猪蓝耳病病毒RT-LAMP快速可视化检测方法的建立

为建立能快速检测猪蓝耳病病毒（PRRSV）的检测方法,本研究根据基因库中PRRSV非结构蛋白NSP2基因的保守序列,设计了一套特异性环介导等温扩增（RT-LAMP）引物,建立了PRRSV的RT-LAMP可视化检测方法。该方法的敏感性可达10 copies RNA,高于常规PCR方法 10倍;全部反应可以在50 min内完成;可通过肉眼观察钙黄绿素（calcein）颜色变化直接判定结果;对其它常见病原体的检测结果均为阴性,同时应用实时浊度仪对反应体系浊度及浊度的变化速率进行实时测量,结果相一致。结果表明建立的RT-LAMP方法简便、快速、灵敏、特异,可用于PRRSV感染的快速检测。

Development of a Revetse Transctiption Loop-mediated Isothermal Amplification Assay for Visual Detection of Porcine reproductive and Respiratory Syndrome Virus

To establish a rapid method for detection of Porcine reproductive and respiratory syndrome virus（PRRSV）, a loop-mediated isothermal amplification（RT-LAMP） assay was developed with a set of primers designed according to the conserved sequence of the non-structural protein gene 2（Nsp2） of PRRSV in the GenBank. Under the optimized conditions, the amplification reactions could be completed in 50 min in a water bath at 62℃. The results showed that the assay was specific to PRRSV with a detection limit of 10 copies, which was higher than that of the routine PCR, and that no cross-reactions with the other pathogens of pig were detectatble. In addition, the positive result was visible in the presence of calcein. in the same reaction system using real-time turbidity and the rate of change in real-time measurements, the results are consistent. These data suggest that the established RT-LAMP assay is a simple sepsitive and specific method for rapid detection of PRRSV in clinical samples.