Nonrepetitive nested PCR
 

My HIV nested PCR is nonrepetitive. Since HIV is a RNA virus, I start with a one-step RT-PCR then do a nested PCR. I expect low reproductibility when starting with 2 copies of RNA, but high reproductibility with higher viral loads.

Sample A (1613 copies) was positive one day and negative twice the next day. When diluted 1/3 in water, it was positive in all 3 PCR tests! Sample B (>34 copies) was positive in all PCRs, whether diluted or not. Sample C (> 4 copies) was negative twice, but positive twice if diluted. When RNA from sample C was prepared again, all PCR were negative, whether diluted or not.

Can someone explain? How to evaluate the sensitivity of my PCR ? Thanx for your help or comments...