1. Rapid and effective method for preparation of fecal specimens for PCR assays
- Author
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Qinyuan Lou, Stephen D. Allen, Joseph F. Fitzgerald, Jean A. Siders, Sonny K. F. Chong, and Chao-Hung Lee
- Subjects
Microbiology (medical) ,Clostridium difficile toxin B ,Biology ,medicine.disease_cause ,biology.organism_classification ,Molecular biology ,Enterobacteriaceae ,Polymerase Chain Reaction ,Bacterial Typing Techniques ,Sepharose ,Feces ,fluids and secretions ,Enterotoxigenic Escherichia coli ,Spin column-based nucleic acid purification ,medicine ,Sample preparation ,Centrifugation ,Escherichia coli ,Research Article - 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.
- Published
- 1997