猪繁殖与呼吸综合征病毒SYBR Green I荧光定量RT-PCR方法的建立

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 Green I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 TCID_(50) 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.

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

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 TCID_(50) 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 specifici-ty,and could be further applied to PRRSV clinical diagnosis and research.