PCR结合反向膜杂交快速鉴定临床常见念珠菌方法学的建立

To apply PCR-Reverse membrane hybridization for rapid detection of clinical main pathologic Candida

目的建立PCR结合反向膜杂交技术快速检测临床常见致病念珠菌的方法。方法根据真菌rDNA高度可变区设计白色念珠菌、热带念珠菌,近平滑念珠菌,都柏林念珠菌,光滑念珠菌、克柔念珠菌的种特异性探针,加尾后固定在尼龙膜上,生物素标记的通用引物扩增转录间隔区ITS1,利用反向膜杂交技术快速检测念珠菌种群。结果以上六种念珠菌均能扩增出大小为218 bp的条带,每张膜上只有与靶序列对应的探针点有阳性结果,其他探针点均为阴性。正常人类基因组DNA及临床常见的几种细菌DNA扩增结果均为阴性。结论 PCR结合反向膜杂交技术方法学的建立,可以快速检测临床常见致病的念珠菌。该方法特异性高,耗费时间短。可以快速、准确的为临床抗真菌药物的使用提供指导依据。

Objective To establish rapid methods to detect and identify clinical main pathologenic Candida by using PCR-Reverse membrane hybridization.Methods Six species-specific oligonucleotide probes（Candida albicans,Candida tropicalis,Candida parapsilosis,Candida dubliniensis,Candida glabrata,Candida krusei）were designed based on ITS1 and added multimer T tails so as to inoculated on membrane.The DNA of the Candida spp were amplified with biotin labeled universal primers based on rDNA.The PCR products were detected and identified by hybridization with the probes on the membrane.Results 218 bp of PCR products were amplified in all the 6 species of candida.Each species-specific probe only hybridized with its target molecules among the 6 species candidas.Amplification and hybridization results were negative in Normal human genome DNA and several common clinical bacterials.Conclusion The PCR reverse hybridization membrane was proved to be a fast,sensitive and specific technology,it only needed 3 or 4 hours and was potential for clinical application.