基于PRRSV ORF5基因TaqMan qPCR检测方法的建立及遗传变异分析  

Establishment of TaqMan qPCR detection assay and analysis of genetic variation based on porcine reproductive and respiratory syndrome virus ORF5 gene

作　　者：张帅[1] 赵云环 刘莹[1] 翟刚 郭禹 刘涛[1] 左玉柱[1,2] 范京惠 ZHANG Shuai;ZHAO Yunhuan;LIU Ying;ZHAI Gang;GUO Yu;LIU Tao;ZUO Yuzhu;FAN Jinghui(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding,Hebei 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001

出　　处：《中国兽医学报》2022年第6期1122-1130,共9页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(19226622D);河北省农业产业技术体系生猪创新团队资助项目(HBCT2018110207)。

摘　　要：为了快速、高效、准确地检出猪繁殖与呼吸综合征病毒(PRRSV),并进一步了解河北部分地区的流行毒株遗传变异情况,本研究根据PRRSV经典、高致病性和NADC30代表毒株ORF5基因保守序列设计特异性引物和探针,建立了一种通用型TaqMan qPCR检测方法以实现对猪场的全面筛查,并对筛查出的阳性样品ORF5基因扩增测序后进行遗传进化分析。结果显示:该方法与PRV、PCV2、PEDV、CSFV、TGEV、RV均无交叉反应,特异性强;对重组质粒pMD19T-PRRSV标准品的最低检测下限为2.19×10^(1)拷贝/μL,敏感性高;组内和组间变异系数均小于1%,重复性好;在临床样品检测中较普通PCR有更高的检出率;扩增出的5株PRRSV流行毒株均为美洲型毒株,5株PRRSV流行毒株之间核苷酸序列同源性为82.9%~99.8%,氨基酸序列同源性为82.1%~99.0%;氨基酸序列与国内外已报道的代表株相比在GP5抗原表位及高变区分别存在着不同的差异。结果表明:河北部分地区当前流行毒株复杂多样,优势毒株正从高致病性毒株逐步向NADC30类毒株过渡,提示持续监测PRRSV流行毒株变异动态的必要性,为PRRSV的防控、临床诊断及掌握遗传变异规律提供依据。In order to detect the porcine reproductive and respiratory syndrome virus(PRRSV)quickly,efficiently and early and to further understand the genetic variation of the epidemic strains in some areas of Hebei,a universal TaqMan qPCR detection method was established by designing specific primers and probes based on the conservative sequence of the ORF5 gene of the PRRSV classic,highly pathogenic and NADC30 representative strain to achieve a comprehensive screening of pig farms,then the ORF5 whole gene of the positive sample was amplified,sequenced and analyzed for genetic evolution.The results show that:the method has no cross reactivity with PRV,PCV2,PEDV,CSFV,TGEV,RV,and has strong specificity;The minimum detection limit for the recombinant plasmid pMD19 T-PRRSV standard is 2.19 × 10^(1) copies/μl,which has high sensitivity;The coefficient of variation within and between groups is less than 1 %,indicating that the method has good repeatability;It has a higher detection rate than ordinary PCR in the detection of clinical samples;The 5 amplified PRRSV epidemic strains are all American type strains,with nucleotide sequence homology ranging from 82.9% to 99.8%,and amino acid sequence homology ranging from 82.1 % to 99.0%;Compared with the representative strains reported at home and abroad,the amino acid sequence has different differences in the epitope and hypervariable region of GP5.The results show that the current epidemic strains in some areas of Hebei are complex and diverse,and they are gradually transitioning from highly pathogenic strains to NADC30 strains,suggesting that we must continue to monitor the mutation dynamics of the epidemic strains of PRRSV,which is the prevention,control,and clinical practice of PRRSV provide basis for diagnosis and mastering the law of genetic variation.

关 键 词：猪繁殖与呼吸综合征病毒 ORF5基因 TaqMan qPCR 遗传变异分析 

分 类 号：S855.3[农业科学—临床兽医学]