Optimization of viability qPCR for selective detection of membrane-intact Legionella pneumophila

Although a number of viability qPCR assays have been reported to selectively detect signals from membrane-intact Legionella pneumophila, the efficient suppression of amplification of DNA from dead membrane-compromised bacteria remains an ongoing challenge. This research aimed at establishing a new oligonucleotide combination that allows for a better exclusion of dead Legionella pneumophila on basis of the mip gene. Propidium monoazide (PMA) was chosen as viability dye. An oligonucleotide combination for the amplification of a 633 bp sequence was established with 100% specificity for different Legionella pneumophila strains compared with 17 other Legionella species tested. Apart from increasing amplicon length, the study aimed at optimizing dye incubation time and temperature. An incubation temperature of 45 degrees C for 10 min was found optimal. Dye treatment of heat-killed bacteria in the presence of EDTA improved signal suppression, whereas deoxycholate also affected signals from live intact bacteria. Suppression of signals from heat-treated bacteria was found to be approx. twice as efficient compared to a commercial kit, although the detection sensitivity is superior when targeting short amplicons. With a limit of detection of 10 genome copies per PCR well and a 6-log signal reduction of bacteria killed at 80 degrees C, the assay appears useful for applications where pathogen numbers are not limiting and where the priority is on the distinction between intact and damaged Legionella pneumophila for the evaluation of hygienic risk and of disinfection efficiency.