Real-time pcr - (Oct/13/2015 )

Recently I have submited a manuscript and one of the reviewer have comment on the aspects below:

1. PCR amplifies DNA from live as well as dead bacteria and in short term batch cultures is no opportunity for dilution of killed or slow growing microbes due to wash out.  It is possible some of the DNA from easily digestible bacteria could be degraded but I do not think we have an idea of how much that may occur.  I would like the authors to discuss some of these aspects as it certainly has an influence on the conclusions that readers may make.

Please someone help me to answer this question.

Thank you.

The best way of addressing this is head on by quantifying the live bacteria in your sample. You could do this with a colony count of diluted culture, and an estimate for the amount of DNA per cell (depending on doubling rates, perhaps 1.5 - 2 genomes per cell, different depending on the location of your amplified sequence relative to the origin of replication in the genome. By comparing this amount to the qPCR amount, you can estimate the percentage of live vs. dead degrades DNA.