Detection of Clostridium difficile related toxin gene by Allglo probe-multiplex real-time PCR

Abstract

Objective To establish a rapid, specific and sensitive assay based on multiplex real-time PCR integrated with Allglo probes for detection and identification of Clostridium difficile related toxin genes, and investigate the situation of diarrhea patients infected by toxigenic C.difficile in Hangzhou. Methods The primer pairs and Allglo probes that were specific to tcdA,tcdB gene of C.difficile were designed, and then the PCR amplification condition was optimized, the specificity, low of limitation and reproducibility were evaluated.A total of 180 diarrheal stool specimens were collected from August to December 2012 from Hangzhou First People′s Hospital.Finally, the multiplex real-time PCR results were compared with the results obtained by direct sequencing. Results The low of limitation was 10 CFU/ml.The coefficients of variation for tcdA gene were 2.30%, 0.42%, 0.81% respectively, and the coefficients of variation for tcdB gene were 1.91%, 1.02%, 1.18%, respectively.All of them were less than 5%.There were 26 positive in 180 diarrhea stool specimens，the positive rate was 14.4%.Among the positive specimens, 23 stool specimens were tcdA and tcdB positive (23/26, 88.5%), and then only two stool specimens were tcdA positive to and tcdB negative (2/26, 7.7%), only one stool specimen was tcdA negative and tcdB positive to was detected (1/26, 3.8%).Then, all the specimens were detected by using a direct sequencing method, and the sequences were aligned with standard.The above results were in correspondence with the results of the real-time PCR. Conclusions The results indicated that it was the main characteristics for C.difficile strains with tcdA and tcdB toxin genes infecting diarrheal patients in Hangzhou, Zhejiang.The assay established here could be used in clinic for rapid, accurate detection of toxigenic C.difficile.（Chin J Lab Med,2014,37:32-35） Key words: Clostridium difficile; Bacterial toxins; Enterotoxins; Cytotoxins; Fluorescence; Polymerase chain reaction