Rapid and effective method for preparation of fecal specimens for PCR assays.
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.
Lou
Q
Q
Department of Pediatrics, Indiana University School of Medicine, Indianapolis 46202, USA.
Chong
S K
SK
Fitzgerald
J F
JF
Siders
J A
JA
Allen
S D
SD
Lee
C H
CH
D015373:Bacterial Typing Techniques; D005243:Feces / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*