A型塞内卡病毒、口蹄疫病毒和猪捷申病毒3重TaqMan荧光定量PCR检测方法的建立  

Development and application of a triplex TaqMan fluorescent quantitative PCR assay for simultaneous detection of Senecavirus A,foot-and-mouth disease virus and porcine teschovirus

作　　者：甘诗琪 魏千禾 倪语晨 倪健波 赵秀玲 董婉玉 周莹珊 王晓杜 GAN Shiqi;WEI Qianhe;NI Yuchen;NI Jianbo;ZHAO Xiuling;DONG Wanyu;ZHOU Yingshan;WANG Xiaodu(China-Australia Joint Laboratory for Animal Health Big Data Analytics/Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,College of Animal Science and Technology&College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China;Technical Center of Ningbo Customs,Ningbo Key Laboratory of Port Biological and Food Safety Testing,Ningbo,Zhejiang 315000,China)

机构地区：[1]浙江农林大学动物科技学院动物医学院浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程实验室/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]宁波市口岸生物与食品安全检测重点实验室宁波海关技术中心,浙江宁波315100

出　　处：《中国兽医学报》2025年第1期22-29,共8页Chinese Journal of Veterinary Science

基　　金：国家级大学生创新创业训练计划资助项目(202310341032);宁波市公益性科技计划资助项目(2022S009)。

摘　　要：针对A型塞内卡病毒(Senecavirus A,SVA)、口蹄疫病毒(foot-and-mouth disease virus,FMDV)和猪捷申病毒(porcine teschovirus,PTV)核酸的保守区域,设计了用于构建TaqMan荧光定量PCR体系的引物和探针。以含有这3种病毒核酸保守序列的重组阳性质粒标准品为模板,建立了3重TaqMan荧光定量PCR方法,并对该体系进行了优化,确定了该方法的特异性、重复性、灵敏性及标准曲线,同时建立了混合感染模型。此外,利用所构建的3重TaqMan荧光定量PCR方法对临床样品进行了检测。结果显示,该方法能够特异性检测SVA、FMDV和PTV 3种病毒,与其他病原体无交叉反应;检测SVA、FMDV和PTV的最低浓度均可达到1×10~1拷贝/μL;组间和组内变异系数均小于5%;在126份样品中未检出这3种病毒。上述结果表明,该方法具有较强的特异性、高灵敏性和良好的稳定性,可用于临床检测。Primers and probes were designed based on the conserved regions of Senecavirus A(SVA),foot-and-mouth disease virus(FMDV),and porcine teschovirus(PTV)and used to develop a TaqMan fluorescent quantitative PCR method for detecting the above-mentioned three viruses.The triplex fluorescent quantitative PCR system was developed using recombinant positive plasmids containing conserved sequences of the three viruses as templates.After optimizing the conditions,the specificity,sensitivity,repeatability,standard curve,and mixed infection model were evaluated,and the constructed triplex fluorescenl quantitative PCR was used for clinical detection.The results showed that this method could specifically detect SVA,FMDV and PTV without cross-reactivity with other pathogens with the minimal detection concentrations for SVA,FMDV,and PTV as low as 1×101 copies/μL,respectively.The coefficients of variation within and between groups were less than 5%,Furthermore,none of the three viruses were detected in 126 samples.The above results indicate that this method is highly specific,sensitive,and stable,making it suitable for clinical detection.

关 键 词：A型塞内卡病毒 口蹄疫病毒 猪捷申病毒 荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]