Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes

We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false-positive results with universal primers for 16S rRNA, contaminating DNA had to eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNase I and was followed by DNase inactivation at 94 degrees C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After Optimization of PCR conditions for each polymerase, Deep-Vent Exo-(R) polymerase (New England Biolabs, Beverly, MA), and super-Taq(R) Polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq(R) polymerase (Perkin-Elmer Cetus, Norwalk, CT), Ampli-Taq LD(R) polymerase (Perkin-Elmer Cetus) or Deep-vent(R) polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.