微流控芯片环介导等温扩增技术检测3种猪圆环病毒  

Microfluidic Chip Loop-mediated Isothermal Amplification for Detecting Three Porcine Circoviruses

作　　者：石艳萍 邓飞[1] 周丽媛[1] 李丽[1] 邵靓[1] 陈斌[1] 张孟思 邱明双 陈弟诗[1] Shi Yanping;Deng Fei;Zhou Liyuan;Li Li;Shao Jing;Chen Bin;Zhang Mengsi;Qiu Mingshuang;Chen Dishi(Sichuan Center for Animal Disease Prevention and Control,Chengdu 610041,Sichuan,China)

机构地区：[1]四川省动物疫病预防控制中心,四川成都610041

出　　处：《中国动物检疫》2024年第2期58-64,共7页China Animal Health Inspection

基　　金：四川省科技计划重点研发项目(2023YFN0021)。

摘　　要：为建立快速区分3种猪圆环病毒(PCV2、PCV3和PCV4)的现场检测方法,采用微流控芯片环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),收集3种猪圆环病毒临床阳性样本进行核酸提取,与市场上3种猪圆环病毒荧光探针法检测试剂盒进行灵敏度、特异性和重复性同步比对。结果显示:微流控芯片LAMP法在3种猪圆环病毒联检测试中具有非常高的灵敏度,可以在30 min内,实现不低于荧光定量PCR法的敏感性;与非洲猪瘟病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪细小病毒临床阳性样本均无交叉反应,特异性好;测试3种猪圆环病毒重复性Ct值变异系数(CV)均在2%以下,稳定性好。结果表明:3种猪圆环病毒微流控芯片快速联检技术特异性好、灵敏度高、重复性强,检测速度快,环境要求低,可以满足现场检测的要求,适用于养猪场等场所的猪圆环病毒现场快速检测。本方法的建立为猪相关病原体的现场快速核酸检测提供了有力工具。In order to rapidly distinguish three types of porcine circoviruses(PCV2,PCV3 and PCV4)at field level,clinical positive samples infected with the three circoviruses were extracted for nucleic acids for microfluidic chip loop-mediated isothermal amplification,the results of which were synchronously compared with those of three commercial fluorescent probe kits in the aspects of sensitivity,specificity and repeatability.The results indicated that the microfluidic chip loop-mediated isothermal amplification method was highly sensitive in joint-detection of the three viruses,with the sensitivity no lower than fluorescent quantification PCR within 30 minutes,failed to crossly react with clinical positive samples infected with African swine fever virus(ASFV),classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV)and porcine parvovirus(PPV),indicating good specificity;and all the coefficients of variation(CVs)of the repeated Ct values after detection of the three viruses were below 2%,showing good stability.In conclusion,the microfluidic chip method for joint-detection of the three viruses was characterized with the advantages of good specificity,high sensitivity,strong repeatability,rapid detection and low requirements for environment,and appropriate for rapid on-site detection of porcine circoviruses on swine farms and relevant premises.The established method could be used as a strong tool for rapid on-site detection of relevant pig pathogens.

关 键 词：猪圆环病毒 微流控芯片 环介导恒温扩增 快速检测 核酸检测 

分 类 号：S852.23[农业科学—基础兽医学]