不同基因型PRRSV鉴别诊断多重RT-PCR检测方法的建立  

Establishment of Multiple PCR for Identification of Multiple Genotypes of PRRSV

作　　者：贾钦瑞 闫乙鑫 罗志鹏 徐通 陈弟诗[2] 周远成 朱玲[1] JIA Qinrui;YAN Yixin;LUO Zhipeng;XU Tong;CHEN Dishi;ZHOU Yuancheng;ZHU Ling(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Animal Disease Prevention and Control Center,Chengdu 610041,China;Sichuan Huasun Animal Biological Product Co,Ltd.,Chengdu 610299,China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]四川省动物疫病预防控制中心,成都610041 [3]四川华神兽药生物制品有限公司,成都610299

出　　处：《四川农业大学学报》2025年第2期452-457,478,共7页Journal of Sichuan Agricultural University

基　　金：四川省“十四五”川猪重大科技专项(021ZDZX0010-3);四川省科技计划重点研发项目(2023YFN0021);国家生猪技术创新中心(NCTIP-MY23006)。

摘　　要：【目的】针对PRRSV的多基因型特性,旨在建立一种灵敏、特异且稳定的多重RT-PCR检测方法。【方法】根据GeneBank数据库中公布的ORF1、ORF6和Nsp2基因保守序列分别设计合成3对特异性引物,分别以PRRSV欧洲株(PRRSV-1)、高致病性毒株(HP-PRRSV)、美洲经典株(Classic-PRRSV)和NADC30-like株cDNA为模板进行PCR扩增并构建重组质粒,通过对反应体系和反应条件的优化建立不同基因型PRRSV鉴别诊断多重RT-PCR检测方法。对该方法进行特异性、灵敏性、重复性和准确性验证。【结果】优化过后的cDNA模板为7.5μL,引物为0.5μL,退火温度为56℃;该方法与猪传染性胃肠炎病毒(TGEV)、猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV-2)无交叉反应,特异性强。对PRRSV-1、NADC30、HP-PRRSV、Classic-PRRSV株的最低检测下限分别为3.93×10^(2)、4.52×10^(2)、3.70×10^(2)和4.58×10^(2)copies/μL,灵敏性较高。分别以10^(4)copies/μL的混合阳性质粒和4种单一阳性质粒为模板,进行PCR扩增,重复性较好。对来自四川各地的118份临床样本进行检测,总符合率达到98%。【结论】该方法能够精确鉴别PRRSV的不同基因型毒株,具有良好特异性、灵敏性、重复性和准确性,在临床样品中鉴别不同基因型PRRSV毒株的方面具有较高的应用价值。【Objective】This study aim to develop a sensitive,specific and stable multiples RT-PCR method for the multiple genotypes of porcine reproductive and respiratory syndrome virus(PRRSV).【Method】Three groups of specific primers were designed and synthesized based on conserved sequences of the ORF1,ORF6,and Nsp2 genes,which were previously published in the GeneBank database.The cDNA of the European strain(PRRSV-1),the highly pathogenic strain(HP-PRRSV),the American Classic Strain(Classic-PRRSV)and the NADC30-like strain were used as templates for PCR amplification and recombinant plasmid construction.A multiplex RT-PCR assay for the differential diagnosis of various PRRSV genotypes was established via optimizing the reaction system and conditions.The assay was subsequently validated for specificity,sensitivity,and accuracy.【Result】The optimized cDNA template volume was determined to be 7.5μL,with a primer volume of 0.5μL and an annealing temperature of 56℃.The assay exhibited no cross-reactivity with Transmissible Gastroenteritis Virus(TGEV),Pseudorabies Virus(PRV),Porcine Parvovirus(PPV)and Porcine Circovirus Type 2(PCV-2),demonstrating high specificity.The detection limits for PRRSV-1,NADC30,HP-PRRSV,and lassic-PRRSV strains were 3.93×10²,4.52×10^(2),3.70×10^(2),and 4.58×10^(2)copies/μL,respectively,indicating high sensitivity.PCR amplification employing a mixed positive plasmid template of 10^(4)copies/μL and four single positive plasmid templates demonstrated good reproducibility.Analysis of 118 clinical samples from Sichuan Province showed a high overall compliance rate of 98%.【Conclusion】The assay can accurately identifies different genotypes of PRRSV strains with high specificity,sensitivity,reproducibility,and accuracy.Consequently,this assay has significant potential for application in the clinical.

关 键 词：PRRSV欧洲株 高致病性毒株 美洲经典株 NADC30-like株 多重RT-PCR 

分 类 号：S858.28[农业科学—临床兽医学]