空肠弯曲杆菌SYBR GreenⅠ荧光定量PCR检测方法的建立  

Development of a SYBR GreenⅠFluorescence Quantitative PCR Assay for Campylobacter jejuni

作　　者：张风荣 冯之航 王艳 陈翔 王辰 杨新奥 杨金保 Zhang Fengrong;Feng Zhihang;Wang Yan;Chen Xiang;Wang Chen;Yang Xin'ao;Yang Jinbao(Shandong Vocational Animal Science and Veterinary College,Weifang,Shandong 261061,China;Technical Center of Shanghai Customs,Shanghai 200135,China;Shanghai Customs,Shanghai 200135,China;Qingdao Agricultural University,Qingdao,Shandong 266109,China)

机构地区：[1]山东畜牧兽医职业学院,山东潍坊261061 [2]上海海关动植物与食品检验检疫技术中心,上海200135 [3]上海海关,上海200135 [4]青岛农业大学,山东青岛266109

出　　处：《中国动物检疫》2023年第3期85-89,共5页China Animal Health Inspection

基　　金：潍坊市科研创新平台-潍坊市宠物疾病防控工程技术研究中心项目。

摘　　要：空肠弯曲杆菌(Campylobacter jejuni)是一种人兽共患病病原菌,给养殖业带来了较大经济损失。为实现对空肠弯曲杆菌的快速检测,针对其MapA基因序列设计特异性引物,优化反应条件和反应体系,建立了空肠弯曲杆菌SYBR GreenⅠ荧光定量PCR检测方法,并对该方法的特异性、敏感性、重复性进行了评估。结果显示:建立的SYBR GreenⅠ荧光定量PCR检测方法具有良好的特异性,仅能特异性扩增出空肠弯曲杆菌,对胎儿空肠杆菌、大肠杆菌、金黄色葡萄球菌、沙门氏菌无交叉反应;敏感性较强,最低检测限为6.42 copies/μL;以5个稀释度的重组质粒为模板分别进行组内和组间重复性试验,发现组内和组间Ct值变异系数(CV)均小于2.50%,说明该方法具有良好的可重复性。结果表明,本研究建立的SYBR GreenⅠ荧光定量PCR具有良好的特异性、敏感性及重复性,可用于空肠弯曲杆菌的快速诊断和流行病学调查。As one of zoonotic pathogens,Campylobacter jejuni(CJ)could bring huge economic loss to animal industry.In order to rapidly detect it,specific primers were designed based on its MapA gene sequence,followed by optimization of the reaction conditions and reaction system,a SYBR greenⅠfluorescence quantitative PCR assay for CJ was established,followed by evaluation on its specificity,sensitivity and repeatability.The results showed that the established method,with good specificity,not only specifically amplified CJ,but also failed to crossly react with fetal jejunobacteria,Escherichia coli,Staphylococcus aureus and Salmonella;its minimum detection limit reached 6.42 copies/μL due to its strong sensitivity;intra-group and inter-group repeatability tests were carried out using the recombinant plasmid with 5 dilutions as a template,and it was found that both the coefficient of variation(CV)of Ct values were less than 2.50%,indicating a good repeatability.In conclusion,the established method was with good specificity,sensitivity and repeatability,which could be used for rapid diagnosis and epidemiological investigation of CJ.

关 键 词：空肠弯曲杆菌 SYBR GreenⅠ荧光定量PCR 特异性 敏感性 重复性 

分 类 号：S855.1[农业科学—临床兽医学]