中国兽医科学2024，Vol.54Issue(2)：174-184,11.DOI:10.16656/j.issn.1673-4696.2024.0023
产气荚膜梭菌多重TaqMan荧光定量PCR检测方法的建立
Establishment of a multiplex TaqMan qPCR method for detection of Clostridium perfringens
摘要
Abstract
To establish a method for rapid detection and typing of five toxin genes cpa,cpb,etx,itx and cpe of Clostridium perfringens(C.perfringens).Specific primers and TaqMan probes were designed based to the cpa,cpb,etx,itx and cpe toxin gene sequences of C.perfringens published in NCBI database.By con-structing standard positive plasmids,optimizing reaction conditions,constructing standard curves,and performing specificity,sensitivity,repeatability and clinical sample detection tests,a multiplex TaqMan fluorescence quantitative PCR(qPCR)detection method was established that can simultaneously detect five toxin genes.The multiplex TaqMan qPCR assay was established with a good linearity of its amplification curve with R2＞0.99 and 90％＜E＜110％,with a high specificity that this method was no cross-reactivity with different pathogens,with a high sensitivity that the lower limit of detection for all its five positive plasmids was 102 copies/μL,and with a good repeatability that the coeffi-cients of variation were all less than 2％.Thirty samples were tested by using this method,and the re-sults showed a total of 30 positive samples,including 11 strains of C.perfringens type A,1 strain of C.perfringens type C and 18 strains of C.perfringens type D.The multiplex TaqMan qPCR detection method for C.perfringens established in this experiment provides a convenient,rapid and efficient technical support for the diagnosis,prevention and control of C.perfringens disease.关键词
产气荚膜梭菌/毒素基因/多重TaqMan荧光定量PCRKey words
Clostridium perfringens/toxin gene/multiplex TaqMan qPCR分类
农业科技引用本文复制引用
王景松,郭亚男,王健霖,李继东,王建东..产气荚膜梭菌多重TaqMan荧光定量PCR检测方法的建立[J].中国兽医科学,2024,54(2):174-184,11.基金项目
宁夏回族自治区重点研发项目(2021BEF02026,2022BBF03024) （2021BEF02026,2022BBF03024）
银川市2023推进产学研融合助力农业高质量发展项目 （）
