基于盖他病毒nsP3基因的TaqMan荧光定量RT-PCR检测方法的建立及初步应用  

Establishment and preliminary application of a fluorescence quantitative TaqMan RT-PCR detection method based on the nsP3 gene of Getah virus

作　　者：魏新宇 王铭 杜炳辰 史智宾 杨德成[2] 王靖飞[1,2] WEI Xin-yu;WANG Ming;DU Bing-chen;SHI Zhi-bin;YANG De-cheng;WANG Jing-fei(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第9期910-915,929,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然这基金(32272980);黑龙江省自然科学基金(YQ2021C038);中国博士后基金面上项目(2019M650925)。

摘　　要：为建立一种灵敏、特异且高效的检测盖他病毒(GETV)的TaqMan荧光定量RT-PCR方法,本研究根据GETV的nsP3基因设计特异性引物及探针,以GETV (SC483株) cDNA作为模板,扩增目的基因并克隆至p ET-29a载体中,构建重组质粒标准品p ET29a-nsP3并经PCR和测序鉴定。基于该质粒标准品,采用方阵法优化引物、探针浓度以及退火温度,初步建立了一种基于GETV nsP3基因的TaqMan荧光定量RT-PCR检测方法,并绘制标准曲线。以GETV (SC483株、SC266株)、辛德毕斯病毒(SINV)、猪流感病毒(H3N2、SIV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪繁殖和呼吸障碍综合征病毒(PRRSV)等病毒的基因组RNA反转录为cDNA作为模板,利用本实验建立的TaqMan荧光定量RT-PCR方法检测,结果显示,仅GETV为阳性,SINV、SIV、TGEV、PEDV、PRRSV均为阴性,表明该方法特异性较强;分别以10倍倍比稀释(3.07×10^(-1)拷贝/μL~3.07×10^(8)拷贝/μL)的重组质粒标准品p ET29a-nsP3作为模板,利用本研究建立的方法和普通RT-PCR方法分别检测,结果显示,本研究建立的方法对重组质粒标准品的检测限为30.7拷贝/μL,敏感性是普通RT-PCR的100倍,敏感性较高;分别以同一时间及不同时间提取的不同浓度的质粒标准品作为模板,利用本研究建立的荧光定量RT-PCR进行组内与组间的重复性试验检测,结果显示,该方法的组内组间变异系数均小于2%,重复性较好。利用本实验建立的TaqMan荧光定量RT-PCR方法与普通RT-PCR方法分别对来源于GETV感染小鼠的84份组织样品中的病毒核酸进行检测,结果显示,该TaqMan荧光定量RT-PCR方法对GETV检出率为95.24%(80/84),而普通RT-PCR对GETV的检出率为67.86%(57/84)。二者的阳性符合率达100%。本研究首次基于GETV nsP3基因建立了TaqMan荧光定量RT-PCR检测方法,该方法特异性强、敏感性高、重复性好,为GETV的检测提供了有效工具。To develop a sensitive,specific,and efficient real-time PCR method for the detection of Getah virus(GETV),specific probes and primers were designed targeting a conserved region in the gene of nsP3 of GETV.The nsP3 gene was amplified with the cDNA of GETV SC483 strain as the template and then cloned into the pET29a vector to prepare construct the recombinant plasmid,and identification by PCR and sequencing standard.After a series of optimization of the reaction conditions such as the probe concentration and annealing temperature,a fluorescence quantitative RT-PCR for detecting the nsP3 gene of GETV was established and then used to draw the standard curve.This method showed no cross-reactivity with other swine RNA viruses,including sindbis virus(SINV),swine influenza virus(SIV)(H3N2),transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV),and porcine reproductive and respiratory syndrome virus(PRRSV),which only react with GETV showing good specificity.The serially diluted recombinant plasmicl standard(3.07×10^(-1) copies/μL-3.07×10^(8) copies/μL)of GETV was used as a template,and detected by the method established in this study and the ordinary RT-PCR method.The results showed that the minimum detection concentration by this method was 30.7 copies/μL,and the sensitivity was about 100 times higher than that of the common RT-PCR,which was highly sensitive.The coefficients of variation(CV)of the intra-and inter-batch repetitive tests were less than 2%,which showed this method had good repeatability.The method and a common RT-PCR were simultaneously employed to detect GETV in the tissues samples collected from the experimentally infected mice.The results showed that the positive rate determined by the this method was 95.24%(80/84),which was higher than that obtained with the common RT-PCR(67.86%)(57/84).The positive conformity rate was 100%.Together,we established a TaqMan fluorescence probe RT-PCR assay based on the nsP3 gene,which has good specificity,high sensitivity,and good repeatabili

关 键 词：盖他病毒 荧光定量RT-PCR nsP3 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]