I have inoculated serum sample on mosquito cell lines and confirmed virus isolation by NS1Ag ELISA. After that i have done PCR for serotyping but its not working. What must be the reason to have negative PCR on same culture supernatent which is positive for NS1 Ag?
Hi Sunita,
It can be a problem with the virus nucleic acid isolation. Have you measured virus RNA by spectrophotometry. There can be problem with the reverse transcription. Before you use the cDNA in Real Time PCR I suggest to run a classical PCR reaction (with NTC, NAC and positive control) to reveal that any amplification can occur. If the PCR is suboptimal, you can run a touchdown PCR to maximize specific PCR product. So there are manny levels from RNA to cDNA can be a problem. I suggest to step back to the viral RNA prep then go step-by step to QPCR level.
Best,
Tamas
Hello, I also approve with the previous comment. I think that your RNA isolation step need more optimization.
Hi Sunita,
Are you sure the virus is actively replicating or is it possible that the antigen you detected was just shed off the initial inoculum? In addition to the earlier answers, I assume you have taken care to use appropriate primers for reverse transcription.
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