产气荚膜梭菌α毒素荧光定量PCR检测方法的建立及初步应用  

Establishment and Preliminary Application of a Fluorescence Quantitative PCR for Clostridium perfringens Alpha Toxin

作　　者：杨夷平 李海利 杨帆 杨康 段进刚 张国新 李斌 马春江 Yang Yiping;Li Haili;Yang Fan;Yang Kang;Duan Jingang;Zhang Guoxin;Li Bin;Ma Chunjiang(Hami Center for Animal Disease Prevention and Control,Hami,Xinjiang 839000,China)

机构地区：[1]哈密市动物疫病预防控制中心,新疆哈密839000

出　　处：《中国动物检疫》2023年第11期95-99,共5页China Animal Health Inspection

基　　金：2021年新疆哈密市人才发展专项(2022SNGGNT032)。

摘　　要：为利用TaqMan荧光定量PCR技术建立一种快速、敏感、特异的产气荚膜梭菌α毒素检测方法,根据产气荚膜梭菌各型之间共同的生物学特性,以编码产气荚膜梭菌α毒素的基因序列作为靶序列,设计1对引物和探针,分别以A、B、C、D4个型的产气荚膜梭菌DNA为模板,通过优化引物、探针浓度和扩增条件,建立了TaqMan荧光定量PCR方法,同时对该方法的特异性、灵敏度和重复性进行了验证。结果显示:当上下游引物浓度和探针浓度均为0.25μmol/L时,Ct值最低;该方法与大肠杆菌、巴氏杆菌、沙门氏菌等细菌均无交叉反应,具有良好的特异性;灵敏度高,对A型产气荚膜梭菌基因组DNA的最低检测限为9.1pg/μL;重复性好,组内及组间试验标准差均小于0.4;对2023年收集的24份样品进行检测,发现本方法的阳性检出率(20.83%)与商品试剂盒一致。综上,本研究建立了一种快速、灵敏、稳定、特异的TaqMan荧光定量PCR方法,可对产气荚膜梭菌α毒素进行快速检测,为防控产气荚膜梭菌病提供了有力的技术支撑。In order to establish a rapid,sensitive and specific method for detecting alpha toxin of Clostridium perfringens using TaqMan fluorescence quantitative PCR assay,based on common biological characteristics of all types of Clostridium perfringens,a pair of primers and a probe were designed with using the gene sequence encoding alpha toxin as the target sequence.A,B,C and D types of Clostridium perfringens DNA were used as templates to establish a TaqMan fluorescence quantitative PCR assay through optimizing the concentration of primers and probes and amplification conditions,meanwhile,its specificity,sensitivity and repeatability were evaluated.The results revealed that the Ct value was the lowest when the concentration of primers and probe were all 0.25μmol/L;the method had good specificity without any cross-reaction with Escherichia coli,Pasteurella and salmonella;and good sensitivity with the minimum detection limit of 9.1 pg/μL for genomic DNA of Clostridium perfringens type A;and good repeatability with standard deviations of less than O.4 for both intra group and inter group experiments;it was found that,after detection of 24 clinical samples collected in 2023,the positive detection rate of the established method(20.83%)was consistent with that of commercial reagent kits.In conclusion,a rapid,sensitive,stable and highly specific TaqMan fluorescence quantitative PCR assay was well established in the study,which could be used to quickly detect Clostridium perfringens alpha toxin,providing a powerful technical support for the prevention and control of Clostridium perfringens disease.

关 键 词：产气荚膜梭菌 Α毒素 TaqMan荧光定量PCR 检测 

分 类 号：S852.62[农业科学—基础兽医学]