猪繁殖与呼吸综合征病毒SYBR GreenⅠ荧光定量RT-PCR方法的建立

Development of SYBR Green Ⅰ Fluorescence Quantitative RT-PCR Assay for Detection of Porcine Reproductive and Respiratory Syndrome Virus

根据文献合成1对针对PRRSV毒株ORF6基因高度保守序列的引物，建立用于检测PRRSV的SYBR GreenⅠ荧光定量RT～PCR诊断方法。提取PRRSV—LC病毒RNA，经所建立的荧光定量RT—PCR方法扩增，可获得特异的扩增曲线，而日本乙型脑炎病毒（JEV）、猪伪狂犬病毒（PRV）、猪细小病毒（PPV）和猪圆环病毒（PCV）进行同条件检测，结果为阴性；方法可检测到1 TCID50 PRRSV，比琼脂糖凝胶电泳敏感度高10～100倍。结果表明，所建立的PRRSV荧光定量RT—PCR诊断方法特异性好、敏感性高，可进一步应用于猪繁殖与呼吸综合征的临床诊断和科学研究。

According to the reference, a pair of primers based on highly conservative ORF6 gene sequence of PRRSV were synthesized. The quantitative SYBR Green Ⅰ RT - PCR method for detecting PRRSV was established. PRRSV RNA was extracted and then amplified using the Fluorescence Quantitative （FQ） RT-PCR. Specific amplification curve was obtained as expected. Under the same conditions, however, the results of the detection of JEV,PRV,PPV and PCV by the FQ RT-PCR were all negative. The FQ RT- PCR could detect the least 1 TCID50 virus, and the sensitivity was 10 - 100 times higher than that of Agarose Gel Electrophoresis. The results indicate that the FQ RT - PCR method in this research has high sensitivity and specificity, and could be further applied to PRRSV clinical diagnosis and research.