Use of the Polymerase Chain Reaction for the Specific and Direct Detection of Clostridium dijficile in Human Feces

Abstract

The polymerase chain reaction was used for the detection of Clostridium difficile, the etiologic agent of antibiotic-associated colitis. An upstream primer identical to a coding region (segment I) of the C. difficile 16SrRNA gene and a downstream primer complementary to a highly conserved region of eubacterial 16S rRNA served to amplify a targeted 270-base-pair fragment of genomic DNA. This technique allowed the detection of as fewas 10 C. difficile organisms among 10⁶Escherichia coli bacteria. This level of sensitivity represents a lOO-fold increase over that of conventional anaerobic culture. C. difficile wasdetected in DNAextracted directly from the stools of 23 patients with antibiotic-associated colitis and from those of four patients with diarrhea whosestoolshad beennegativefor C. difficile whenassessedin a cytotoxicity assay. No amplification products were found in the stools of asymptomatic patients. When detected in stools of symptomatic patients, amplification products of C. difficile were confirmed bySouthern blotting with a nonradioactive, horseradish peroxidase-catalyzed, chemiluminescent probing systemin which biotin-labeled oligonucleotides wereused. This system discriminates between C. difficile and similar organisms, such as Clostridium sordellii and Clostridium bifermentans. The combination of the polymerase chain reaction with enzyme-linked probing results in a faster and more sensitive assay for C. diJficile than standard culture.