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

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 Microbiol

authors

Norton DM,Batt CA

doi

10.1128/AEM.65.5.2122-2127.1999

keywords:

subject

Has Abstract

pub_date

1999-05-01 00:00:00

pages

2122-7

issue

5

eissn

0099-2240

issn

1098-5336

journal_volume

65

pub_type

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