Detection of viable Listeria monocytogenes with a 5' nuclease PCR assay.
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.
Norton
D M
DM
Department of Food Science, Cornell University, Ithaca, New York 14853, USA.
Batt
C A
CA
D001427:Bacterial Toxins; D001431:Bacteriological Techniques; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003851:Deoxyribonucleases; D005069:Evaluation Studies as Topic; D005516:Food Microbiology; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D008089:Listeria monocytogenes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012260:Ribonucleases
D001427:Bacterial Toxins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D003851:Deoxyribonucleases; D012260:Ribonucleases; C038705:hlyA protein, Listeria monocytogenes