鉴别高、低致病性猪繁殖与呼吸综合征病毒二重实时荧光定量RT-PCR检测方法的建立及应用

据GenBank登录的高、低致病性猪繁殖与呼吸综合征病毒（highly and lowly pathogenic porcine reproductive and respiratory syndrome virus，HP/LP-PRRSV）的非结构蛋白2（Nsp2）基因序列设计特异性引物和TaqMan MGB荧光探针，经优化反应条件，建立鉴别HP/LP-PRRSV二重TaqMan MGB实时荧光定量RT-PCR（Real-time fluorescent quantitative RT-PCR）检测方法；对该二重实时荧光定量RT-PCR方法进行了敏感性、特异性和重复性试验；对疑似PRRSV感染临床样品进行了应用检测，同时与建立的HP/LP-PRRSV二重RT-PCR方法进行了对比试验。结果显示，成功建立了鉴别HP/LP-PRRSV的二重实时荧光定量RT-PCR检测方法，HP/LP-PRRSV标准曲线的循环阈值与模板浓度呈良好的线性关系，相关系数分别为0.998和0.997；该方法灵敏度可达10¹拷贝/μL；特异性高，对HP/LP-PRRSV阳性对照扩增呈阳性反应，而对5个对照病原均呈阴性反应；不同浓度的HP/LP-PRRSV重组质粒分别重复扩增3次，重复结果良好；对25份临床疑似PRRSV感染样品进行了应用检测，阳性检出率为92%，较普通二重RT-PCR方法阳性检出率高。

Establishment and Application of Duplex Real-time Fluorescent Quantitative RT-PCR Assay for Identification of Highly and Lowly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

A highly sensitive and specific duplex Real-time TaqMan MGB-fluorescent quantitative RT-PCR (FQ-PCR) assay had been developed to identify and quantitate the highly and lowly pathogenic porcine reproductive and respiratory syndrome virus (HP/LP-PRRSV) infection using PRRSV nonstructural 2 (Nsp2) gene-derived primers and TaqMan fluorescent probe. The sensitivity, specificity and repetition assays of duplex FQ-PCR were tested, and 25 clinic suspicious PRRSV infected samples were detected by the FQ-PCR in contrast to the routine duplex RT-PCR method. The results indicated FQ-PCR assay as well as the quantitative standard curves for HP/LP-PRRSV with good linearities (0.998 and 0.997,respectively) were successfully established. The developed FQ-PCR assay was able to detect as little as 10¹ copies/μL of recombinant plasmid DNA. The specificity assay exhibited that positive signals could be obtained from the HP/LP-PRRSV positive control, but not from the genomic DNA of the other 5 kinds of pathogenic microorganism as the control. The repetition test indicated that the FQ-PCR was reproducible by repeatedly amplifying 3 times of different concentrations of HP/LP-PRRSV recombinant plasmids. 92% positive results from 25 clinic suspicious PRRSV infected samples were obtained, which showed better sensitivity than the detection results of the same 25 suspected samples by duplex RT-PCR.