Abstract:
:A 5' nuclease assay has been developed to detect viable Listeria monocytogenes. The assay targets the hemolysin A (hlyA) transcript, which is found only in L. monocytogenes. The single-tube, reverse transcriptase (RT), fluorogenic probe-based assay was formatted by using Tth DNA polymerase whose activity was modulated by using the manganese-chelating morpholinepropanesulfonic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribed hlyA mRNA. The viability of L. monocytogenes was reduced by heating at various temperatures 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 primers located in the most distal regions of the hlyA transcript appeared to correlate with the number of CFU while primers located more internal on the amplicon overestimated the cell viability. The assay with primers that included the 3' end of the transcript was an accurate indicator of viability as measured by CFU determination or staining with 5-sulfofluorescein diacetate.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Norton DM,Batt CAdoi
10.1128/AEM.65.5.2122-2127.1999keywords:
subject
Has Abstractpub_date
1999-05-01 00:00:00pages
2122-7issue
5eissn
0099-2240issn
1098-5336journal_volume
65pub_type
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