I am working with HPV. But when using MY9/MY11 & GP5+/GP6+ primers in nested approach , the PCR picks up a positive band in known negative samples? Although my MY9/ MY11 primers are not consensus, the false positivity is a issue? Can anyone help me in this? Wheras with PGEMY 9/PGEMY11 set of primers I am getting very nonspecific band along with strong specific amplicon of 450 bp.
hi Heena
Yes these HPV general primers generate a lot of non-specific amplicons. you have to optimize the PCR reactions for HPV. You can try doing the following, one at a time:
i) using a 10 times dilution of the original template for PCR
ii) use less of the primers
iii) check the actual annealing temperature for each primer pair through a gradient PCR
iv) use a hot start Taq polymerase.
You can also try the method described in the following paper:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC334491/pdf/nar00233-0232.pdf
best of luck
Hi Heena,
You can optimize the PCR reactions for HPV by adjusting the temperature and the annealing time.
I recommend reading the paper: PCR Detection of Human Papillomavirus: Comparison between MY09/MY11 and GP51/GP61 Primer Systems. JOURNAL OF CLINICAL MICROBIOLOGY, 0095-1137/97/$04.0010, June 1997, p. 1304–1310
I suggest also the pair of primers FAP 59/FAP64.
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