Lou Q, Chong S K, Fitzgerald J F, Siders J A, Allen S D, Lee C H
Department of Pediatrics, Indiana University School of Medicine, Indianapolis 46202, USA.
J Clin Microbiol. 1997 Jan;35(1):281-3. doi: 10.1128/jcm.35.1.281-283.1997.
We have developed a novel method for the preparation of fecal specimens for PCR assays. Approximately 100 mg of solid stool or 200 microliters of liquid fecal sample was thoroughly suspended in 1 ml of water. Fecal debris was removed by low-speed centrifugation (2,800 x g for 2 min). The supernatant was then boiled for 10 min in a water bath and further clarified by high-speed centrifugation (12,000 x g for 5 min). Fifty microliters of the clarified supernatant was then purified by Sepharose CL-6B spin column chromatography, and a portion of the purified supernatant was used for PCR. By this method, stools containing enterotoxigenic Escherichia coli H10407 were amplified by colonization factor antigen I fimbrial gene PCR, with a sensitivity of 100 organisms per reaction. The method was also effective for processing stool specimens for Clostridium difficile toxin A and B gene PCRs. This method is rapid, effective, and simple to perform and will improve the applications of PCR to stool specimens for diagnostic purposes.
我们开发了一种用于制备粪便标本以进行PCR检测的新方法。将约100mg固体粪便或200微升液体粪便样本充分悬浮于1ml水中。通过低速离心（2800×g，2分钟）去除粪便残渣。然后将上清液在水浴中煮沸10分钟，并通过高速离心（12000×g，5分钟）进一步澄清。接着，取50微升澄清的上清液通过Sepharose CL - 6B旋转柱色谱法进行纯化，纯化后的上清液一部分用于PCR。通过这种方法，含有产肠毒素大肠杆菌H10407的粪便经定居因子抗原I菌毛基因PCR扩增，每个反应的灵敏度为100个菌。该方法对处理用于艰难梭菌毒素A和B基因PCR的粪便标本也有效。此方法快速、有效且操作简单，将改善PCR在粪便标本诊断应用中的效果。
