鸡源产气荚膜梭菌重组酶聚合酶扩增检测方法的建立  

Recombinase polymerase amplification:a detection method for the detection of Clostridium perfringens from chicken

作　　者：许笑 王艳华[1,2] M Umar Zafar Khan 王恒 蔡建平 XU Xiao;WANG Yan-hua;M Umar Zafar Khan;WANG Heng;CAI Jian-ping(Stale Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;JiangSu Co-Innovation Center for Prevention and Control of Animal Infectious Disease and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2019年第12期1475-1483,共9页Chinese Veterinary Science

基　　金：公益性行业(农业)专项(201303044-7)资助

摘　　要：为建立鸡源产气荚膜梭菌(Clostridium perfringens)的快速诊断方法,本研究以产气荚膜梭菌α-毒素的编码基因plc为基础设计、合成引物和探针,并以real-time PCR的方法进行验证,建立了鸡源产气荚膜梭菌的重组酶聚合酶扩增(RPA)检测方法。该方法可在38℃、25 min内对产气荚膜梭菌的α-毒素编码序列进行快速特异性扩增,最低检测限量可达1×10^1copies/μL,灵敏度良好。以此方法检测实验室保存的临床样本,其检测结果与real-time PCR的检测结果一致。上述结果表明,本研究建立的real-time RPA方法在检测鸡源产气荚膜梭菌时具有操作简单、快速灵敏的特点,适用于鸡源产气荚膜梭菌的临床检测。In order to develop a rapid molecular detection method for Clostridium perfringens(C.perfringens) causing chicken necrotic enteritis(NE),a real-time recombinase polymerase amplification(RPA) method based on the α-toxin gene of C.perfringens(Cp PLC) was built,and validated with real-time PCR method as a control.This real-time RPA can complete the specific amplification to Cp PLC at 38 ℃ for 25 min, with lower reaction temperature and shorter run time compared to a common real-time PCR method,as well as high sensitivity in lowest detectable copy number to 1×10^1 copies/μL.It was also found that the results for clinical sample detections with this real-time RPA are highly consistent with the conventional real-time PCR.Our results shows that the real-time RPA detection for C.perfringens established here can be used for detecting C.perfringens related to chicken NE.

关 键 词：产气荚膜梭菌 重组酶聚合酶扩增技术 荧光定量检测 鸡坏死性肠炎 

分 类 号：S852.616[农业科学—基础兽医学]