Does either seminested RT-PCR (w/degenerate primers/Taqman probe) or rRT-PCR ( w/Taqman probe/AF 485331 EF1-α-Cyprinus carpio) have a reliably higher diagnostic/analytical sensitivity than virus isolation for detection of the spring viremia in carp virus in apparently healthy carp populations? If so, is one recommended over the other?
Hi Annie,
if you are sure that your primer pairs are suitable to detect your target of interest a PCR should be a good tool for virus detection. But you are working with degenerated primers, so I asume you try to detect a no highly conserved genomic region. So, in that case you should be aware of the possibility, that the virus you are looking for may show a sequence variance in the primer binding region which will lead to a non-detection. Then virus isolation would be the next step to get more sequence information for optimizing the primers.
I wish you luck!
I feel rRT PCR is always better option than virus isolation. Just do proper validation of your RT PCR or rRT PCR assay before its routine use. Additional benefit of RT PCR is high rate of detection than isolation based detection. If possible go with specific primer probe set rather than degenerate one as though degenrate set will increase assay sensitivity, it might reduce specificity.
You can use PCR for screening. but virus isolation is gold standard. by virus isolation, you can isolate all viruses and after that identify the isolated viruses. on other hand by PCR you work in specific virus. another point is that real time pcr is the better than PCR due to high sensitivity but if you work by real time pcr you should ensure that your test is sensitive enough for using in diagnostic .... it take a lot of work in sensitivity issue including cloning and testing of several dilutions of cloned plasmid and drawing a standard curve.
Hello Annie!
Based on my experience, PCRs are more sensitive than virus isolation. Many viruses need to adapt for in vitro cultivation and sometimes several passaeges are not succesfull. However, PCR is detect only the nucleic acid, and based on that, you can not assess is the virus in a sample infectious. I think the best way is to do both, PCRs and isolation simultaneously. If you want to do screening, just to see is in your sample any virus or not, it is enough to do PCR. But if you want to use a field strains for infection study, you have to try to isolate it as well.
All the best!
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