Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology

6

Weeks in Review

2

Weeks to Publication
Indexed in KCI, KoreaMed, Synapse, DOAJ
Open Access, Peer Reviewed
pISSN 2288-0585 eISSN 2288-6850

Direct Detection of Clostridium difficile Toxin B Gene by Nested PCR in Human Stool Specimens

Original article

Annals of Clinical Microbiology (Ann Clin Microbiol) 2003 June Volume 6, Issue 1, pages 63-68.

Direct Detection of Clostridium difficile Toxin B Gene by Nested PCR in Human Stool Specimens

Hee Kyung Park, Young Mi Lee, Hyun Jung Jang, Cheol Min Kim, Kyungwon Lee, Seok Hoon Jeong, Mooin Park

Institute for Biomedical Research, SJ Hightech Co.; Department of Biochemistry*, Pusan National University College of Medicine, Busan; Department of Diagnostic Medicine** and Research Institute of Bacterial Resistance***, Yonsei University College of Medicine, Seoul; Department of Diagnostic Medicine**** and Internal Medicine*****, Kosin University College of Medicine, Busan, Korea

Abstract

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)

Keywords

Clostridium difficile, nested PCR, Toxin B