Real-Time Multiplex Polymerase Chain Reaction Assay for Rapid Detection ofClostridium difficileToxin-Encoding Strains

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Abstract
Clostridium difficile is considered an important emerging pathogen capable of causing disease in humans and animal species. In our study, we developed and evaluated a multiplex real-time polymerase chain reaction (PCR) assay for the detection of C. difficile genes encoding toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB). The standardized real-time PCR assay for toxin genes of C. difficile was used to screen for toxigenic C. difficile in fecal samples from 71 preweaned calves, 53 retail ground meat samples, and 27 pasteurized milk samples. All samples were also examined for C. difficile using traditional culture techniques to validate the PCR assay. A total of 24 fecal samples (33.80%) were positive for toxigenic C. difficile using either multiplex real-time PCR or culture. Toxin-encoding C. difficile was detected in 23 enriched fecal samples using the multiplex real-time PCR assay and only 15 samples using culture techniques. C. difficile was not detected in ground meat or pasteurized milk by traditional culture or real-time PCR assay. Eleven fecal samples were positive for all 4 toxin genes, suggesting that preweaned calves may be a likely source for toxigenic C. difficile. On the basis of findings of our study, it can be concluded that multiplex real-time PCR carried out on samples enriched for C. difficile is a reliable, sensitive, and specific diagnostic tool for rapid screening and identification of samples contaminated with C. difficile.