d.

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.

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