Japaneses encephalitis (JE) is most common zoonoses caused by Japanese encephalitis virus (JEV) with a high mortality and disability rate. To take timely preventive and control measures, early and rapid detection of JE RNA is necessary. But due to characteristic brief and low viraemia, JE RNA detection remains challenging. In this study, a real-time nucleic acid sequence-based amplification (RT-NASBA) was developed for rapid and simultaneous detection of JEV. Four pairs of primer were designed using a multiple genome alignment of all JEV strains from GenBank. NASBA assay established and optimal reaction conditions were confirmed by using primers and probe on ns1 gene of JEV. The specificity and sensitivity of the assay were compared with RT-PCR by using serial RNA and virus cultivation dilutions. The results showed that JEV RT-NASBA assay was established, and robust signals could be observed in 10 min with high specificity. The limit of dectetion of RT-NASBA was 6 copies per reaction. The assay was thus 100 to 1, 000 times more sensitive than RT-PCR. The cross-reaction was performed with other porcine pathogens, and negative amplification results indicated the high specificity of this method. The novel JEV RT-NASBA assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swine and would be beneficial for public health security.

日本脑炎（JE）是由日本脑炎病毒（JEV）引起的最常见的人畜共患病，其死亡率和致残率很高。为了及时采取预防和控制措施，有必要及早和快速地发现JE RNA。但是由于特征性的短暂和低病毒血症，JE RNA检测仍然具有挑战性。在这项研究中，开发了基于实时核酸序列的扩增（RT-NASBA），用于快速，同时检测JEV。使用来自GenBank的所有JEV株的多基因组比对设计了四对引物。利用引物和JEV的ns1基因探针，确定了NASBA检测方法，确定了最佳反应条件。通过使用系列RNA和病毒培养物稀释液，将测定的特异性和敏感性与RT-PCR进行了比较。结果表明，建立了JEV RT-NASBA检测方法，可以在10分钟内以高特异性观察到稳定的信号。每个反应RT-NASBA的检测限为6个拷贝。因此，该测定的灵敏度是RT-PCR的100到1000倍。与其他猪病原体进行交叉反应，阴性扩增结果表明该方法具有很高的特异性。新型的JEV RT-NASBA检测方法可以用作诊断JEV的有效分子生物学工具，这将有助于监测猪的生殖衰竭疾病，并有利于公共卫生安全。与其他猪病原体进行交叉反应，阴性扩增结果表明该方法具有很高的特异性。新型的JEV RT-NASBA检测方法可以用作诊断JEV的有效分子生物学工具，这将有助于监测猪的生殖衰竭疾病，并有利于公共卫生安全。与其他猪病原体进行交叉反应，阴性扩增结果表明该方法具有很高的特异性。新型的JEV RT-NASBA检测方法可以用作诊断JEV的有效分子生物学工具，这将有助于监测猪的生殖衰竭疾病，并有利于公共卫生安全。