不同引物RT-PCR方法检测猪繁殖与呼吸综合征病毒的比较研究

用3对分别针对猪繁殖与呼吸综合征病毒(PRRSV)的ORF7、ORF5和ORF5的PCR引物N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2进行RT-PCR,检测PRRSV,从其敏感性、特异性和临床样品检出率等方面进行比较,在此基础上进一步建立一步法RT-PCR检测方法。结果显示:3对引物对PRRSV均有很高的特异性;应用N1/N2引物病毒最低检测量为7.9 TC ID50,而AdGP5.1/AdGP5.2引物和RFLP5.1/RFLP5.2引物PCR最低检测量为79 TC ID50;运用N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物分别进行RT-PCR扩增检测临床样品,PRRSV检出率分别为28/48、27/48和25/48,且用AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物检测的阳性样品,用N1/N2引物检测也都呈阳性。运用N1/N2引物,通过一步法RT-PCR成功地从PRRSV S1株中扩增出374 bp的目的基因片段。结果表明,用N1/N2引物扩增PRRSV目的基因,其敏感性和临床样品检出率更高,更适合临床样品PRRSV的检测。

Comparative study on the detection of porcine reproductive and respiratory syndrome virus with different primers in RT-PCR

According to the genome sequence of Porcine reproductive and respiratory syndrome virus(PRRSV),three pairs of primers(N_1/N_2、AdGP5.1/AdGP5.2、 RFLP5.1/RFLP5.2) were designed and the RT-PCR methods were developed to amplify the corresponding gene fragments.Their sensitivity、specificity and positive rate of the samples were detected and compared.One step RT-PCR was also constructed for the detection of PRRSV.The results showed that: ①Three pairs of primers could be used to specifically amplify the gene fragments of PRRSV;②The RT-PCR using primers N_1/N_2 was able to detect at least 7.9 TCID_(50) PRRSV,while the RT-PCR using primers AdGP5.1/AdGP5.2 or primers RFLP5.1/RFLP5.2 could detect only 79 TCID_(50) PRRSV;③Among forty-eight clinical samples,twenty-eight samples were identified as positive to PRRSV by RT-PCR using primers N_1/N_2,and twenty-seven were positive by using primers AdGP5.1/AdGP5.2 and twenty-five positive by using primers RFLP5.1/RFLP5.2.Furthermore,the expected 374bp fragment was also amplified by one step RT-PCR method with the primer N_1/N_2.It indicated that the RT-PCR with the primers N_1/N_2 had higher sensitivity for PRRSV than that with the other two pairs of primers,and it could be used to detect PRRSV in clinical samples.