Nucleic acid-based diagnostics (RT-nested PCR and Real time PCR) Yan LIU

Transcription

1 Nucleic acid-based diagnostics (RT-nested PCR and Real time PCR) Yan LIU Changchun Veterinary Research Institute China Ministry of Agriculture [OIE Reference Laboratory]

2 FAT (Fluorescent antibody test) drit (Direct rapid immunohistochemistry test ) Sequencing and Genotyping RNA Extraction One-step real time PCR Data Analysis Reverse Transcription Nested PCR Electrophoresis RTCIT (Rabies tissues-culture inoculation test) (ELISA MIT (mouse MIT inoculation (, ELISA test) ELISA (Enzyme Linked MIT (, Immunosorbent ELISA Assay)

3 N protein is an essential component of nucleocapsid that wraps RNA genome, G protein forms individual spikes on the surface of viral envelope and function in virus attachment and penetration. In all these five proteins, N protein is the most conserved among diverse virus, so it is regarded as the target for rabies virus detection.

4 experimental procedure -- PCR

5 p Viral RNA extraction p cdna synthesis p Nested PCR p One-step real time PCR p Troubleshooting

6 Viral RNA extraction Reliable diagnosis of rabies can only be performed from the brain in a lab. Saliva Salivary gland 丘脑 海马 脑桥 延髓 Cell culture brainstem, medulla oblongata, thalamus, cortex, Hippocampus

7 Viral RNA extraction Lyses Binding Washing (DNA protein) silica membrane transfer 630μl lysate + 560μl onto the RNase-free spin cooled Column ethanol GD-Wash RW-Wash DNA remove protein-remove Elution 140μl sample + 560μl lysis buffer Elution RNase-free ddh 2 O RW-Wash repeated skin, clothes, dust, saliva Gloves, Mask, Rnase-free tubes and tips, Rnase-free ddh 2 O

8 Viral RNA extraction OD 260 /OD 280 < 1.9 OD 260 /OD 280 = 1.9~2.1 OD 260 /OD 280 > 2.1 DNA contamination good Part degradation

9 p Viral RNA extraction p cdna synthesis p Nested PCR p One-step real time PCR p Troubleshooting

10 cdna synthesis 2.5mM dntps 4.0 µl Random Primer (50pmol/µL) 1.5 µl Oligo(dT) (50pmol/µL) 0.5 µl M-MuLV Reverse transcriptases 1.0 µl 5 M-MuLV buffer 4.0 µl RNases inhibitors 1.0 µl Template (RNA) + DEPC-H 2 O 8.0 µl

11 cdna synthesis Reaction condition 42 for 90 mins 95 for 5 mins hold at 4 or keep at -20

12 p Viral RNA extraction p cdna synthesis p Nested PCR p One-step real time PCR p Troubleshooting

13 Nested PCR Suitable for 7 lyssavirus species N gene 1 st round products 845bp (-19~0, 807~825) 2 nd round products 371bp (-6~15, 345~367)

14 Nested PCR 1 st Round primer pair: N ATG TAA CNC CTC TAC AAT GG-3 N GCC CTG GTT CGA ACA TTC T-3 2 nd Round primer pair: RVN371 F 5 -ACA ATG GAK KCT GAC AAR ATT G-3 RVN371 R 5 -CCT GYY WGA GCC CAG TTV CCY TC-3 1 st round 845bp 2 nd round 371bp

15 Nested PCR 1 st round 2 nd round PCR master mix 12.5 μl PCR master mix 12.5 μl N127 (20pmol/μl) 1.0 μl RVN371F (20pmol/μl) 1.0 μl N829 (20pmol/μl) 1.0 μl RVN371R (20pmol/μl) 1.0 μl ddh 2 O 9.5 μl ddh2o 9.5 μl Template cdna 1.0 μl Template cdna (First round PCR product ) 1.0 μl Total volume 25 μl Total volume 25 μl PCR master mix:ex-taq (5U/μl), Taq Buffer (Mg 2+ Plus), dntps

16 Nested PCR Reaction condition Pre-denaturation denaturation annealing extension 94 for 2mins 94 for 30s 56 for 30s 72 for 40s 72 for 10mins Hold at 4 1 cycle 35 Cycles 1 cycle 1 st round PCR and 2 nd round PCR use the same condition

17 Nested PCR Gel electrophoresis 5 µl of PCR product + 1 µl 6x loading buffer 1% agarose gel with Super GelRed (fluorescence dye) 120 V for 20mins

18 Nested PCR Positive control (+ve) Negative control (-ve) Reading data First round (845bp) UV Light Second round (371bp)

19 p Viral RNA extraction p cdna synthesis p Nested PCR p One-step real time PCR p Troubleshooting

20 qrt-pcr VS. RT nested-pcr? One-step qrt-pcr: 1. Sensitive and precision: quantitated the target gene by the Ct during the exponential phase of the reaction. 2. High specificity: Specific primers and specific probe. 3. Simple and practicable: suitable for detecting in the clinics, simplify the operation and shorten the experiment time (3-4 hours). 4. Most dogs, domesticated animal and terrestrial animal carry genotype 1 rabies, so one-step qrt-pcr is sufficient and fast. False positive :could easily happen due to potential sources of contamination. RT nested-pcr Suitable for detecting the virus in bat or other wide animal, which might carry different varients, other than genetype 1.

21 one-step qrt-pcr VS. two-step qrt-pcr Two-step qrt-pcr : to detect multiple messages from a single RNA sample. It also allows the archiving of cdna for further analysis. One-step qrt-pcr: to detect large numbers of samples and help minimize carryover contamination, since tubes do not need to be opened between cdna synthesis and amplification. Since the entire cdna sample is amplified, one-step qrt-pcr is more sensitive, down to 0.1 pg total RNA.

22 One-step real time PCR Amplification segments and primers Cover all strains? Primer pair: RVFQ For: 5 ATG TAA CAC CYC TAC AAT G 3 RVFQ Rev:5 GCA GGG TAY TTR TAC TCA TA 3 genotype 1 Probe: 5 FAM (reporter) -ACA AGA TTG TAT TCA AAG TCA ATA ATC AG-TAMRA 3 Probe (81-109) Amplication segment (55-164)

23 One-step real time PCR Taqman probe No fluorescence Reporter Quencher Taq polymerase + PacMan =Taqman 5 3 Probe Taq

24 2 x Quant One Step Probe qrt-pcr Master mix 25 µl Quant RTase (for one step) 0.7 µl HotMaster Taq polymerase (2.5U/µL) 2 µl RVFQ For (20pmol/µL) 2 µl RVFQ Rev (20pmol/µL) 1 µl RVFQ Probe (5pmol/µL) 1 µl RNase free dh2o 8.3 µl Total volume 40 µl

25 One-step real time PCR Reaction condition 42 C 30 mins 92 C 2 mins 92 C 30s 55 C 30s 72 C 20s collect fluorescence 1 cycles 40 cycles Hold at 4 C

26 One-step real time PCR Amplification plot Rn p Amplification curve 2,000,000 Plateau p Threshold p Ct value (threshold cycle number) 1,000,000 Threshold Ct Log-Linear phase 0 Baseline No template PCR cycle number

27 One-step real time PCR Ct and template concentration Log-Linear phase:the amount of PCR product is proportional to the amount of starting template that was initially added. High concentration of DNA(RNA) template get small Ct. A linear regression relationship between log of RNA(DNA) template concentration and Ct. Initial RNA template concentration is confirmed by TCID 50 test.

28 One-step real time PCR Result reading Positive control Negative control Sample 1 Sample 2 Sample 3 Positive control curve: 0<Ct<26 Negative control curve: No Ct Test validity

29 One-step real time PCR Result reading Positive control Negative control Sample 1 Sample 2 Sample 3 Positive : 0<Ct<32 (sample 2) Negative : No Ct (sample 1) If Ct > 32 : Repeat assay for verifying (sample 3)

30 p Viral RNA extraction p cdna synthesis p Nested PCR p One-step real time PCR? p Troubleshooting

31 What is the common contamination factors? between samples or positive control to the samples open lid contamination aerosol contamination (48,000 copies) the residual on the gloves the PCR product from early reaction RNase contamination from skin or dust

32 How to avoid contamination by the layout of the equipment? Maintain separated areas for nucleic acid sample preparation, PCR set-up, and PCR amplification. RNA Extraction Mix Preparing Template Preparing PCR Amplication + Gel Running Risk of infection

33 Automated High-Throughput Nucleic acid Extraction Platform.

34 Real-time PCR: How does criteria of Ct value 32 for positive sample set? Dilution (TCID 50 /100 µl) Ct None RTCIT TCID 50 virus stock was diluted at least 5 times. Each dilution was performed for realtime PCR and RTCIT to map a standard curve. Standard curve shows threshold cycle (Ct) on the y-axis and the starting quantity of RNA or DNA target on the x-axis. Ct 32 is corresponding to the TCID 50 of which RTCIT just turn negative from positive. Threshold cycle (Ct) Starting quantity Figure 1. Example of a standard curve of real-time PCR data.

35 Thanks for your attention! If you have any further question about PCR, let s discuss during the practise. We will be around with you for any help.