Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR

Abstract

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat-and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10 4 -to 10 5 -fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

“…This technique was initially employed by Nogva et al (43) and Rudi et al (55,56) to differentiate between viable and dead cells of Escherichia coli, Salmonella spp., Listeria monocytogenes, and Campylobacter jejuni. Since then, the EMA-qPCR approach has been used to discriminately quantify viable cells of other bacterial types (8,41,51,57,64). The standardized protocol initially established by Nogva et al (43) and Rudi et al (55,56) was not suitable to effectively discriminate live from dead Legionella cells.…”

Detection of <i>Legionella</i> Species in Environmental Water by the Quantitative PCR Method in Combination with Ethidium Monoazide Treatment

“…EMA-treated DNA is not a target for amplification by PCR, and therefore viable Legionella are selectively detected by PCR when combined with EMA treatment. The effect of EMA treatment on Legionella detection by PCR has been published Chang et al, 2009;Chen et al, 2010;Delgado-Viscogliosi et al, 2009;Qin et al, 2012 . In this paper, we report a comparison of the detection of Legionella spp. by the plate culture method, quantitative PCR qPCR method and qPCR method in combination with EMA treatment EMA-qPCR .…”

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…Interestingly, PCR in clinical applications may have a higher PPV (than for environmental samples) despite the lower sensitivity with nonrespiratory specimens (379). Two potential remedies for the low PPV with samples from environmental sources include reverse transcription-PCR, amplifying labile RNA targets present in metabolically active bacteria, and the use of a cell-impermeant chemical, such as ethidium monoazide (EMA) or propidium monoazide (PMA), to inhibit PCR amplification from nonviable cells or extracellular nucleic acids (385)(386)(387)(388)(389)(390). Notably, neither alternative protocol alone will discriminate VBNC legionellae; however, this cell population still poses a potential human health risk (391)(392)(393)(394).…”