Reagent decontamination to eliminate false-positives in Escherichia coli qPCR.
The application of real-time quantitative PCR (qPCR) for the detection of low concentrations of Escherichia coli as well as universal 16S rDNA has been hindered by false-positives due to endogenous contamination of PCR reagents with E. coli and other bacterial DNA. We optimized a DNase I decontamination method to eliminate false-positives in a qPCR assay targeting the uidA gene in E. coli. In contrast to previous methods reported in the literature, our decontamination method did not cause PCR inhibition. We determined that residual DNase I activity was the cause of the inhibition in the previous methods, and eliminated it by ensuring complete inactivation prior to qPCR. DNase inactivation was accomplished by adding dithiothreitol (DTT) and then heating for 30 min at 80 degrees C. The optimized DNase method was compared to another decontamination method, ultrafiltration, and to untreated controls. We detected contamination in 85% of the untreated commercial PCR master mix samples at a level of about 10 copies per well (12.5 microL of master mix). Both decontamination methods could eliminate up to 100 copies of added contaminant DNA and did not cause PCR inhibition, resulting in a reduction of the detection limit to 10 copies per reaction well.
Silkie
Sarah S
SS
Civil and Environmental Engineering Department, University of California, Berkeley, CA 94720-1710, USA.
Tolcher
Matthew P
MP
Nelson
Kara L
KL
D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis* / Q000235:genetics; D003666:Decontamination / Q000379:methods*; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D005189:False Positive Reactions; D006358:Hot Temperature; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014462:Ultrafiltration
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D012336:RNA, Ribosomal, 16S; D003850:Deoxyribonuclease I; D004229:Dithiothreitol