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Nonrepetitive nested PCR

RT PCR repetitive

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#1 gonz



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Posted 16 October 2012 - 05:33 AM

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...

#2 Trof


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Posted 16 October 2012 - 11:10 AM

If you generaly get better amplification when diluted it means the sample contained inhibitors, that are reduced by dilution. The fact that some samples work undiluted and some not may mean nonreproducible isolation or different growth conditions (something added to medium, or if that's a patient sample, it can differ from patient to patient).

Other thing is if your amplification is very low in overall, there can be small numbers problem. In such high dillution of samples, sometimes you may pipet a copy of template and sometimes not.
For example sample C has so little copies (btw how you know the exact number?) that it can be easily lost.

Are you only evaluating positive/negative PCR or do you look at overall amount? Those positive even undilluted, was there difference in the amount of product between dilutions?

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#3 phage434



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Posted 17 October 2012 - 04:45 AM

Or your dilution water has contaminating DNA in it. Negative control!

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