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Applied and Environmental Microbiology, February 2002, p. 799-806, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.799-806.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Optimization of Reverse Transcriptase PCR To Detect Viable Shiga-Toxin-Producing Escherichia coli{dagger}

S. C. McIngvale, D. Elhanafi, and M. A. Drake*

Department of Food Science, North Carolina State University, Raleigh, N.C. 27695-7624

Received 22 June 2001/ Accepted 26 November 2001

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.

* Corresponding author. Mailing address: Department of Food Science, North Carolina State University, Box 7624, Raleigh, NC 27695-7624. Phone: (919) 515-2951. Fax: (919) 515-7124. E-mail: mdrake{at}unity.ncsu.edu.

{dagger} This is paper number FSR 01-44 in the Journal Series of the Department of Food Science, North Carolina State University, Raleigh, N.C.

Applied and Environmental Microbiology, February 2002, p. 799-806, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.799-806.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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