Boondeekhun H S, Gurtler V, Odd M L, Wilson V A, Mayall B C
Department of Microbiology, Heidelberg Repatriation Hospital, Victoria, Australia.
J Med Microbiol. 1993 May;38(5):384-7. doi: 10.1099/00222615-38-5-384.
A rapid assay was developed for detection of the Clostridium difficile enterotoxin gene in stool specimens by means of the polymerase chain reaction (PCR). The PCR primers amplified a 63-bp repetitive sequence of the enterotoxin gene, thereby generating a distinctive ladder pattern of DNA bands following electrophoresis. Crude DNA extracts from stools containing C. difficile produced one (63-bp) or more bands of the characteristic ladder. Of 172 stool specimens from 58 patients, 37 gave positive results by culture (15 specimens) or cytotoxin assay (36 specimens). When 36 available "positive" specimens were tested by the PCR assay, 34 (94%) gave positive results--24 by direct testing, and 10 after extraction of DNA by the Qiagen procedure. Insufficient material of the remaining two specimens was available for DNA extraction. Of 21 stools "negative" for C. difficile by culture or cytotoxin assay, one gave a positive result by PCR and seven produced atypical bands. The rapid PCR detection technique for C. difficile was more sensitive than standard culture, and of a sensitivity similar to cytotoxin testing. The method has the potential for adoption in routine laboratory practice.
开发了一种通过聚合酶链反应（PCR）检测粪便标本中艰难梭菌肠毒素基因的快速检测方法。PCR引物扩增了肠毒素基因的一段63碱基对的重复序列，从而在电泳后产生独特的DNA条带阶梯模式。含有艰难梭菌的粪便粗提DNA提取物产生一条（63碱基对）或更多条特征性阶梯条带。在来自58名患者的172份粪便标本中，37份通过培养（15份标本）或细胞毒素检测（36份标本）得出阳性结果。当用PCR检测法检测36份可用的“阳性”标本时，34份（94%）呈阳性结果——24份直接检测呈阳性，10份经Qiagen方法提取DNA后呈阳性。其余两份标本没有足够的材料用于DNA提取。在21份经培养或细胞毒素检测对艰难梭菌呈“阴性”的粪便中，1份经PCR检测呈阳性，7份产生非典型条带。艰难梭菌的快速PCR检测技术比标准培养法更敏感，且灵敏度与细胞毒素检测相似。该方法有可能应用于常规实验室实践。
