基于ORF7基因的猪繁殖与呼吸综合征病毒RT-RAA检测方法的建立

Establishment of RT-RAA Method for Detection of PRRSV Based on ORF7 Gene

根据GenBank公布的ORF7基因保守序列设计了特异性引物,建立了PRRSVORF7基因反转录-重组酶介导等温扩增(RT-RAA)荧光法快速检测方法。结果表明,RT-RAA荧光法在42℃、20min内即可检测到PRRSV特异性扩增曲线,最低检测浓度为9.9×10^(6)copies/μL;与PRVBartha-K61、PCV、CSFV、PRRSVGD、PRRSVXH-GD、PRRSVGM2、PRRSVCH-1a病毒均无交叉反应。分别对157份样品进行荧光RT-RAA法与PCR方法的检测进行比对,结果显示,荧光RT-RAA法敏感性为94.0%,特异性为100%。建立的PRRSV荧光RT-RAA法操作简便,特异性强,灵敏度高。

Specific primers were designed according to the conserved sequence of ORF7 gene published in GenBank,and a rapid detection method for reverse transcription recombinase mediated isothermal amplification(RT-RAA)fluorescence of PRRSV ORF7 gene was established.The specific amplification curve of PRRSV could be detected by RT-RAA fluorescence method within 20 min at 42℃.The lowest detection limit was 9.9×10^(6)copies/μL;There was no cross-reactivity with PRV Bartha-K61,PCV,CSFV,PRRSV GD,PRRSV XH-GD,PRRSV GM2,PRRSV CH-la strains.The fluorescence RT-RAA method and PCR method were compared with 157 samples,and the results showed that the fluorescence RT-RAA method had a sensitivity of 94.0%and a specificity of 100%.The PRRSV fluorescence RT-RAA method established in this study is easy to operate,has strong specificity and high sensitivity,and can quickly and effectively detect clinical samples.