Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler.

Journal of Clinical Microbiology
Simon D BélangerMichel G Bergeron

Abstract

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing Escherichia coli (STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes stx(1) and stx(2), respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (n = 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n = 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for stx(1) and/or stx(2). Compared with the culture results, both the sensitivity an...Continue Reading

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Citations

Jan 28, 2003·Drug Discovery Today·François J Picard, Michel G Bergeron
Oct 2, 2002·Current Opinion in Microbiology·Maurice Boissinot, Michel G Bergeron
Mar 1, 2012·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Triinu Koressaar, Maido Remm
Oct 9, 2003·Applied and Environmental Microbiology·Karen C JinnemanStephen D Weagant
Nov 6, 2004·Applied and Environmental Microbiology·Yuli SongSydney M Finegold
Jul 9, 2004·Applied and Environmental Microbiology·Freddie H SharkeyRoger Marchant
Jan 19, 2006·Clinical Microbiology Reviews·M J EspyT F Smith
Dec 15, 2010·Journal of Clinical Microbiology·Marc Roger CouturierLinda Chui
Feb 8, 2003·Journal of Clinical Microbiology·Simon D BélangerMichel G Bergeron
Jun 16, 2005·Journal of Clinical Microbiology·Ching-Fang HsuTzu-Ming Pan
May 16, 2003·Clinical Chemistry and Laboratory Medicine : CCLM·Klara AbravayaJohn Robinson
Nov 13, 2003·Médecine sciences : M/S·Maurice Boissinot, Michel G Bergeron
Sep 4, 2012·International Journal of Food Microbiology·Choreh FarrokhOlivier Cerf
Apr 5, 2011·Ecotoxicology and Environmental Safety·S RamR Shanker
May 15, 2010·The Journal of Molecular Diagnostics : JMD·Linda ChuiMatthew W Gilmour
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Feb 19, 2005·International Journal of Food Microbiology·Zhu FuThomas L Kieft
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Mar 31, 2004·Canadian Journal of Microbiology·Anil K Deisingh, Michael Thompson
Jul 7, 2007·Journal of Microbiological Methods·Tim SchuurmanYvonne T H P van Duynhoven

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