Hello Researchers. I am conducting a study on the determination of WNV on mosquito samples. I run nested PCR. Bands were shown to almost all samples during carrying out gel electrophoresis. After I sent the PCR product for sequencing service for 5 random samples, and the sequencing result for 4 out of 5 appears to be 100% percentage identification of my targeted virus (WNV). While one sample shows 98% as the highest percentage identification. Hence, I would like to know whether my results are true positive (valid) or false positive? May I know how to determine the false positive result (any criteria or book/journal reference) as my future reference?
My targeted primer is between capsid and pre-membrane.
Thank you for your thought and suggestion.
Hi Natasha,
Hello, it is not the sequencing which will indicate if they are true or false positives but the controls that you should imperatively introduce ; PCR without RT, RT-PCR without RNA samples (just with the reagents)). You will need a lab dedicated to the preparation of oligo, reagents ... a lab dedicated to the extraction of RNA / DNA from samples / a place for RT / never use the same pipettes for loading PCRs on gels and starting the RT-PCR or DNA/RNA extraction.... There is several ways to avoid contamination from PCR products (use of dUTP and uracil-DNA N-glycosylase...) taht will diminish the contamination.
You'll need controls in your PCR to rule out contamination (aka "false positives"). Check the peaks on your 98% match sample. I'm guessing it has a few ambitious base calls. Clean it up by removing the lower quality parts of the data and I bet you'll see a higher match.
Katie A Burnette does it means I rerun PCR with the only control sample (add water as template)?
thank you for the response Dr
Natasha Jafar Ali I did see you have a "-ve control" at the very first lane on the gel. It did not show a band. That is good. What was in your -ve control tube? Reaction buffer without DNA or just water? The 98% identity might be just some bases are not correct as mentioned Kati.
thank you Dr. Yuan-Yeu Yau .. I add water as template in my -ve control tube. How about the 100% percentage identification. Does it common to produce 100% as percentage identification?
Thank you Dr for the respone
Natasha Jafar Ali Ok, so your negative control has no bands. That is a good news. 100% is possible. Why do you think it is not possible? Is there a reason to let you expect that they should not 100% match? Just curious.
Dr. Yuan-Yeu Yau , it is because there’s another researcher told me it could be something wrong if the match is 100% with the targeted virus. Besides, my most concern is, one of the 100% percentage identification is the sequencing of my positive control. does it possible? Thank you Dr
How long are your sequencing reads and what region are they amplifying? If you are sequencing a short, conserved region then I would expect to find 100% matches to known sequence.
Why did the other researcher tell you that there is "something wrong" if you got 100% match to your known control sequence?
Are you expecting to find novelty or a known virus?
Hello Dr Traci L Testerman , I run MyTaq OneStep for 1st round PCR and MyTaqRedmix for my 2nd round PCR (I run nested PCR for my study).
Dr Katie A Burnette , I amplify the conserved region of WNV (capsid and premembrane region). The length of my sequencing results are between 250-259 bp. the other researcher suspected something wrong because almost all my samples show band during gel electrophoresis and the nucleotide of my sequencing result almost similar.
It is a known virus which had found on the host. It also found on the vector once in 1960 but no continuous study on the determination of the virus on vector was conducted
Natasha Jafar Ali Well, that is the definition of 'a conserved region', isn't it? Very likely to get high degree of identical sequence (PCR product) from the region.
- Badges
- Science method