实时荧光定量PCR法检测产气荚膜梭菌  

Real-time quantitative PCR for rapid detection of Clostridium perfringens

作　　者：李林海[1] 石玉玲[1] 陈丽丹[1] 曾兰兰[1] 王露霞[1] 孙朝晖[1] 何洁静[1] 陈建芸[1] 杨永泉[1] 曾桦[2] 岳枫 

机构地区：[1]广州军区广州总医院检验科,广州市510010 [2]中山大学附属第二医院检验科,广州市510120 [3]广东省武警总队医院检验科,广州市510507

出　　处：《中华实用诊断与治疗杂志》2011年第7期656-659,共4页Journal of Chinese Practical Diagnosis and Therapy

基　　金：全军医药卫生"十一五"重大专项(08Z009)

摘　　要：目的:运用实时荧光定量PCR法检测产气荚膜梭菌,为早期快速诊断气性坏疽提供新方法。方法:以产气荚膜梭菌16s rDNA基因序列为模板,在其保守区域设计特异性引物与探针,将PCR扩增所得产物片段克隆,作为定量检测的标准品,绘制标准曲线。并对荧光定量PCR体系与反应条件进行优化,验证方法的特异性、敏感性、重复性及可行性。结果:实时荧光定量PCR法对产气荚膜梭菌的检测具有高度特异性,与创伤弧菌等24种相关细菌等均无交叉反应;检测灵敏度以纯菌计数达9×102cfu/mL,相当于9 cfu/反应;反应体系有较高稳定性;整个操作过程仅需3 h;300例创伤可疑分泌物样本的荧光定量PCR检测结果与细菌培养结果一致。结论:实时荧光定量PCR法特异、灵敏、快速,适用于产气荚膜梭菌的临床检测及突发事件的批量检测。Objective To detect Clostridium perfringens with real-time quantitative PCR technique to provide a new tool for diagnosing gas gangrene early.Methods The gene sequences of Clostridium perfringens downloaded from the genebank were aligned using the biologic software and the specific primers and probe were designed in the conserved region of the 16s rDNA gene from Clostridium perfringens.The primers,probe and the reactive condition were optimized to improve the sensitivity,specificity,repetitiveness and feasibility of the assay.Results Only Clostridium perfringens possessing 16s rDNA gene generated fluorescent signals,and no cross-reaction was observed with 24 other associated bacteria.The reactive system had a high stability.The sensitivity achieved was 9×102cfu/ml,i.e.9 cfu/per reaction.It took only three hours to do the real-time quantitative PCR.The results of 300 cases of suspected wound secretion detected by the real-time quantitative PCR were the same with bacteria cultivation.Conclusion The real-time quantitative PCR assay is a quick,sensitive and specific tool for usual monitoring and emergent examination of Clostridium perfringen.

关 键 词：实时荧光定量PCR 产气荚膜梭菌 气性坏疽 16s RDNA 

分 类 号：R440[医药卫生—诊断学]