基因Ⅰ型乙脑病毒分离鉴定及荧光定量PCR诊断方法建立
发布时间：2019-05-08 11:57
【摘要】：目的：流行性乙型脑炎是危害人类健康比较严重的虫媒病毒。尽管目前乙脑的发病率有所下降,但发病区域仍呈现不断扩大的趋势。加强乙脑的监测,就可以评估该地区乙脑的流行状况。蚊子是JEV的主要传播媒介,对蚊子的流行性乙型脑炎病毒和虫媒病毒进行分子流行病学监测,对疾病预防具有防范意义。方法：首先将云南省景洪县采集15份蚊虫样品研磨接种BHK-21和C6/36单层细胞,待出现细胞病变。提取病毒RNA,并反转录cDNA进行PCR鉴定将目的基因克隆到pMD18-T载体,送上海生工测序。同时进行盖塔病毒、辛德毕斯、基孔肯雅病毒的PCR鉴定,回收并测序。在此基础上构建了乙脑基因Ⅰ型标准质粒,建立了基于SYBR Green Ⅰ染料的Real-time PCR检测方法,选取Ct值制作标准曲线。结果：乙脑PCR扩增目的基因经测序验证为乙脑基因Ⅰ型,然后构建应用荧光定量PCR标准质粒。研究建立的标准曲线能够准确定量目的基因,线性相关度很高,呈现良好的重复性,熔解曲线为单一峰型,未出现非特异性扩增。SYBR Green Ⅰ Real-time PCR与传统方法相比灵敏度要高10倍,而且与猪蓝耳病毒等,均不发生非特异性扩增,具有良好的特异性。结论：鉴定了基因Ⅰ型乙脑病毒,并构建了快速检测乙脑Ⅰ型的检测方法。通过实验初步验证了其具备良好的灵敏性和重复性,为以后乙脑的流行病学调查奠定了基础。
[Abstract]:Objective: Japanese encephalitis (Japanese encephalitis) is a serious insect-borne virus which is harmful to human health. Although the incidence of je has declined at present, but the area of the disease is still showing an expanding trend. The epidemic situation of je in this area can be evaluated by strengthening the surveillance of je. Mosquitoes are the main vectors of JEV. Molecular epidemiological surveillance of epidemic Japanese encephalitis virus and arbovirus is of great significance for prevention of diseases. Methods: fifteen mosquito samples were collected from Jinghong County of Yunnan Province and inoculated with monolayer cells of BHK-21 and C _ 6c _ (36). The virus RNA, was extracted and reverse transcribed cDNA was used to identify the target gene. The target gene was cloned into pMD18-T vector and sent to Shanghai for sequencing. At the same time, PCR identification, recovery and sequencing of Gaeta virus, Sindbis virus and Kikungunya virus were carried out. On the basis of this, the standard plasmid of je gene type I was constructed, the Real-time PCR detection method based on SYBR Green 鈪，
本文编号：2471893
[Abstract]:Objective: Japanese encephalitis (Japanese encephalitis) is a serious insect-borne virus which is harmful to human health. Although the incidence of je has declined at present, but the area of the disease is still showing an expanding trend. The epidemic situation of je in this area can be evaluated by strengthening the surveillance of je. Mosquitoes are the main vectors of JEV. Molecular epidemiological surveillance of epidemic Japanese encephalitis virus and arbovirus is of great significance for prevention of diseases. Methods: fifteen mosquito samples were collected from Jinghong County of Yunnan Province and inoculated with monolayer cells of BHK-21 and C _ 6c _ (36). The virus RNA, was extracted and reverse transcribed cDNA was used to identify the target gene. The target gene was cloned into pMD18-T vector and sent to Shanghai for sequencing. At the same time, PCR identification, recovery and sequencing of Gaeta virus, Sindbis virus and Kikungunya virus were carried out. On the basis of this, the standard plasmid of je gene type I was constructed, the Real-time PCR detection method based on SYBR Green 鈪，
本文编号：2471893
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