Toxigenic strains of Clostridium difficile are causative agents of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis. The toxigenicity is routinely assayed by using highly sensitive cell cultures. We used a simple and rapid polymerase chain reaction (PCR) assay to differentiate toxigenic and nontoxigenic strains of C. difficile. Two sets of oligonucleotide primer pairs derived from nonrepeating sequences of the toxin A gene were used to amplify 546-and 252-bp DNA fragments. A primer pair derived from repeating sequences of the toxin A gene was used to amplify a 1,266-bp DNA product. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 35 cytotoxic strains of C. difficile tested generated the expected amplified DNA. In contrast, none of the 26 noncytotoxic strains tested gave positive results. Although the toxins of C. difficile have been demonstrated to cross-react serologically with the toxins of Clostridium sordellii, we did not detect any amplified DNA in two cytotoxic strains or seven noncytotoxic strains of C. sordellii. PCR was negative in all 30 strains of 20 other Clostridium species. Southern hybridization of HindIll-digested genomic DNA by use of subgenomic probes showed a single hybridization band in toxigenic strains but not in nontoxigenic strains. PCR appears to be a sensitive and specific assay for the rapid identification of toxigenic C. difflcile. Nontoxigenic C. difficile appeared to lack the C. difflcile toxin A gene. Clostridium difficile is known as a major cause of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis (2, 13), although many infants and hospitalized patients can be asymptomatically colonized with C. difficile (3, 16). The organism produces at least two toxins: toxin A (a potent enterotoxin) and toxin B (a potent cytotoxin) (1, 24, 25). These toxins are thought to play a major role in the diarrhea and colitis caused by C. difficile. C. difficile-induced diarrhea or colitis is suspected in the patients who develop diarrhea or colitis during or after treatment with antimicrobial agents and can be confirmed by the detection of the toxin(s) or the isolation of toxigenic C. difficile from the stool specimen. The cell culture assay is preferably used to detect the cytotoxin produced by toxigenic C. difficile strains because of its high sensitivity and * Corresponding author. from nontoxigenic strains. Negative PCR results were demonstrated in DNAs of C. sordellii and other Clostridium spp. MATERIALS AND METHODS Bacteria. The bacteria used in this study are listed in Table 1. The toxigenic strain C. difficile VPI 10463 was provided by

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Iodination of highly purified hepatitis A (HAV) virus results in the selective labeling of two viral polypeptides, which are identified as the the VP 1 and VP 2 capsid polypeptides. Based upon the kinetics of labeling, the exposed region of VP 1 appears to be more accessible to iodination, although the ultimate proportion of label present within VP 1 and VP 2 is approximately equal. By utilizing iodinated whole virions, isolated VP 1, VP 2, and the tryptic digest derived from VP 1 and VP 2, binding by heterologous anti-160 S antibody indicated that a significant portion of the antibodies was directed against an epitope on VP 2 that was not affected by denaturation. Identification of the regions exposed for iodination on these two polypeptides was accomplished by tryptic digestion of the isolated polypeptides followed by characterization of the iodinated tryptic peptide by gel filtration and reverse-phase chromatography. The results indicate that tyrosine 100 on VP 2 and a large tryptic peptide composed of amino acids 222 through 260 on VP 1 which contains four tyrosine residues are two regions that are surface-exposed on these molecules.