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Journal of Clinical Microbiology, February 2003, p. 730-734, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.730-734.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Clostridium difficile in Feces by Real-Time PCR

Simon D. Bélanger,1,2 Maurice Boissinot,1,2 Natalie Clairoux,1 François. J. Picard,1 and Michel G. Bergeron1,2*

Centre de Recherche en Infectiologie de l’Université Laval, Centre Hospitalier Universitaire de Québec (Pavillon CHUL),1 Division de Microbiologie, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada2

Received 2 August 2002/ Returned for modification 3 September 2002/ Accepted 17 November 2002

Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is closely related to the production of toxins A and B. Toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the "gold standard." However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficile toxin genes tcdA and tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficile strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than C. difficile (n = 14), including Clostridium sordellii for which the lethal toxin gene sequence is closely related to the toxin genes of C. difficile. Following a rapid (15 min) and simple fecal sample preparation protocol, both tcdA and tcdB were efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of C. difficile. It is rapid, sensitive, and specific and allows detection of C. difficile directly from feces samples.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie, CHUQ (Pavillon CHUL), 2705 Boul. Laurier, Sainte-Foy, Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: Michel.G.Bergeron{at}crchul.ulaval.ca.

Journal of Clinical Microbiology, February 2003, p. 730-734, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.730-734.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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