NDLI: Rapid Detection of Shiga Toxin-Producing Bacteria in Feces by Multiplex PCR with Molecular Beacons on the Smart Cycler

Content Provider	PubMed Central
Author	Bélanger, Simon D. Maurice, Boissinot Ménard, Christian Picard, François J. Bergeron, Michel G.
Copyright Year	2002
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 and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.
Related Links	http://dx.doi.org/10.1128/jcm.40.4.1436-1440.2002
Ending Page	1440
Page Count	5
Starting Page	1436
File Format	PDF
ISSN	00951137
e-ISSN	1098660X
Journal	Journal of Clinical Microbiology
Issue Number	4
Volume Number	40
Language	English
Publisher	American Society for Microbiology
Publisher Date	2002-04-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Microbiology (medical) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Microbiology (medical)

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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