Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Foodborne Pathog Dis. 2010 Jun;7(6):719-26. doi: 10.1089/fpd.2009.0483.
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.
艰难梭菌被认为是一种重要的新兴病原体，能够在人类和动物物种中引起疾病。在我们的研究中，我们开发并评估了一种用于检测艰难梭菌编码毒素 A（tcdA）、毒素 B（tcdB）和二元毒素（cdtA 和 cdtB）的多重实时聚合酶链反应（PCR）检测方法。用于艰难梭菌毒素基因的标准化实时 PCR 检测方法用于筛选来自 71 头未断奶小牛、53 份零售绞碎肉样本和 27 份巴氏杀菌奶样本的产毒艰难梭菌。所有样本也使用传统培养技术检查艰难梭菌，以验证 PCR 检测方法。共有 24 份粪便样本（33.80%）使用多重实时 PCR 或培养均为产毒艰难梭菌阳性。使用多重实时 PCR 检测方法在 23 份富集粪便样本中检测到编码毒素的艰难梭菌，而仅在 15 份样本中使用培养技术检测到。传统培养或实时 PCR 检测均未在绞碎肉或巴氏杀菌奶中检测到艰难梭菌。11 份粪便样本均对所有 4 种毒素基因呈阳性，表明未断奶小牛可能是产毒艰难梭菌的一个潜在来源。基于我们研究的结果，可以得出结论，对艰难梭菌富集样本进行多重实时 PCR 是一种可靠、敏感和特异性的诊断工具，可用于快速筛选和鉴定污染艰难梭菌的样本。
