Establishment of multiplex PCR for the rapid identification and toxin detection of Clostridium difficile strains / 中华微生物学和免疫学杂志

ABSTRACT

Objective To design a multiplex PCR for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. MethodsThree pairs of primers were designed for the amplification of a species-specific internal fragment of the tpi( triose phosphate isomerase) gene, an internal fragment of the tcdB ( toxin B) gene, and an internal fragment of the tcdA ( toxin A) gene. Twenty-one standard strains including Clostridium difficile ATCC 9689 and 47 isolates of Clostridium difficile were applied for the assessment of detection limit, specificity and detections of the multiplex PCR, respectively. Toxin A and Toxin B of 47 isolates were analyzed by ELISA. ResultsThe detection limit for DNA concentration of the multiplex PCR was 0.5 pg/μl. The specificity was determined to be 100％. Among the results of 47 isolates detected by multiplex PC R, 37 strains were tpi ( + )/tcdA (+)/tcdB ( + ), 10 strains were tpi ( + )/tcdA (-)/tcdB ( - ). Tpi ( + )/tcdA ( - )/tcdB ( + ) was not found. The toxin detection of 47 isolates by ELISA showed that 20 isolates were positive and 27 isolates were negative. Twenty isolates of toxin (+) by ELISA were all tpi( +)/tcdA( +)/tcdB(+) by multiplex PCR. ConclusionThe multiplex PCR method combined diagnosis and toxigenic type characterization contributes to the diagnosis for Clostridium difficile infection.