猪轮状病毒TaqMan实时荧光定量RT-PCR检测方法的建立与应用  

Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR for detection of porcine rotavirus

作　　者：柳佳佳 梁海英[1] 曾智勇[1] 汤德元[1] 王彬[1] 叶泥 边孟婷 黄书 田红利 潘向英 LIU Jiajia;LIANG Haiying;ZENG Zhiyong;TANG Deyuan;WANG Bin;YE Ni;BIAN Mengting;HUANG Shu;TIAN Hongli;PAN Xiangying(College of Animal Science,Guizhou University,Guiyang 550025,China)

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》2024年第2期162-168,共7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：根据猪轮状病毒VP6基因序列保守区设计特异性探针和引物,建立一种特异、灵敏、通用、高效检测Po RV的实时荧光定量RT-PCR检测方法。扩增目的片段为173 bp,以构建的重组质粒p MD19-T-VP6为模板进行反应程序的优化。结果显示,该方法的标准曲线为y=-3.1139x+39.298,R^(2)=0.9937,E=109.5%;用该方法检测猪常发传染病病原时结果均为阴性;对标准品质粒的最低检测限为1.32 copies/μL;批内、批间变异系数分别小于0.600%、1.300%,该方法特异性强、灵敏度高、重复性好。利用该方法检测114份临床腹泻病料,阳性检出率(39/114)优于常规RT-PCR(22/114),39份阳性样品经测序鉴定均为Po RV,扩增VP7基因后成功构建22份阳性质粒,测序结果显示共检出6种G型Po RV,检出率依次为9.09%(G3)、22.72%(G4)、18.18%(G5)、31.82%(G9)、4.54%(G11)、9.09%(G26)。上述结果表明,本试验建立了一种快捷、准确检出Po RV的Taq Man探针通用型实时荧光定量RT-PCR方法,对该病的诊断、病原监测、流行病学调查具有重要意义。Specific probes and primers were designed according to the conserved region of porcine rotavirus VP6 gene sequence to establish a specific,sensitive,universal and efficient real-time fluorescence quantitative RT-PCR assay for PoRV detection.The amplified target fragment was 173 bp,and the reaction procedure was optimized with the constructed recombinant plasmid pMD19-T-VP6 as the template.The results show that the standard curve of this method isy=-3.1139x+39.298,R^(2)=0.9937,andE=109.5%.The results were negative when the method was used to detect the pathogens of common infectious diseases in pigs.The minimum detection limit of standard quality particles was 1.32 copies/μL.The coefficient of variation within and between batches were less than 0.600%and 1.300%,respectively.The method had strong specificity,high sensitivity and good repeatability.114 samples for clinical diarrhea were detected,and the positive detection rate of this method(39/114)was better than that of conventional RT-PCR(22/114).All 39 positive samples were identified as PoRV by sequencing,and 22 positive plasmids were successfully constructed after VP7 gene was amplified.The sequencing results showed that 6 types of G-type PoRV were detected,and the detection rates were 9.09%(G3),22.72%(G4),18.18%(G5),31.82%(G9),4.54%(G11)and 9.09%(G26),respectively.The above results indicated that an universal TaqMan real-time fluorescence quantitative RT-PCR method has been established in this experiment,which is of great significance for the diagnosis,pathogen monitoring and epidemiological investigation of porcine rotavirus.

关 键 词：猪轮状病毒 TAQMAN探针 荧光定量RT-PCR 

分 类 号：S852.659.4[农业科学—基础兽医学]