Background: Clostridium difficile is the major cause of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC). The aim of this study was to develop the nested PCR assay for direct detection of toxigenic C. difficile in stool specimens and to evaluate the usefulness of the method.

Methods: Specificity of newly designed primers are tested with 36 reference strains of intestinal flora. Lower detection limit of nested PCR for B toxin gene in C. difficile was determined using 10-fold serial dilutions of C. difficile ATCC 9689. One hundred and two clinical stool samples were cultured for detection of C. difficile on cycloserine-cefoxitin- fructose agar and the PCR assay for detection of toxin B gene in C. difficile isolates was performed. Nested PCR assay for direct detection of toxin B gene in clinical samples was also performed.

Results: Nested PCR assay showed negative amplification results in intestinal floras except C. difficile ATCC 9689. Lower detection limit of nested PCR for toxin B gene was 10⁴ CFU/mL. Sensitivity of nested PCR assay compared to culture method was 100% (29/29), and the specificity was 68% (50/73).

Conclusion: Nested PCR assay showed high sensitivity in direct detection of toxin B gene in C. difficile isolates even after administration of metronidazole, so the assay could be used in initial diagnosis and follow-up tests of AAD and PMC. (Korean J Clin Microbiol 2003;6(1):63-68)