猪流行性腹泻病毒荧光定量RT-PCR检测方法的建立  

Establishment of fluorescent quantitative RT-PCR for detection of porcine epidemic diarrhea virus

作　　者：万娟 梁海英[1] 曾智勇[1] 汤德元[1] 王彬[1] 张婧旭 柳佳佳 边孟婷 黄书 WAN Juan;LIANG Hai-ying;ZENG Zhi-yong*;TANG De-yuan;WANG Bin;ZHANG Jing-xu;LIU Jia-jia;BIAN Meng-ting;HUANG Shu(College of Animal Science,Guizhou University,Guiyang 550025)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《中国兽医科学》2022年第11期1367-1372,共6页Chinese Veterinary Science

基　　金：贵州省科技支撑计划项目[黔科合支撑(2021)一般162项目]。

摘　　要：为建立一种快速、特异、灵敏检测猪流行性腹泻病毒(PEDV)的荧光定量RT-PCR方法,参比NCBI中登录的S基因高度保守核苷酸序列,设计1对针对PEDV S基因的特异性引物和探针,建立TaqMan荧光定量RT-PCR方法。结果显示,建立的方法与猪圆环病毒2型、猪圆环病毒3型、猪繁殖与呼吸综合征病毒、猪轮状病毒、猪传染性胃肠炎病毒、猪德尔塔冠状病毒不存在交叉反应,具有较强的特异性;最低检测限度为1.0 copies/μL,比普通RT-PCR方法灵敏100倍;批内、批间重复性试验的变异系数分别在1.0%、2.8%以下,具有较好的重复性;对临床疑似样品进行检测,荧光定量RT-PCR方法与普通RT-PCR方法总符合率为80%,阳性符合率为100%。结果表明,建立的TaqMan荧光定量RT-PCR检测方法特异性强、灵敏性高、重复性好。In order to establish a rapid,specific and sensitive fluorescent quantitative RT-PCR method for the detection of porcine epidemic diarrhea virus(PEDV),a pair of specific primers and probes were designed according to the highly conserved nucleotide sequence of S2 gene in NCBI,and Taq Man fluorescent quantitative RT-PCR method was established.The results showed that there was no cross reaction between the established method and porcine circovirus type 2,porcine circovirus type 3,porcine reproductive and respiratory syndrome virus,porcine rotavirus,porcine transmissible gastroenteritis virus and porcine delta coronavirus.The minimum detection limit is 1.0 copies/μL.It is 100 times more sensitive than ordinary RT-PCR.The coefficient of variation of intra batch and inter batch repeatability experiments were less than 1.0%and 2.8%respectively,showing good repeatability.The total coincidence rate between fluorescent quantitative RT-PCR and ordinary RT-PCR was 80%,and the positive coincidence rate was 100%.The results showed that the established Taq Man fluorescence quantitative RT-PCR method had strong specificity,high sensitivity and good repeatability.

关 键 词：猪流行性腹泻病毒 TAQMAN探针 荧光定量RT-PCR方法 

分 类 号：S852.659.6[农业科学—基础兽医学]