title: [Using the real-time PCR assay to establish TaqMan-MGB probe for rapid identification of Clostridium difficile and its toxin].
abstract: OBJECTIVE
  To develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin.
  
  
  METHODS
  TaqMan 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors: Department of Clinical Laboratory, Aerospace Center Hospital, Beijing 100049, China.
authors: Donghua
authors: D
authors: Shao
authors: Department of Clinical Laboratory, Aerospace Center Hospital, Beijing 100049, China.
authors: Na
authors: N
authors: Ji
authors: Department of Clinical Laboratory, Aerospace Center Hospital, Beijing 100049, China. Email: htyysdh2006@qq.com.
authors: Guowei
authors: G
authors: Liang
authors: Department of Clinical Laboratory, Aerospace Center Hospital, Beijing 100049, China.
authors: Jing
authors: J
authors: Liu
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification*; D001427:Bacterial Toxins / Q000302:isolation & purification*; D016360:Clostridioides difficile / Q000302:isolation & purification*; D004768:Enterotoxins / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile