【作者】 曾志磊；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2008， 博士

【摘要】 目的:本研究拟建立一步法实时RT-PCR检测Nipah病毒(Nipah virus,NiV)、Hendra病毒(Hendra virus,HeV)和西尼罗病毒(West nile virus,WNV)的方法,用于三种嗜神经病毒的快速、准确、敏感、特异的定量检测,为大规模流行病学监测提供一种简便易行的检测手段,并使用所建立的嗜神经病毒检测方法,对采集自中国重庆、四川、贵州和广西地区的猪外周血样本进行NiV和HeV检测,以获得我国西部地区的NiV和HeV的病毒流行病学资料,评估我国西部地区的生物安全现状。方法:1根据GenBank提供的基因序列,针对三种嗜神经病毒基因的保守区域(NiV N基因、HeV N基因和WNV E基因),设计并合成特异性引物和荧光标记TaqMan探针。从澳大利亚动物健康实验室(AAHL)及中国军事医学科学院(AMMS)惠赠的分别含有目的基因的质粒出发,利用PCR方法扩增获得目的基因,克隆至pBS-T载体进行体外转录,将得到的RNA纯化并使用RiboGreen方法进行定量后作为阳性标准品。分别建立三种嗜神经病毒的一步法实时RT-PCR方法并分析敏感性和特异性。2自2007年6月起,采集自中国西部地区重庆、四川、贵州和广西省市饲养的猪外周血标本320份,使用密度梯度离心法分离外周血淋巴细胞,应用Trizol法提取细胞总RNA。用已建立的NiV和HeV一步法实时RT-PCR检测方法,对RNA样本进行NiV和HeV检测。对阳性样本进行PCR产物序列鉴定、序列分析等研究,在可能的情况下进行病毒分离培养,并提交流行病学调查报告。结果:1对所设计的NiV和HeV引物和探针序列,登录美国国立卫生研究院网站(http://www.ncbi.nlm.nih.gov/BLAST/)进行Blast检索,所设计的引物和探针可以与所有已公布的NiV病毒株(DQ508095,AF376747,AF212302,AY029768,AY029767,AJ627196,AJ564623,AJ564622,AJ564621,AY858110和AY988601)序列完全匹配。不与Hendra病毒株(AF017149)产生阳性结果。本研究建立NiV和HeV一步法实时RT-PCR方法的最低检出限即敏感性分别为1.7×10~2copies/μl和2.6×10~1copies/μl,标准曲线的定量范围分别为1.7×10~2～1.7×10~6copies/μl、2.6×10~1～2.6×10~7copies/μl,相关系数R~2分别为0.9998和0.9896,不与其他病毒发生非特异反应。2对所设计的WNV引物和探针序列,登录美国国立卫生研究院网站(http://www.ncbi.nlm.nih.gov/BLAST/)进行Blast检索,所设计的引物和探针可以与已公布的WNV病毒株((EU394703、AY262283、DQ377178、DQ374650、AF404753、AF481864、EF571854、EF530047、EU155484、EF657887、DQ983578、EF431693、DQ666448等)列完全匹配,不与其他病毒匹配。本研究建立WNV一步法实时RT-PCR检测方法的最低检出限即敏感性为3.6×10~2copies/μl,标准曲线的定量范围为3.6x10~7～3.6×10~2copies/μl,相关系数R~2为0.97534,不与其他病毒发生非特异反应。3对采集自中国西部地区(重庆、四川、贵州和广西)饲养的猪的样本进行NiV和HeV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。结论:1本研究构建了NiV N基因、HeV N基因和WNV E基因核酸定量检测用的RNA标准品,并建立了NiV、HeV和WNV三种嗜神经病毒的一步法实时RT-PCR检测方法。该方法检测快速,具有较好的灵敏性和特异性,不易出现污染引起假阳性结果,适于流行病学研究和病毒性疾病的诊断。2初步流行病学研究提示我国西部地区(重庆、四川、贵州、广西)饲养的猪中可能不存在NiV和HeV感染,上述地区出现NiV和HeV爆发的可能性较小。更多还原

【Abstract】 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.更多还原