猪瘟病毒实时荧光定量RT-PCR

摘要

根据GenBank公布的猪瘟病毒基因组5'非编码区基因序列进行同源性比较分析,选择保守序列区作为扩增区域,设计1对特异性扩增引物,通过优化反应条件,建立了一个用于猪瘟病毒快速定量检测的SY13R GreenⅠ荧光定量RT-PC R方法.试验结果表明该方法重复性好,反应批内循环阈值差异不显著.与猪繁殖与呼吸综合征病毒、伪狂犬病病毒和猪圆环病毒2型等猪源病毒无交叉反应,具有高度的特异性,而且灵敏度高,最小检出量为2×10(2)病毒基因组拷贝数.利用此方法对15例临床样本进行检测,其结果与兔体交又免疫试验一致,表明此方法可作为猪瘟实验室快速诊断和疫情监测的一种快速、准确、简便的检测工具.%To establish a real-time fluorogenetic quantitative RT-PCR assay for detection of classical swine fever virus (CSFV), the 5' non-translated region sequences of CSFV in GenBank were aligned and a pair of specific primers was designed from the conserved sequence within 5' non-translated region.The reactive conditions were optimized to improve the sensitivity and specificity of the assay.The results of reproducibility of this assay were reliable and the intra assay variations were not significant.The specificity test proved that this assay had a high specificity which could not detected PRRSV,PRV and PCV2.The assay also proven to be specific,and the detection limit was up to 2 × 102 copies.The accuracy of real-time fluorogenetic quantitative RT-PCR was evaluated by testing 15 clinical samples.The results of real-time fluorogenetic quantitative RT-PCR were consistent with results of rabbit-cross reaction test,which suggested that real-time quantitative RT-PCR assay may be used as a powerful tool for repaid detection of CSFV.