A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction
[摘要] Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrheaand pseudomembranous colitis. Detection of C. difficile by anaerobicbacterial culture and/or cytotoxicity assays has been largely replaced by rapidenzyme immunoassays (EIA). However, due to the lack of sensitivity of stoolEIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patientssuspected of having C. difficile-associated disease were prospectivelycollected. Three testing modalities were evaluated, including enriched culture,cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identifiedclinical specimen were analyzed. The sensitivities of stool real-time Pcr were95% against cepheid Xpert C. difficile and 93% against enriched culturerespectively, with a specificity of 97% and 94%. The lower limit of detectionof the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Directdetection of C. difficile toxin genes in stool samples by real-time Pcrshowed performance comparable to enriched culture. Real-time PCR of DNA fromstool samples is a rapid and cost-effective diagnostic modality for patientsthat should facilitate appropriate patient management.
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[效力级别] [学科分类] 分子生物学,细胞生物学和基因
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