PCR is a sensitive method for detecting presence of DNA in both research and diagnostics. Polymerases used in PCR are frequently contaminated with E.coli DNA. Contaminating DNA may cause reduced sensitivity and false positives when small amounts of bacterial DNA are targeted. Other sources of contamination might be dNTP’s, buffer components and primers / probes, as well as DNA introduced during handling. Detection and typing of bacteria can be done by using broad-range primers targeting conserved regions i.e. in the 16S or 23S rDNA gene. The method is very sensitive to bacterial DNA contamination and traces of bacterial DNA can be found in most master mixes and polymerases. When using qPCR for detection or quantification of minor amounts of bacterial DNA, contaminating E.coli DNA may cause false positive results.
|Figure 1: The presence of E.coli 23S DNA in 2x probe master mixes from various suppliers was quantified by using water instead of template (NTC) and following the manufacturer’s instructions. The figure shows plots from several separate experiments. Traces of E.coli 23S DNA were found in all master mixes tested, with Cq values generally ranging from 30 – 35.|
To solve this problem, we have developed a simple and accurate PCR Decontamination kit which removes contaminating DNA in master mixes, without reduction of PCR sensitivity.