OBJECTIVETo develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin.

METHODSTaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi) of C. difficile strains and the toxins A(TcdA), B(TcdB) and truncated toxin A(TcdAT). Sensitivity, specificity and anti-interference ability of these methods were estimated, as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay.

RESULTSThe detection limits of tpi were 6×10⁻² CFU/µl and 6 × 10⁻¹ CFU/µl in the non-oxin producing and toxin producing strains, respectively. The coefficients of variability(CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3% . The CVs of intra-assay and inter-assay for the detection limit of tpi, tcdA, tcdB and tcdAT in the toxin producing strain were 3.0% and 3.4%, 2.9% and 3.2%, 5.3% and 5.7%, 2.7% and 2.8%, respectively. No interference was detected from other genus or species in clostridium. From 50 clinical samples, thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique, in which 3 were dubious and 2 were negative under VIDAS.

CONCLUSIONThe newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis, clinically.