Detection of viable Listeria monocytogenes with a 5 ' nuclease PCR assay

A 5' nuclease assay has been developed to detect viable Listeria monocytoge nes, The assay targets the hemolysin A (hlyA) transcript, which is found on ly in L. monocytogenes, The single-tube, reverse transcriptase (RT), fluoro genic probe-based assay was formatted by using Tth DNA polymerase whose act ivity was modulated by using the manganese-chelating morpholinepropanesulfo nic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribed hlyA m RNA, The viability of L. monocytogenes was reduced by heating at various te mperatures and times up to a maximum of a 9-D inactivation. The location of the primer had a pronounced effect on the utility of the assay, and primer s located in the most distal regions of the hlyA transcript appeared to cor relate with the number of CPU while primers located more internal an the am plicon overestimated the cell viability. The assay with primers that includ ed the 3' end of the transcript was an accurate indicator of viability as m easured by CFU determination or staining with 5-sulfofluorescein diacetate.