Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR.
Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.
Klaschik
Sven
S
Klinik und Poliklinik für Anästhesiologie und Spezielle Intensivmedizin, Universität Bonn, Sigmund Freud Strasse 25, D-53105 Bonn, Germany. klaschik@ukb.uni.bonn-de
Lehmann
Lutz E
LE
Raadts
Ansgar
A
Hoeft
Andreas
A
Stuber
Frank
F
D016477:Artifacts; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D003666:Decontamination / Q000379:methods*; D003851:Deoxyribonucleases / Q000378:metabolism; D004866:Equipment Contamination / Q000517:prevention & control*; D019544:Equipment Failure Analysis / Q000379:methods; D005189:False Positive Reactions; D007202:Indicators and Reagents / Q000378:metabolism / Q000528:radiation effects; D008730:Methoxsalen / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000187:drug effects / Q000379:methods* / Q000528:radiation effects; D011786:Quality Control; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D007202:Indicators and Reagents; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes; D008730:Methoxsalen