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 PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNaseI, 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-polymerase (New England Biolabs, Beverly, MA), and super-Taq polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq polymerase (Perkin-Elmer Cetus, Norwalk CT), Ampli-Taq LD polymerase (Perkin-Elmer Cetus) or Deep-vent 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.
Hilali
F
F
Laboratoire de Microbiologie Médicale, Institut Gustave-Roussy, Villejuif, France.
Saulnier
P
P
Chachaty
E
E
Andremont
A
A
D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005189:False Positive Reactions; D006361:Heating; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D011933:Reagent Kits, Diagnostic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic