马病毒性动脉炎病毒数字PCR方法的建立  

Establishment of a Digitial RT-PCR Detection Method for Equine Arteritis Virus

作　　者：梁歆歆 王科珂 蒋刚强 王艳 徐军 陈凯云 肖媛媛 白梅花 LIANG Xin-xin;WANG Ke-ke;JIANG Gang-qiang;WANG Yan;XU Jun;CHEN Kai-yun;XIAO Yuan-yuan;BAI Mei-hua(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832000,China;Urumqi Customs Technology Center,Urumqi,Xinjiang,830063,China;Shanghai Customs Animal,Plant and Food Inspection and Quarantine Technology Center,Shanghai,200120,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]乌鲁木齐海关技术中心,新疆乌鲁木齐830063 [3]上海海关动植物与食品检验检疫技术中心,上海200120

出　　处：《动物医学进展》2024年第12期8-13,共6页Progress In Veterinary Medicine

基　　金：海关总署科研项目(2022HK126);省部级青年科学基金项目(2022D01B08)。

摘　　要：在马病毒性动脉炎病毒(EAV)基因组序列的高度保守区域ORF7上设计引物和探针,对反应体系中的条件进行优化,验证其灵敏性、特异性、重复性,建立了马病毒性动脉炎病毒数字PCR方法(digital PCR,dPCR)。结果显示,建立的EAV数字PCR方法在退火温度为58℃、引物终浓度为0.9μmol/L和探针终浓度为0.6μmol/L的反应条件下效果最优;该方法与马泰勒虫、马疱疹病毒1型、马疱疹病毒4型、马流感病毒均无交叉反应;最低检测限为0.16 copies/μL;组内及组间变异系数均在2.5%之内;通过对234份临床样本进行dPCR检测,阳性检出率为15.81%,高于实时荧光RT-PCR方法检出率(14.1%)。结果表明,建立的dPCR能快速、精准定量EAV在样本中的病毒载量,可用于马病毒性动脉炎病毒的早期检测。This article designed primers and probes on the highly conserved region ORF7 of the genome sequence of Equine arteritis virus(EAV),optimized the conditions in the reaction system,verifies its sensitivity,specificity,and repeatability,and established a digital PCR(dPCR)method for equine arteritis virus.The results showed that the established EAV digital PCR method had the best reaction conditions at an annealing temperature of 58℃,a primer final concentration of 0.9μmol/L and probe final concentration of 0.6μmol/L;This method has no cross reactivity with Theileria equi,EHV-1,EHV-4,and EIV;The minimum detection limit is 0.16 copies/μL;The coefficient of variation of intra and intergroups is within 2.5%;by conducting digital PCR detection on 234 clinical samples,the positive detection rate was 15.81%,which is superior to the real-time fluorescence RT-PCR method with a detection rate of 14.1%.This method can quickly and accurately quantify the load of EAV in the samples,and can be used for early detection of Equine arteritis virus.

关 键 词：马病毒性动脉炎 ORF7 数字PCR 

分 类 号：S852.652[农业科学—基础兽医学]