title: Optimization of reverse transcriptase PCR to detect viable Shiga-toxin-producing Escherichia coli.
abstract: The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing Escherichia coli (STEC) was investigated. Four primer sets, each targeting a specific region in the slt-II operon, were evaluated for their stringency and specificity for slt-II mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of E. coli slt-II mRNA is useful for detection of viable STEC in ground beef.
authors.affiliation: Department of Food Science, North Carolina State University, Raleigh, N.C. 27695-7624, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: McIngvale
authors.forename: D
authors.initials: D
authors.lastname: Elhanafi
authors.forename: M A
authors.initials: MA
authors.lastname: Drake
mesh_terms: D000818:Animals; D002417:Cattle; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D008461:Meat Products / Q000382:microbiology; D012329:RNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022621:Shiga Toxin / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D022621:Shiga Toxin