| ObjectiveThis research aimed to establish nucleic acid testing techniques for detecting Nipah virus (NiV), Hendra virus (HeV) and West nile virus (WNV), so that these neurotropic viruses RNA in field specimens or laboratory materials can be characterized rapidly and specifically and quantitated, which would provide an easy way for epidemiological surveillance. For further study, the method was used for identification of NiV and HeV in peripheral blood collected from domestic pigs in Chongqing, Sichuan, Guizhou, and Guangxi provinces, in order for obtaining the epidemiologic data of NiV and HeV in China's western region and assessing of the biosafety status in these areas.Methods1 Specific primers and TaqMan probes were designed in the conserved region of virus genome, NiV N gene, HeV N gene and WNV E gene respectively. Conserved region gene contained in plasmids offered by Australian Animal Health Laboratory and The Academy of Military Medical Sciences was inserted into pBS-T vector and transcribed in vitro to produce RNA, which would be used as standards in PCR quantification. One-step real-time RT-PCR methods for detecting these three viruses were established independently and the sensitivity and specificity were assessed.2 Peripheral blood samples of 320 domestic pigs were collected from the China's western region (Chongqing, Sichuan, Guizhou and Guangxi) since June, 2007. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of NiV and HeV with one-step real-time RT-PCR methods established before. Sequence identification and analysis were performed for positive PCR products. Virus isolation and culture were proceeded for positive samples, and epidemiologic reports were submitted.Results1 We performed Blast search to check specificity of designed primers and probes of NiV and HeV on the website of American National Institute of Health(NIH). Primer sets and probes could match perfectly with all NiV strains published before, and there was no cross-hybridization with HeV or other unwanted sequences. The minimum detection limits (MDL) of one-step real-time RT-PCR for NiV and HeV were 1.7×10~2 copies/μl and 2.6×10 copies/μl respectively, the quantitative range of standard curve was 1.7×10~2～1.7×10~6 copies/μl(R~2 0.9998) for NiV, and 2.6×10～2.6×10~7 copies/μl(R~2 0.9896) for HeV. Nonspecific PCR amplification with other virus was not discovered.2 We also performed Blast search for specificity assessment of the primers and probes for WNV on the website of American NIH. The designed primer set and probe could perfectly match with above 70 WNV strains. There was no cross-hybridization with other unwanted sequences The MDL of one-step real-time RT-PCR methods for WNV was 3.6×10~2 copies/μl. The quantitative ranges of standard curve were 3.6×10~7～3.6×10~2 copies/ul and R~2 was 0.97534. Nonspecific PCR amplification with other virus was not discovered.3 Nucleic acid detections searching for Ni V and HeV were successfully performed in domestic pig blood samples collected from China's western regions (Chongqing, Sichuan, Guizhou, Guangxi), using One-step real time RT-PCR. We found no "takeoff points" in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.Conclusions1 We established one-step real-time RT-PCR methods for detecting NiV, HeV and WNV successfully. The methods are sensitive and reliable and allow rapid detection and quantitation of these neurotropic viruses in field and experimental materials used for epidemiological surveillance and specific diagnosis. 2 Until now, we found no infections of either NiV or HeV in domestic pig blood samples collected from China's western regions(Chongqing, Sichuan, Guizhou, Guangxi), which suggest a lower possibility of outbreaks of Nipah disease and Hendra disease in these regions in the near future.
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