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Rapid and effective method for preparation of fecal specimens for PCR assays.

By Q Lou, S K Chong, J F Fitzgerald, J A Siders, S D Allen and C H Lee

Abstract

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

Topics: Research Article
Year: 1997
OAI identifier: oai:pubmedcentral.nih.gov:229557
Provided by: PubMed Central
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