弓形虫半巢式PCR实时荧光检测方法的建立  

Development of a New Semi-nested PCR Real-time Fluorescence Technology for Detection of Toxoplasma gondii

作　　者：陈梦陶 朱龙佼 刘海燕 许文涛[2,3] CHEN Meng-Tao;ZHU Long-Jiao;LIU Hai-Yan;XUWen-Tao(School of Public Health,North China University of Science and Technology,Tangshan 063210,China;Department of Nutrition and Health,Key Laboratory of Food Precision Nutrition and Quality Control,China Agricultural University,Beijing 100083,China;College of Food Science&Nutritional engineering,Key Laboratory of Transgenic Biological Safety Evaluation(Food Safety)/Food Science and Nutrition Engineering,Beijing Laboratory of Food Quality and Safety,China Agricultural University,Beijing 100083,China;Research Center for Sports Nutrition and Eudainomics,Tianjin University of Sport,Tianjin 301617,China)

机构地区：[1]华北理工大学公共卫生学院,唐山063210 [2]中国农业大学营养与健康系/食品精准营养与质量控制教育部重点实验室,北京100083 [3]中国农业大学食品科学与营养工程学院转基因生物安全性评价(食品安全)重点实验室/食品质量与安全北京实验室,北京100083 [4]天津体育学院运动营养与健康组学研究中心,天津301617

出　　处：《农业生物技术学报》2023年第12期2665-2673,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2022YFF0607900);北京市科技计划(Z221100007122004);北京市景观休闲农业创新团队项目(BAIC09-2023);河北省重点研发计划(21372801D);青年人才托举工程项目(BYESS2022133)。

摘　　要：弓形虫(Toxoplasma gondii)是一种全世界分布的人畜共患寄生虫,在猪(Sus scrofa)和小型家畜中的慢性感染率较高,能够经食源性传播感染人类,对免疫功能较弱的个体会造成极大的危害。本研究根据GenBank公布的弓形虫529 bp基因序列(DQ779191.1)设计了一组特异性引物,基于SYBR GreenⅠ染料建立了半巢式PCR实时荧光法对弓形虫进行检测。第一轮扩增利用外引物扩增弓形虫529 bp重复序列,第二轮则结合SYBR GreenⅠ染料利用内引物对529 bp基因片段进行实时荧光扩增,其最终产物片段长度为260 bp。在优化后的最佳反应条件下,该方法的检测限为18.4 fg/μL,灵敏度较高;与细粒棘球绦虫(Echinococcus granulosus)、猪肉绦虫(Taenia solium)基因组均无交叉反应,特异性较强。因此,本研究成功建立了灵敏度高、特异性强的半巢式PCR实时荧光检测方法,为监测家畜及肉类食品中弓形虫感染情况提供了有效的技术手段。Toxoplasma gondii is a worldwide zoonotic parasite with a high chronic infection rate in pigs(Sus scrofa)and small ruminants.It can cause great harm to individuals after being infected with immunocompromised people through food-borne transmission.In this study,a set of specific primers were designed according to the 529 bp gene sequence of T.gondii(DQ779191.1)published in GenBank,and a seminested PCR real-time fluorescence method was established based on SYBR GreenⅠdye to detect T.gondii.In the first-round of amplification,the 529 bp repetitive sequence of T.gondii was amplified by external primers,and then the 529 bp gene fragment was amplified by real-time fluorescence amplification with the internal primer of the second-round of amplification combined with SYBR GreenⅠdye,and the final product fragment length was 260 bp.Under the optimized reaction conditions,the detection limit of this method was 18.4 fg/μL with high sensitivity.There was no cross reaction with the genomes of Echinococcus granulosus and Taenia solium,and the specificity was sound.Therefore,this study successfully established a new semi-nested PCR real-time fluorescence detection method with high sensitivity and specificity,which provides an effective technical means for monitoring T.gondii infection in livestock and meat products.

关 键 词：半巢式PCR SYBR GreenⅠ染料 弓形虫 实时荧光检测 

分 类 号：S828[农业科学—畜牧学]