Using the real-time PCR assay to establish TaqMan-MGB probe for rapid identification of Clostridium difficile and its toxin
10.3760/cma.j.issn.0254-6450.2014.05.024
- VernacularTitle:TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立
- Author:
Donghua SHAO
1
;
Na JI
;
Guowei LIANG
;
Jing LIU
Author Information
1. 航天中心医院检验科
- Keywords:
Clostridium difficile;
TaqMan-MGB probe;
Real-time PCR;
Bacteria genus gene;
Toxin gene
- From:
Chinese Journal of Epidemiology
2014;(5):576-580
- CountryChina
- Language:Chinese
-
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 interferance 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. Conclusion 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.
