Estimation of inherent bacterial DNA contamination in a qPCR master mix: concerns about background DNA of reagents

Abstract

BACKGROUND: The present study estimated the total inherent DNA contamination of a commercially available qPCR (quantitative Real-Time polymerase chain reaction) master mix. A previous step of DNase treatment with both DNase I and a double strand specific DNase failed to remove the background contamination, even with varying enzymatic concentrations and incubation times. METHODS: Absolute quantification by qPCR was done with a 16S rRNA gene standard qPCR curve from genomic DNA of Escherichia coli, using a previously unopened vial of a commercial SYBR green master mix to estimate the total inherent bacterial contamination of the mix. Additionally, qPCR with primers specific for the rpoB gene of Enterobacterales was performed with a clinical serum sample with unknown bacterial load. RESULTS: The estimated mean (standard deviation) of bacterial DNA of the master mix was 171.211 (21.140) E. coli equivalent genomes per µL of master mix. The clinical sample had a higher C<inf>T</inf> value compared with the non-template control. This could be attributable to an inhibitory effect of human DNA in the serum with an expected low quantity of bacterial DNA. CONCLUSIONS: Estimation of the background bacterial DNA of molecular grade reagents is strongly suggested as a validation measure before sample analysis for bacterial quantification studies by qPCR, in particular when the expected bacterial DNA load is low.