Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis.
Rapid microbiological methods (RMMs) as an alternative to conventional cultivation-based bioburden analysis are receiving increasing attention although no single technology is currently able to satisfy the needs of the health care industry. Among the RMMs, quantitative PCR (qPCR) seems particularly suited. Its implementation is, however, hampered by false-positive signals originating from free DNA in PCR reagents or from dead cells in the samples to be analysed. In this study, we assessed the capability of propidium monoazide (PMA) to inactivate exogenous DNA in PCR reagents and thus to minimise its impact in bioburden analysis. PMA is a membrane-impermeant dye that intercalates into DNA and covalently binds to it upon photoactivation leading to strong inhibition of PCR amplification. PMA is currently used mainly for treatment of microbiological samples to exclude signals from membrane-compromised cells, but is also very useful for suppression of exogenous DNA signals. In addition to testing the effect of different PMA concentrations on non-template controls and target DNA, we demonstrate the effect of amplicon length on the exclusion of background amplification. Targeting a 1,108-bp 16S rRNA gene fragment using universal bacterial primers and PCR reagents treated with 5 μM PMA resulted in complete suppression of signals from exogenous DNA within 50 cycles of amplification, while a limit of detection of 10 copies of Escherichia coli genomic DNA per PCR reaction was achieved. A combined PMA treatment of sample and PCR reagents furthermore improved the selective detection of live cells making this method appear a highly attractive RMM.
Cranfield School of Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK.
Franz
F
Schnetzinger
Youwen
Y
Pan
Andreas
A
Nocker
D001386:Azides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005189:False Positive Reactions; D007364:Intercalating Agents / Q000378:metabolism; D050296:Microbial Viability; D008828:Microbiological Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D012680:Sensitivity and Specificity
D001386:Azides; D004791:Enzyme Inhibitors; D007364:Intercalating Agents; C533957:propidium monoazide; D011419:Propidium