GGG for both sequences. 7. The sequence for this fragment should be 389 nt long. Look at the bottom left of the

window where it says “Site#” and type “389” then press the Enter key. Nucleotide 389 will be highlighted in white in the middle of the window. Click on the grey bar above the nucleotide to verify the nucleotide #.

8. To crop the sequences to the proper length, click on nt 390 for both sequences. Scroll to the end. Holding down the shift key, click on the last nt. The nts to be cut for both sequences should now be blue. Click on the X to delete the selected nucleotides. The final 6 nts should now be GGGYGAG where Y can be a C or a T.

9. The alignment window should now show 2 sequences, 389 nt in length. If there are no errors or artifacts, the sequences should be identical. Use the slider bar at the bottom of the screen to scroll though the sequences noting positions where the 2 nucleotides are not identical. Look at the grey bars above the nucleotides. There will be an asterisk at every residue where there is agreement between the nucleotides of the 2 sequences.

10. Check that both sequences stop and start at the correct nucleotides. 11. Resolve all mismatches as done for fragment 1. Cut one the sequences as done for

fragment 1. Go to Data>Export Alignment and save the sequence as a Fasta file (virus name_frag2.fas).

Combining the 2 fragments to make the full 739 nt window 1. There should now be 1 sequence for fragment 2 in the alignment window. Click the Insert

Sequence icon , browse to the folder containing the fragment 1 .fas file made above; select the .fas file for fragment 1 and open it. 2. Click on the first nucleotide of the fragment 2 sequence. Scroll to the end of the fragment 2 sequence, hold down the shift key and click on the last nucleotide of the fragment 2 sequence. Go to Edit<Copy. 3. Click in the space after the last nucleotide of the Fragment 1 sequence. Go to Edit<Paste. 4. The completed sequence should be 739 nucleotides in length. Scroll to the end of the combined fragments to check for the proper length.


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5. Select the Fragment 2 sequence and delete it, leaving a single complete sequence in the window. 6. Rename the sequence by double clicking in the sequence name box (virus name_739). 7. Export the 739 sequence as a .fas file (virus name_739.fas). Alignment of sample sequence with reference sequences and phylogenetic analysis

1. If the completed 739 sequence is not in the alignment window, locate the file and open it. If more than one PCR product was sequenced, all edited sequences may be imported to be analyzed in one tree. Alternatively, each sample can be analyzed in a separate tree.

2. Go to Import Sequences and browse to find the fasta file containing the 32 WHO rubella reference sequences. Highlight and import. Check the alignment carefully to make sure that the new sequence aligns with the reference sequences. If there is a problem with the alignment, check the chromatograms again.

3. Go to Data>Phylogenetic Analysis. When asked whether the sequence is protein-coding nucleotide data, click on Yes.

4. The Alignment window can be minimized (if you want to save the alignment file, choose Export>Fasta format or MEGA format).

5. Click on the MEGA 5.05 window. Three squares will be shown in the window, one labeled TA, one labeled Close data, and one a series of lines (represents the alignment file). Clicking on TA shows the data explorer, which displays variable nucleotide positions. Minimize this window.

6. Proceed to Phylogenetic analysis. Choose: Phylogeny>Construct Test/Neighbor Joining Tree(s). Use default settings. Click on Compute. A new window will open showing the tree.

Printing a phylogenetic tree In the tree window, go to File>Print in a Sheet. In the tree window, click on Caption. A window with a figure legend and references for publication will open. You may use this information for publications and your records. To print the caption, click on the print icon. Saving a phylogenetic tree in PowerPoint In the tree window, choose Image, then copy to clipboard then paste into PowerPoint. In PowerPoint, select the tree and then use the Ungroup command twice to generate a tree that can be edited. The labels of the different taxa can now be edited. After importing the tree into Powerpoint, close the window. Click OK to discard the results. Interpretation of a phylogenetic tree The horizontal lines depict nucleotide differences between strains as indicated by the scale at the bottom of the tree. The differences between two isolates can be calculated by adding up the length of the horizontal bars that connect them. Vertical bars are added by the program to facilitate visualization but they do not indicate nucleotide differences. The nodes show where the different genotypes branch off. If an unknown sequence branches off the same node as a reference strain, it belongs to the same genotype. Because several of the reference strains were isolated many years ago, more recent isolates of the same genotype can display considerable sequence variation. Always check that the overall topology of the tree is intact. For example, genotype 2B sequences should branch off the same central node. BLAST Search The goal of a BLAST search is to find sequences in GenBank that are closely related to the sample sequences. This may provide additional information beyond that which is obtained by

GW

螢光標示

Should be "Edit>Insert Sequence from File"

user

螢光標示

Should be "Data>Export Alignment"


5

comparing an unknown sequence to sequences of reference viruses. For example, if the new sequence is identical to a rubella virus sequence which has been recently deposited in GenBank, a BLAST search will find it. However, the set of rubella virus sequences in GenBank is currently far less robust than the set of measles virus sequences deposited there. For example, there are rubella virus sequences in GenBank which contain errors or have been assigned the wrong genotype by the depositor; therefore BLAST results should be interpreted with caution. In order to carry out a BLAST search, your computer must be able to access the internet.

1. Open an internet browser and type the address www.ncbi.nlm.nih.gov/BLAST. 2. Open the .fas file of the new sequence and copy the sequence using the Microsoft text

editor, highlight the sequence only and copy. 3. On the BLAST webpage, paste the sequence into the field Enter accession numbers. 4. Select nucleotide blast 5. Under database click on ‘others’ and choose the Nucleotide collection (nr/nt), which

includes measles sequences. 6. At the bottom of the page, click on ‘BLAST’. After a brief wait, the results page will be

displayed (see below).

7. Scroll down to the table. Look at the columns query coverage and max identity. Query

coverage should be 100%, indicating that the retrieved sequences cover the entire 450 nucleotide sequencing window. Max identity should be as close to 100% as possible.

8. Look at the column description. Not all descriptions will include a genotype, but those that do should list the same genotype that you have identified in your phylogenetic analysis. The most closely related sequences will be listed at the top.

http://www.ncbi.nlm.nih.gov/BLAST


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9. Below the table are sequence comparisons of your query sequence with the sequences listed in the table. You can check for the number of nucleotide differences between your query sequence and the retrieved sequences. Click on the square button at the top of each comparison to choose the retrieved sequence for further analysis.

10. Click on get selected sequences at the top of the first sequence comparison. The

sequences will then be listed. 11. Click on display settings at the top left and select FASTA (txt) and click Apply. The

FASTA file will be displayed. 12. On the desktop, open the Microsoft text editor. Copy the FASTA file from the BLAST

web site and paste it into the open text file. Save the file with the extension .fas. 13. This file can be edited to add more sequences or change the sequence names. The file

can also be imported into the alignment editor in MEGA to generate a phylogenetic tree as described above.

Reporting

Mandatory: To fulfill WHO accreditation requirements, LabNet Laboratories must submit genotype information to WHO within 2 months of completion of the sequence. The Measles-Rubella Genotype Database at WHO/HQ (Sharepoint) is a limited access database. The genotype and additional epidemiologic and virologic information must be reported. To get access, contact Dr. Featherstone ([email protected])

A database for rubella sequences similar to MeaNS is under construction, but is not accessible at present.

Highly recommended: Submission to GenBank. GenBank is a repository of sequences that allows all users to share and and compare sequences. BLAST searches use the GenBank database. Without continued submission to GenBank BLAST searches will not return current sequences. Submission to GenBank does not constitute publication.

o Direct submission to GenBank can be made using these programs:


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Bankit (www.ncbi.nlm.nih.gov) Sequin (www.ncbi.nlm.nih.gov)

o CDC can help to prepare sequences for submission.

http://www.ncbi.nlm.nih.gov/


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Agarose Gel Electrophoresis Purpose The following protocol is to be used for the visualization of PCR products. This protocol can be used for the products of measles genotyping RT-PCR, rubella genotyping RT-PCR and rubella diagnostic RT-PCR. Many different gel casting and gel electrophoresis systems are commercially available. The protocol must be adapted to the materials available in the laboratory. Other protocols for gel electrophoresis are acceptable if they provide adequate resolution of bands in the range of 150-800 base pairs. Reagents and materials needed

• 1X TBE (Tris-borate EDTA) buffer or 1X TAE (Tris-acetate EDTA) buffer

• Agarose (e.g. Invitrogen Ultra-Pure, catalog number 15510-027)

• De-ionized water

• Gloves

• Lab coat

• Ethidium Bromide (EtBr) at a concentration of 10µg/ml or GelRed (Biotium, # 41003)

• Loading dye containing bromophenol blue (e.g. Promega, catalog number G190A)

• Molecular weight marker (e.g. 100 bp ladder, Invitrogen, catalog number 15628-019) Equipment needed

• Agarose gel casting tray and comb(s)

• Agarose gel electrophoresis box

• Camera

• Graduated cylinder

• Heat-resistant glove


• Microwave-safe glass beaker or flask

• Parafilm

• Power supply

• Scale

• UV transilluminator Precautions

• Gel electrophoresis is a post amplification procedure and should not be performed in the same room where the RT-PCR reactions are set up. Do not share equipment (including lab coats) between pre-PCR and post-PCR rooms.

• Ethidium Bromide is a carcinogen. Always wear gloves while preparing and working with EtBr-containing agarose gels. Dispose of all waste and gel matter containing EtBr in the proper EtBr waste containers.

• To avoid working with EtBr it may be replaced with GelRed. GelRed is a non-carcinogenic dye which is at least as sensitive as EtBr for visualizing DNA bands in agarose gels. It can be used with the same UV transilluminators and cameras used for gels stained with EtBr.

Assay Protocol There are many different gel casting systems and gel electrophoresis boxes available. The volume of gel solution as well as voltage and running time must be determined based on the available equipment.


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1. Weigh appropriate amount of agarose. For PCR products of measles and rubella genotyping RT-PCR and for products of rubella diagnostic RT-PCR 2% agarose gels will work well. Add 1 X TBE buffer (TAE is also acceptable).

2. Melt agarose in a microwave for laboratory use only. Avoid over-heating, as the agarose will bubble all over the inside of the microwave.

3. Add 1 µl of Ethidium bromide solution (10µg/ml) per 50 ml of melted agarose (or add 0.5 µl GelRed) and swirl gently.

4. Pour into casting tray until agarose is half way up the tines of the comb and allow gel to solidify.

5. Remove gel from casting tray. Place in electrophoresis box with enough 1X TBE (or TAE) to cover gel. Gently remove comb.

6. Load the appropriate amount of a molecular weight marker into the first well (e.g. 5 µl Invitrogen 100 base pair ladder premixed with loading dye).

7. Place 2 µl of loading dye onto parafilm. Mix 5 µl of sample with the 2 µl of loading dye, pipet up and down until they are thoroughly mixed. Place all into an empty well of the gel. Repeat for all samples.

8. Set electrophoresis for 5-10 V/cm if using TBE buffer (1-5V/cm for TAE). Run gel until examination by UV light shows that the marker bands are well separated.

9. Visualize DNA by UV light. It is best to use a UV transilluminator. Photograph gel to record results.

Analysis of results

• The bands of the marker should be clearly visible.

• The negative controls (water control, RNA from uninfected cells) should not show any DNA bands. If there are bands in the negative controls, this indicates contamination of reagents with measles RNA. In this case, the RT-PCR must be repeated with fresh reagents.

• The positive control should show one band of the expected size (see below). If there is no band in the positive control, the RT-PCR did not work and must be repeated.

• The samples either show no bands (negative result) or a band of the expected size (see below, positive result).

• If the desired PCR products are present, purify PCR products from the remaining reaction.


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Example gels Example gel for Measles Genotyping RT-PCR 1% agarose gel stained with GelRed and visualized by ultraviolet illumination. The positive control is synthetic measles RNA with insert (see below). Primers MeV214 and MeV216 amplify an 854 base pair (nt) amplicon from the synthetic control RNA. The PCR product for a patient sample is 634 base pairs (nt) in size.

Lane 1: 100 base pair ladder molecular weight marker (Invitrogen) Lane 2: Synthetic MeV RNA (MeV-N3in) with insert = 854 base pair fragment Lane 3: wild-type Measles RNA = 634 base pair fragment Lane 4: negative (water) control

Schematic of measles positive control RNA with insert The location of the genotyping PCR product is indicated. The real-time PCR product is amplified from a different part of the synthetic RNA and is therefore not affected by the insert.

Example gel for Rubella Genotyping RT-PCR 1.5% agarose gel stained with GelRed and visualized by ultraviolet illumination. The positive control is synthetic rubella RNA (see below).

220 nt insert

Measles N gene

genotyping PCR product of control RNA

real-time RT-PCR product nt 426 nt 1764 nt 1522 nt 1521


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Examples of the 2 RT-PCR genotyping fragments and the diagnostic fragment made from wild-type and synthetic RNA are shown below. The wild-type size for fragment 1 should be 450 nucleotides (nt) while the control product should be 370 nt. The wild-type size for fragment 2 should be 633 nt while the control product should be 553. The wild-type diagnostic fragment should be 185 nt while the control product should be 215 nt.

Schematic of rubella positive control RNA with insertion and deletion Synthetic Rubella E1 RNA showing the locations of the 30 nt insertion and 2 deletions.

8633 9112

8945 9577

E1 gene

2-amplicon: fragment 1

fragment 2

8258 9700

8731 9469

Molecular Window (8731-9469; 739-nt)

8696 8806

9179 9265

30-nt insertion

Documents

RNA Extraction from Clinical Samples, Cell Culture Lysates, … · CDC protocols for the molecular epidemiology of measles virus and rubella virus; version of 05/19/2012 2 Controls