For my thesis on the same HPV L1 region sequencing for detecting genotypes , I have used My09/11 & GP5+/6+ primers for amplification on Nested PCR, but I couldn't find sequences other than 16, while same sample showed positive for 16, 18 & other High Risk genotypes on Real Time PCR (Geneproof). Does Nested PCR with My09/11 & GP 5+/6+ can not detect other genotypes other than 16?
Nested consensus PCR is tricky. Consensus primers generally do not amplify all types equally efficiently, particularly if they are in the same sample, in which case the most efficiently amplified type may 'out-compete' less efficiently amplified types. Depending on the biases, nesting could amplify the bias or neutralise it, but since HPV primers are usually optimised for HPV16, a bias towards this type would be expected.
Another problem is that nested PCR is extremely prone to contamination. I would say that to be sure your results are contamination-free, one of three samples should be a negative control, preferably processed with the samples.
Noneheless, I do find your results suprising, as other authors detect a wide range of types with these primers.
My advice is: don't multiplex. Both these PCRs have sufficient sensitivity alone.
If you do find that you have a contamination problem, you might like to try the PT13 primers that I designed: Jenkins A, Allum AG, Strand L and Aakre RK. Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus realtime PCR. J Virol Methods, 187: 345-351 (2013). These target a different region of the L1 gene. And don't worry about the patent, it has lapsed.
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