摘要
为建立一种快速同时检测猪繁殖与呼吸综合征病毒(PRRSV)和猪鼻支原体(Mhr)的方法,根据GenBank中已登录的PRRSV ORF 7基因序列和Mhr P 37基因序列,设计2对特异性引物,通过对扩增条件进行优化,建立了能够同时检测PRRSV和Mhr的双重PCR方法。该方法可以同时扩增出PRRSV的234 bp和Mhr的737 bp特异性片段,该方法特异性好,不与其他常见猪病病原体发生交叉反应,对PRRSV和Mhr重组质粒标准品的最低检出量分别为420拷贝/μL和180拷贝/μL。用85份临床病料对本试验双重PCR技术和单项PCR技术进行对比验证,结果显示,两者的总符合率为100%。表明所建立的双重PCR特异性强,重复性好,敏感性高,为PRRSV和Mhr的快速检测提供了有效的技术支持。
To develop a rapid duplex PCR assay for simultaneous detection of porcine reproductive and respiratory syndrome(PRRSV)and Mycoplasma hyorhinis(Mhr),two pairs of specific primers were designed according to the ORF 7 gene of PRRSV and P 37 gene of Mhr.A duplex PCR was successfully established by optimizing the duplex PCR amplification conditions.The specific PCR products of 234 bp for PRRSV and 737 bp for Mhr were simultaneously amplified from the nucleic acid of PRRSV and Mhr by the duplex PCR,but no PCR products were amplified from genome of porcine parvovirus.The detection limit was 420 copies/μL for PRRSV and 180 copies/μL for Mhr,respectively.85 clinical samples were comparatively detected.The data showed that the duplex PCR method was 100%coincidence with the single PCR.The results indicate that the duplex PCR is characterized by high sensitivity,specificity and stability,which provides effective support for differentiating the clinical samples with PRRSV and Mhr or coinfection.
作者
修晓娜
李天芝
于新友
XIU Xiao-na;LI Tian-zhi;YU Xin-you(Yantai Research Institute,China Agricultural University,Yantai 264670,China;Shandong LVDU Bio-technology Co.Ltd.,Binzhou 256600,China)
出处
《中国兽医杂志》
CAS
北大核心
2020年第5期16-19,24,共5页
Chinese Journal of Veterinary Medicine
基金
中国农业大学烟台研究院校内科研基金项目(YT201401)。
关键词
猪繁殖与呼吸综合征病毒
猪鼻支原体
双重PCR
porcine reproductive and respiratory syndrome
Mycoplasma hyorhinis
duplex PCR
