Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic Clostridium difficile in Human Stools

Background: Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation. Methods: TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC). Results: The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA(+)TcdB(+), TcdA(-)TcdB(+), and TcdA(-)TcdB(-) types), TcdB-producing strains (TcdA(+)TcdB(+) and TcdA(-)TcdB(+) types), and TcdA-producing strains (TcdA(+)TcdB(+) type), respectively, with a lower detection limit of 10(3) cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA(+)TcdB(+) strain in six specimens and TcdA(-)TcdB(-) strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 10(4.5) cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota. Conclusion: Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.