Rapid and simultaneous detection of Japanese encephalitis virus by real-time nucleic acid sequence-based amplification.

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.

Development of sandwich Enzyme-Linked Immunosorbent Assay for the detection of porcine epidemic diarrhea virus in fecal samples.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in new-born piglets with subsequent economic losses to swine industry. In the current study, gene encoding of 381aa-792aa spike protein (S1) with the main epitope relative to virus neutralization of PEDV was amplified by RT-PCR and inserted into vector pET-30A(+). The plasmid was transferred into Escherichia coli BL21 (DE3). Meanwhile, recombinant protein expression was induced by isopropy1-ß-galactopyranoside (IPTG). After denaturation and renaturation of inclusion bodies, the S1 protein was obtained by using purified recombinant S1 protein in immunized female BALB/c mice. Monoclonal antibodies (MAb) against S1 protein, named 4C7 by hybridoma technique were gained successfully. The result showed that MAb can specifically respond to S1 protein and PEDV via ELISA, Western bolt and immunofluorescence assay methods. A sandwich ELISA (S-ELISA) was established by using the captured monoclonal antibodies 4C7. The sensitivity and specificity were compared between S-ELISA and RT-PCR, which showed similar sensitivity and specificity. This work indicated that S-ELISA would be a significant tool alongside a specific diagnostic reagent for PEDV in future.