【摘要】：目的：流行性乙型腦炎是危害人類健康比較嚴(yán)重的蟲媒病毒。盡管目前乙腦的發(fā)病率有所下降,但發(fā)病區(qū)域仍呈現(xiàn)不斷擴(kuò)大的趨勢。加強(qiáng)乙腦的監(jiān)測,就可以評估該地區(qū)乙腦的流行狀況。蚊子是JEV的主要傳播媒介,對蚊子的流行性乙型腦炎病毒和蟲媒病毒進(jìn)行分子流行病學(xué)監(jiān)測,對疾病預(yù)防具有防范意義。方法：首先將云南省景洪縣采集15份蚊蟲樣品研磨接種BHK-21和C6/36單層細(xì)胞,待出現(xiàn)細(xì)胞病變。提取病毒RNA,并反轉(zhuǎn)錄cDNA進(jìn)行PCR鑒定將目的基因克隆到pMD18-T載體,送上海生工測序。同時進(jìn)行蓋塔病毒、辛德畢斯、基孔肯雅病毒的PCR鑒定,回收并測序。在此基礎(chǔ)上構(gòu)建了乙腦基因Ⅰ型標(biāo)準(zhǔn)質(zhì)粒,建立了基于SYBR Green Ⅰ染料的Real-time PCR檢測方法,選取Ct值制作標(biāo)準(zhǔn)曲線。結(jié)果：乙腦PCR擴(kuò)增目的基因經(jīng)測序驗證為乙腦基因Ⅰ型,然后構(gòu)建應(yīng)用熒光定量PCR標(biāo)準(zhǔn)質(zhì)粒。研究建立的標(biāo)準(zhǔn)曲線能夠準(zhǔn)確定量目的基因,線性相關(guān)度很高,呈現(xiàn)良好的重復(fù)性,熔解曲線為單一峰型,未出現(xiàn)非特異性擴(kuò)增。SYBR Green Ⅰ Real-time PCR與傳統(tǒng)方法相比靈敏度要高10倍,而且與豬藍(lán)耳病毒等,均不發(fā)生非特異性擴(kuò)增,具有良好的特異性。結(jié)論：鑒定了基因Ⅰ型乙腦病毒,并構(gòu)建了快速檢測乙腦Ⅰ型的檢測方法。通過實驗初步驗證了其具備良好的靈敏性和重復(fù)性,為以后乙腦的流行病學(xué)調(diào)查奠定了基礎(chǔ)。
[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，

本文編號：2471893