Dewi Lokida Tangerang District Hospital - hkki.org · Introduction Advantages PCR detects the presence of non-culturable or fastidious organisms and pathogens with low concentration

Dewi LokidaTangerang District Hospital

Outlines Introduction

Principles of PCR

Layout of PCR Laboratory Room

Reagents in PCR Workflow

PCR Workflow

PCR Optimization

Application of molecular diagnostic in infectious deseases

Introduction

Molecular diagnostic of infectious diseases means to detect the genome of pathogen(s) in clinical specimens.

The genomes of pathogen: DNA: bacteria, parasites, fungi DNA or RNA: viruses

As DNA/RNA is contained within cells, no intact (culturable) organisms are required

Different species are identified based on a species-specific sequence within DNA

The most established technique is polymerase chain reaction (PCR), which is an amplification of a specific sequence in the DNA

Introduction

Advantages PCR detects the presence of non-culturable or fastidious organisms and

pathogens with low concentration within clinical specimens when conventional methods cannot.

PCR by passes the need to culture, thus is useful to identify pathogens requiring high biosafety level (e.g: B anthracis)

PCR is quite fast (1 – 2 day to get result), thus assists in treatment management and prevention of outbreak

Introduction

Disadvantages Rooms Equipments Reagents Optimization Special skill

Principles of PCR

By Enzoklop - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=32003643

PCR Variants

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

o template = RNA

o RNA is first converted into cDNA, through reverse transcription step (using reverse transcriptase enzyme).

o cDNA is then amplified in the PCR cycle

Principles of PCR

Layout of a PCR Laboratory

X

Clean room

• Prepare PCR master mix

• Must be kept ‘clean’ (free of DNA or specimens)

Extraction room

• Do DNA extraction from specimens

• Add DNA into PCR master mix

Detection room

• Run PCR • Visualize the PCR product

• A PCR laboratory requires minimum 3 separate rooms to facilitate the PCR workflow

• The direction of movement during PCR must be one-way, which is from clean room to extraction room, and end up in the detection room

• Each room requires dedicated lab coats, micropipette ect, gloves, bin ect which cannot be exchanged with each other


Cleanroom Extraction room Detection room

Minimum Equipment in a Clean Room

laminar

freezer

refrigerator

• Laminar: preparation of master mix

• Refrigerator: keep working solution

• Freezer: keep stock solution

• Micropippette and tips• Dedicated lab coat• Disinfectant • The temperatures of freezer and

refrigerator must be recorded routinely• The laminar must be calibrated

routinely


micropippette

Minimum Equipment in an Extraction Room

BSC, refrigerator, micropippette, biohazard bin

centrifuge, heat block

• BSC: DNA extraction, protect lab workers from infectious materials

• Biohazard bin: layered with biohazard bag, dedicated to contain infectious wastes

• Refrigerator: keep working solution and specimens (within a working day)

• Centrifuge and heat block: assist in DNA extraction steps, which require centrifugation and heating of specimens

• Micropippette & tips• Dedicated lab coats• The temperatures of freezer and refrigerator must be

recorded routinely• The laminar must be calibrated routinely


• PCR conventional machines• Callibrated routinely to keep the temperature accurate

• ProFlex PCR System [Thermo Fisher Sci.]

PCR Instrument

• Gel preparation apparatus• Electrophoresis apparatus

• Gel Imaging system

UV illumination (Gel Doc)

Electrophoresis & Gel Imaging

GelDoc EZ System

Layout of a PCR LaboratoryMinimum Equipment in a Detection Room (Conventional PCR)

Minimum Equipment in a Detection Room (Conventional PCR)

gel imaging system

electrophoresis apparatus

beaker glass, stirring block, magnetic stirrer

microwave digital balance

gel preparation apparatus

PCR conventional machine


ABI 7500 Fast Roche Lightcycler 480


Minimum Equipment in Real time PCR room


• Master Mix content: 1. dNTPs (dATP+dTTP+dCTP+dGTP)

2. Polymerase enzymes : Taq Polymerase

3. MgCl24. Proprietary buffer

• Specific primer

PCR Kit

Go TaqGreen Master Mix [Promega]

• Proteinase-K and lysis buffer (AL): break down cell wall/cell membrane & organelles to release the DNA

• Wash buffer (AW1 & AW2): wash impurities from DNA

• Elution buffer (AE):

to preserve DNA in freezing condition (-80o C)• Product extraction :

DNA templateadd to master- mix in the extraction room

• Each run must have positive & negative controls

• Mix the reaction well before run the PCR cycles

DNA Extraction Kit

QIAampDNA Extraction Kit [Qiagen]


• Agaroseand TBE: migrating media• Ethydium bromide

• DNA ladder: mark the size of PCR product

(in base-pair/bp)

Electrophoresis & Gel Imaging

GelDoc EZ System [BioRad]


• Aim: to obtain pure DNA, free of cells component (PCR inhibitors)

• Steps:

cell lysis, DNA wash, DNA elution• Add template DNA to master mix

DNA Extraction

• Aim: to prepare PCR reaction correctly (free of contamination)

• Steps

Prepare reaction mix containing the Master Mix and a pair of specific primer in the clean room

PCR Reaction Preparation

PCR Workflow

PCR Workflow

• Aim: to run the PCR cycles (denature, anneal, extend)

in a thermal cycler (PCR instrument)]Run PCR

• Aim:Separate the PCR product according to size

in a gel and visualize the gel with

UV illumination (Gel Doc)• Steps:

load the PCR product in agarose gel, run the electrophoresis,

take the gel image

Visualize PCR Product

PCR Variants

Real time PCR / qPCR

o Visualization of the increase in the amount of DNA as it is amplified during PCR cycle (“real time”); amplicon is labelled with fluorescence (SYBR Green, or TaqMan probes)

o no need for gel electrophoresis to view PCR result

o Result is expressed as Ct (cycle threshold) = the cycle at which amplicon crosses detection threshold

Applied Biosystems 7500 Fast Real Time Instrument [Thermo Fisher Scientific]

PCR Workflow

No Patogen Metode PCRGene Fragment target/Primer

References Protokol

1 Dengue

Real time NS-5 fragment

Hue KD, Tuan TV, Thi HT, Bich CT, Anh HH, Wills BA, et al. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma. J VirolMethods.

Nested (serotype)

PreM/Envelope fragment

Lanciotti RS1, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.J ClinMicrobiol. 1992 Mar;30(3):545-51.

2 Rickettsia

Rickettsia Sp47 kDA outer membrane protein (omp_gene)

Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, Richards AL. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. The American journal of tropical medicine and hygiene. 2004;70(4):351-6.

Ortientiatsutsugamushi

47 kDA outer membrane protein (omp_gene)

R. typhi Omp geneHenry KM et al. Development of quantitative realtime PCR assay to detect Rickettsia typhi and Rickettsia felis the causative agents of murine typhus and flea-born spotted fever. Molecular and CelularProbes .2007 (21):17-23.

R. felis Omp gene

3 Leptospira Realtime rrs-gene

Thaipadungpanit J, Chierakul W, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Boonslip S, et al. Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study. PLoS One 2011;6(1):e16236.

PCR OPTIMIZATION

No Patogen Metode PCRGene Fragment target/Primer

References Protokol

4 Chikungunya Realtime NS5 gene

Lanciotti RS1, Kosoy OL, Laven JJ, Panella AJ, Velez JO, Lambert AJ, Campbell. 2007. Chikungunya virus in US travelers returning from India, 2006.GL.Emerg Infect Dis. 2007 May;13(5):764-7.

5 Hanta virus Conventional L-segmentBoris Klempa,et.al. 2006. Hantavirus in African Wood Mouse, Guinea.Emerg Infect Dis. 12(5): 838–840.doi: 10.3201/eid1205.051487.

6 S. typhii Conventional V1 region of flagellin gene

M. Hatta & Henk L Smits.2007. Detection of Salmonella Ty[hi by nested polymerase chain raection in blood, urine and stool samples. Am. J. Trop. Med. Hyg., 76(1), 2007, pp. 139–143.

7 S. paratyphii Conventionalputative fimbrial protein (stkG) gene

Chandra Bhan Pratap, Gopal Kumar, Saurabh Kumar Patel, Vijay K Shukla, Kailash Kumar5 Tej Bali Singh, and GopalNath. 2014. Mix-infection of S. Typhi and ParaTyphi A in Typhoid Fever and Chronic Typhoid Carriers: A Nested PCR Based Study in North India.J Clin Diagn Res.; 8(11): DC09–DC14.doi: 10.7860/JCDR/2014/9167.5107

8 16 S Realtime16s small ribosomal RNA -V1-V3 region)

Thaipadungpanit J, Chierakul W, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Boonslip S, et al. Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study. PLoS One 2011;6(1):e16236.

PCR OPTIMIZATION

No Patogen Metode PCR References Protokol

9 HHV6 Conventional

Li-Min Huang, Pei-Fen Kuo, Chin-Yun Lee, Jen-Yang Chen, Mei-Ying Liu, Czau-SiungYang. 1992. Detection of Human Herpesvirus-6 DNA by Polymerase Chain Reaction in Serum or Plasma. Journal of Medical Virology 38: 7-10.

10 Flavi virus Conventional

Phylogeny of the Genus Flavivirus. Goro-Jen.Chang, K.Richard Tsuchiya, Nick Karabatsos and C Bruce Cropp. Journal of Virology, Jan. 1998, p. 73–83

11 16 S Conventional

Demetrio L Valle Jr, Jeannie I Andrade, Esperanza C Cabrera, Windell L Rivera. Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia. Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 105(2): 117-122, March 2010

PCR OPTIMIZATION

No Pathogen MetodeTarget

gene/primerRefference Protocol

12 Parecho virus

TaqmanProbe

RealtimeRT-PCR

Jansen RR, Schinkel J, Koekkoek S, Pajkrt D, Beld M, de Jong MD, Molenkamp R. 2011.et al. Development and evaluation of a four-tube real time multiplex pcrPCRassay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts. J Clin Virol . 2011;51: (3):179-18585.

13 Boca virus

14 Entero Virus

TaqmanProbe

RealtimeRT-PCR

5-UTR region

Beld M, Minnaar R, Weel J, Sol C, DamenM, van der Avoort H, et al. Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control. J Clin Microbiol. 2004;42(7):3059-64.

PCR OPTIMIZATION



15Streptococcus pneumoniae

Taqman Probe

Realtime RT-PCR

pneumolysin(ply) gene

Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, KaczmarskiEB. Simultaneous detection of Neisseria meningitidis, Haemophilusinfluenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. Journal of clinical microbiology. 2001;39(4):1553-8.

16 Hemophillus Infuenzacapsulation(bexA) gene

17Chlamidophilla / Chlamydia pneumoniae

ompA gene

Heddema ER, Pannekoek Y, LangerakAA, Beld M, Duim B. Development of an internally controlled Taqman based PCR assay for the detection of Chlamydia pneumoniae in the Lightcycler 2.0 system. Ned Tijschr Med Microbiol. 2004;12(s1):s61

PCR OPTIMIZATION



18 Chlamydia psittaci

Taqman Probe

Realtime RT-PCR

ompA gene

Heddema ER, Beld MG, de Wever B, Langerak AA, Pannekoek Y, Duim B. Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler2.0 system. Clin Microbiol Infect. 2006;12(6):571-5.

19 Bordetella pertusisinsertion sequences (IS) gene

Reischl U, Lehn N, Sanden GN, Loeffelholz MJ. Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii. J Clin Microbiol. 2001;39(5):1963-6.

PCR OPTIMIZATION



20 Legionella pneumoniaeTaqmanProbe

RealtimeRT-PCR

mip gene

Wilson DA, Yen-Lieberman B, Reischl U, Gordon SM and . Procop GW. Detection of Legionella pneumophila by Real-Time PCR for the mip Gene J. Clin. Microbiol. July 2003 vol. 41 no. 7 3327-3330. doi: 10.1128/JCM.41.7.3327-3330.2003

21Mycoplasma pneumoniae

P1 cytadhesingene

Pitcher D, Chalker VJ, Sheppard C, George RC, Harrison TG. Real-time detection of Mycoplasma pneumoniae in respiratory samples with an internal processing control. J Med Microbiol. 2006;55(Pt 2):149-55.

PCR OPTIMIZATION

PCR works best to detect pathogens in acute specimens, thus is helpful to help patients obtain appropriate treatment

PCR can detect slow-growing pathogens or those that cannot be cultured (either non-cultur-able in medium conventional, or the lab doesn’t have facility culture), as well as pathogens with very low concentration in the specimen

PCR is fast to detect pathogens that are potential to cause outbreak, such as B anthracis or HPAI H5N1, where quick response is required to prevent the spread/severity of outbreak


Realtime PCR can do quantitative assay, therefore allowing further study in relation to disease pathogenesis

The burdens of several diseases are known, but clinicians often lack evidence to diagnose, such as in the case of Chikungunya, RSV, or human herpes virus. PCR helps to clarify the presence/absence of the pathogens.

PCR has helped to reveal pathogens that hosts & vectors are circulating in Indonesia, but no human cases are ever reported (typhus fever, Rickettsiae)


Tangerang hospital molecular lab Avian Influenza : Pilot Project CDC : SARI study NIH : Reference Lab for INARESPOD

(Indonesia Research Partnership on Infectious Desease)

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Dewi Lokida Tangerang District Hospital - hkki.org · Introduction Advantages PCR detects the presence of non-culturable or fastidious organisms and pathogens with low concentration