Designing multiplex real-time PCR for disease diagnosis

Dr. Nurulhasanah OthmanProf Dr. Rahmah Noordin

Institute for Research in Molecular Medicine (INFORMM)

Universiti Sains Malaysia

28th National Science ConferenceBayview Hotel, Penang

16-17th August 2017

Outline

• PCR and history• Real-time PCR features• Designing multiplex real-time PCR• DNA extraction• Troubleshooting• Examples

PCR• Polymerase

chain reaction-Kary B. Mullis (1985)

• A method to copy and synthesize target sequences in a large amount.

https://www.ted.com/speakers/kary_mullis

• 2 most important technologies-a) Taq polymerase enzyme b) Thermal cycler machine.

• 1987, Perkin Elmer developed the first automated thermal cycler machine.

• Regulate the temperature of a reaction, heating or cooling the samples as needed

Real-time PCR• The word “real-time” indicates it can monitor the

amplification while its running.

• 1993- First real-time experiment conducted using EtBrfor quantization and detection by CCD camera.

• 1996- Introduction of TaqMan assay to replace EtBr.

• 1996-1997-The first real-time PCR machine was developed by Applied Biosystem (ABI)

Conventional PCR- Tell us “what”

Real-time PCRTell us “how much ”

Source: Bio-Rad

Real-time PCRConventional PCR

Ct value

Conventional VS Real-time

• It does not require post-PCR analysis ( No electrophoresis)

• Shorten time to obtain result

• Less contamination

• Less complexity for DNA quantitation analysis

Analysis

• Qualitative (presence or absence)• Quantitative (measure the DNA copies no)

Chemistry

• SYBR Green dye, Eva Green…• Hybridization probe: TaqMan Probe

Assay Category

• Real-time Assay (Gene Expression)a) 1 step RT-PCR -RNA b) 2 steps RT-PCR-RNAc) DNA quantification

• Endpoint Assay a) Allelic discrimination b) Plus/Minus

Designing multiplex real-time PCR

1) Model of instrumentHow many detection channels available?1, 2, 3 ??????

Example:1) Develop multiplex to detect 4 different targets2) Choose 4 different fluorophore colours (Depend on model of the instrument)3) ABI- FAM (Green)

VIC (Yellow)NED (Orange)CY5 (Red)

ProbeHydrolysis Probe

Source: ABI

• Taq Man MGB

2. How to design primer and probe?

• Primer 3 –free software• Oligos

IMPORTANT: All primers and probes have almost similar Tm

3. PCR Mastermix

• Choose conventional or ready-made mastermix???

• Total volume use??50, 25, 20, 15 or 10 µl?

• Optimize concentrations ofPrimer and probe

Source: Applied Biosystem

Thermal profile

• 2-steps PCR

• 3-steps PCR

Denaturing Denaturing Annealing/Extension

95°C for 10 min 95°C for 15 sec 60°Cfor 1 min

40-55 cycles

Denaturing Denaturing Annealing Extension

95°C for 10 min 95°C for 15 sec 58°C for 15 sec 72°C for 15 sec

40-55 cycles

• For all optimization steps, multiplexing, background test and testing panel:

1) Pure gDNA from target organisms

2) Plasmid containing target sequences(Cloning PCR product)

PositiveControl

4. Conventional PCR

• Primer working??

4. Primer concentration

100 80 60 40 20 nM

Primer concentration (nM) Mean Ct value

100 25

80 25

60 25

40 27

20 30

Example:

5. Multiplexing and background test

• Test with……

1.single template2. combination of 2 templates3. combination of 3 templates4. combination of 4/all templates….

Ct value showed a little changes in single and combination reactionsExample:Single Ct: 26.79

Combine (2) Ct: 27.1Combine (3) Ct: 27.4…….

6. LoD curve

Su et al., 2009

LoD Ct: 36

LoD Ct: 33

R square > 0.9E efficiency: 80-100%

7. Testing Panel

• Use gDNA from clinical specimens that are expected containing your target diseases

1) Body fluids: Blood, saliva, urine, CNS, tears,sweat..

2) Stools samples3) Tissues4) Buccal swab

8. DNA Extraction

• Conventional method??• Commercial kit???• Automated + commercial kit????

Things to ponder

• Need to remove inhibitor??Yes!!!! If stool samples

• Nature of target organismHave hard shell- egg of wormYes!!!! Must do pre-treatment before extraction (Exmp: Use of mechanical disruption)

Troubleshooting

Sigmaaldrich

Sigmaaldrich

Sigmaaldrich

Application for disease diagnosis

Acknowledgement• INFORMM,USM

Prof Rahmah Noordin Pn Maimunah AhmedPn Noorizan MiswanCik Madihah Basuni

• PPSP,USMProf Zeehaida Mohammed

• PPSK,USMAP. Dr. Lim Boon Huat

• Kementerian Kesihatan MalaysiaDr. Jamail Muhi

• Leiden University Medical CentreDr. Jaco J. Verweij

• National University of MexicoDr. Alfonso Olivos Garcia

Funding:MOHE: FRGS grantEU grant

Hands-on course on

Multiplex Real-Time PCRFrom primer and probe design to data analysis15 – 16 November 2017 | USM Main Campus, Penang

Thank you