高致病性与低致病性猪繁殖与呼吸综合征病毒二重RT-PCR检测方法的建立及应用

为建立高致病性与低致病性猪繁殖与呼吸综合征病毒(HP/LP-PRRSV)快速鉴别诊断方法,本研究根据GenBank已登录的HP-PRRSV与LP-PRRSV的Nsp2基因序列,分别设计了特异性的上游引物(HP-Upper-p1/LP-Upper-p2) 和共用下游引物(Co-Lower-p3)。以HP-PRRSV和LP-PRRSV混合物总RNA为反转录模板,建立了HP-PRRSV和LP-PRRSV的二重RT-PCR检测方法;并进行了特异性、敏感性和重复性试验;利用所建立的检测方法对临床疑似样品进行了检测。结果显示,本试验成功建立了HP-PRRSV和LP-PRRSV二重RT-PCR检测方法,该方法灵敏度高,最低检出限均为100拷贝/μL;重复性好,特异性强,可特异性地扩增HP-PRRSV细胞培养物和LP-PRRSV疫苗毒,但对Marc-145细胞和其他8种病原对照扩增不出任何条带;自25份临床疑似PRRSV感染病料中共检测出了21份PRRSV阳性样品,其中HP-PRRSV阳性样品共计20份,LP-PRRSV阳性样品4份。结果表明,本研究成功建立了HP-PRRSV和LP-PRRSV二重RT-PCR检测方法,可适用于高致病性与低致病性猪繁殖与呼吸综合征的快速鉴别诊断。

Establishment and Application of Duplex RT-PCR Assay for Detection of Highly and Lowly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

A duplex RT-PCR for detection highly and lowly pathogenic porcine reproductive and respiratory syndrome virus(HP/LP-PRRSV) was developed using the primers designed basing on the Nsp2 gene coding the non-structure protein 2 of HP-PRRSV and LP-PRRSV, respectively. The total RNA of standard HP-PRRSV and LP-PRRSV strains was used as the positive control to establish the duplex RT-PCR assay. The sensitivity, specificity and repetition assay of duplex RT-PCR were tested, and samples taken from clinic suspicious PRRSV infected pigs had been testified by the established duplex RT-PCR assay. The results indicated that the duplex RT-PCR assay was successfully established. The specificity and sensitivity of the duplex RT-PCR assay revealed that the threshold of duplex RT-PCR was 100 copies/μL of HP-PRRSV or LP-PRRSV, and no products were amplified from the nucleic acid of Marc-145 cell or the other 8 kinds of pathogenic viral or bacterial microorganism acting as the negative control. The repetition test indicated that the duplex PCR was repeatible. 21 were PRRSV positive detected from 25 clinic suspicious PRRSV infected pigs, while 20 and 4 were HP-PRRSV and LP-PRRSV positive, respectively.The study suggested that the established duplex RT-PCR method was highly specific and sensitive,and suitable for clinic rapid identified diagnosing of HP-PRRSV and LP-PRRSV.