艰难梭菌分离株快速鉴定和毒素检测的多重PCR方法的建立

目的 建立可同时进行艰难梭菌分离株菌种鉴定和毒素检测的多重PCR方法。方法 用于多重PCR中的3对引物分别为艰难梭菌的种特异性的磷酸内糖异构酶(triose phosphate isomerase，tpi)基因、毒素A基因部分序列、毒素B基因部分序列。艰难梭菌ATCC 9689等21株标准菌株和47株临床分离艰难梭菌分别被应用于多重PCR最低检出限、特异性评估试验和验证试验。同时，应用ELISA对47株分离株进行毒素A/B检测。结果 该多重PCR方法可检测到最低DNA浓度为0.5 pg/μ l，特异性为100％。47株艰难梭菌分离株中tpi基因均为阳性，其中毒素基因A(+)/B(+)为37株，毒素基因A（-）/B（-）为10株，未检出毒素基因A（-）/B(+)菌株。47株毒素A/B检测结果为20株阳性、27株阴性。毒素A/B阳性的20株菌均为多重PCR检测毒素基因A(+)/B(+)。结论 成功建立用于艰难梭菌的菌种鉴定和毒素分析结合为一体的多重PCR方法，对临床诊断艰难梭菌感染有着重要应用价值。

Establishment of multiplex PCR for the rapid identification and toxin detection of Clostridium difficile strains

Objective To design a multiplex PCR for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Methods Three 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. Results The 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. Conclusion The multiplex PCR method combined diagnosis and toxigenic type characterization contributes to the diagnosis for Clostridium difficile infection.