PRRSV经典毒株、高致病毒株、类NADC30毒株及类NADC34毒株鉴别RT-PCR检测方法的建立及应用  

Establishment and application of RT-PCR for identification of PRRSV classic strain,highly pathogenic strain,NADC30-like strain and NADC34-like strain

作　　者：蹇艳银 张佳怡 张耕馨 倪民婷 卢淳 师源 王金涛 JIAN Yanyin;ZHANG Jiayi;ZHANG Gengxin;NI Minting;LU Chun;SHI Yuan;WANG Jintao(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国兽医科学》2024年第4期465-471,共7页Chinese Veterinary Science

基　　金：黑龙江省重点研发项目(GA21B004);黑龙江八一农垦大学引进人才科研启动计划项目(XYB201913)。

摘　　要：为提高猪繁殖与呼吸综合征病毒(PRRSV)不同流行毒株的快速鉴别诊断能力,本研究对近几年国内外流行的PRRSV不同毒株的Nsp2核苷酸序列进行比对,设计了1对可用于鉴别4类不同PRRSV毒株的简并引物,建立了鉴别PRRSV经典毒株(C-PRRSV)、高致病毒株(HP-PRRSV)、类NADC30毒株(NADC30-like)及类NADC34毒株(NADC34-like)的RT-PCR检测方法。结果显示,C-PRRSV、HP-PRRSV、NADC30-like、NADC34-like毒株Nsp2基因的扩增片段大小分别为1072 bp、982 bp、679 bp、772 bp,检测限分别为4.70×10 copies/μL、3.34×10^(2)copies/μL、8.57×10 copies/μL、5.71×10^(2)copies/μL,对其他常见猪病病原均未扩增出特异性条带,3次重复试验均能够扩增出均匀一致的目的片段;83份临床组织样品共检出阳性27份,其中NADC30-like毒株7份、NADC34-like毒株20份,与GP5基因检测结果的符合率为97.6%。上述结果表明,本研究建立的PRRSV鉴别RT-PCR检测方法特异性强、灵敏度高、重复性好,可用于C-PRRSV、HP-PRRSV、NADC30-like、NADC34-like毒株的快速鉴别和流行病学调查。In order to improve the ability of the rapid differential diagnosis of different epidem-ic strains of porcine reproductive and respiratory syndrome virus(PRRSV),the Nsp2 nucleotide sequences of the prevalent PRRSV strains in recent years were compared,and the RT-PCR method was established to identify the classical strain(C-PRRSV),the highly pathogenic strain(HP-PRRSV),the NADC30-like strain(NADC30-like)and the NADC34-like strain(NADC34-like).The results showed that the amplified fragment sizes of Nsp2 genes of C-PRRSV,HP-PRRSV,NADC30-1ike and NADC34-like strains were 1072 bp,982 bp,679 bp and 772 bp respectively,and the detection limits were 4.70×10 copies/μL,3.34×10^(2)copies/μL,8.57×10 copies/μL,5.71×10^(2)copies/μL respectively.No specific bands were amplified in other swine diseases,and the target fragment could be amplified uniformly and consistently in 3 replicates.27 samples were positive in 83 clinical tissue samples,including 7 NADC30-like positive samples and 20 NADC34-like positive samples,and the coincidence rate with GP5 gene results was 97.6%.The above re-sults indicated that the RT-PCR method for PRRSV identification established in this study possess good specificity,sensitivity and repeatability,and could be used for rapid identification and epidemiological investigation of C-PRRSV,HP-PRRSV,NADC30-like and NADC34-like strains.

关 键 词：猪繁殖与呼吸综合征病毒 经典毒株 高致病毒株 类NADC30毒株 类NADC34毒株 鉴别 

分 类 号：S852.659.6[农业科学—基础兽医学]