Detection of Toxin B Gene of Clostridium difficile by Polymerase Chain Reaction from Clinical Isolates

Lee, Hyukmin; Kim, Young Ah; Park, Kwang Il; Lee, Kyungwon; Chong, Yunsop

Abstract

BACKGROUND: Clostridium difficile causes antibiotic-associated diarrhea or pseudomembranous colitis by producing of toxins in patients treated with antimicrobial agents. Stool cultures for C. difficile and tests for the presence of its toxin are the most widely used methods for the diagnosis of infection. The aim of this study was to determine the usefulness of polymerase chain reaction for the detection of toxin B gene from C. difficile isolates.
METHODS
In this study, 85 strains of C. difficile were used, which were isolated from stool specimens of patients with suspected antibiotic-associated diarrhea or pseudomembranous colitis from 1987 to 1994 using cefoxitin-cycloserine-fructose agar. DNA of the C. difficile isolates was extracted by boiling and by conventional methods. The primers used for toxin B gene amplification were YT-17, 5'-GGTGGAGCTTCAATTGGAGAG-3' and YT-18, 5'- GTGTAACCTACTTTCATAACACCAG-3'. Amplification products were electrophoresed in a 1% agarose gel containing ethidium bromide and the presence of the 399 bp band was examined under ultraviolet light. The results were compared with those of toxin A detection by PCR and with the results of quantitative cultures.
RESULTS
Toxin B gene was detected in 74% (63/85) of the C. difficile isolates. Toxin B gene was detected in all strains with toxin A gene, but not in the strains without toxin A gene. DNA extraction by boiling and by conventional methods gave the same detection rate. The positive rate of toxin B gene was slightly higher in the strains which were isolated with a higher colony count from stool than nontoxigenic ones.
CONCLUSIONS
The PCR detection of toxin B gene is a useful method for identifying the toxigenic C. difficile strain in the clinical laboratory, and the boiling method is simple for DNA extraction. The use of a toxin test can reduce false positive diagnosis due to the presence of nontoxigenic strains among the isolates.