多重PCR结合核酸侵入反应及纳米金显色技术检测病毒性脑炎脑膜炎病原体

发布时间：2018-02-13 00:20

本文关键词： 病毒性脑炎脑膜炎多重PCR 核酸侵入反应纳米金显色　出处：《中国人兽共患病学报》2017年11期 　论文类型：期刊论文

【摘要】：目的建立一种病毒性脑炎脑膜炎病原体多重PCR结合核酸侵入反应及纳米金显色的检测方法。方法针对几种重要的脑炎脑膜炎病毒(东方马脑炎病毒、西方马脑炎病毒、流行性乙型脑炎病毒、西尼罗病毒和尼帕病毒)保守区基因设计引物,进行多重PCR反应、核酸侵入反应及纳米金显色反应,对多种脑炎脑膜炎病毒同时进行检测,以森林脑炎病毒、圣路易脑炎病毒、基孔肯雅病毒和登革病毒4种病原核酸评价其检测特异性,以体外转录的病毒RNA或扩增的PCR片段评价其检测敏感性,并对流行性乙型脑炎患者临床标本进行检测。结果成功建立了一种脑炎脑膜炎病毒多重PCR结合核酸侵入反应及纳米金显色的检测技术。建立的检测方法可特异的检测目的病原体,且与森林脑炎病毒、圣路易脑炎病毒、基孔肯雅病毒和登革病毒无交叉反应。该方法对不同靶标的检测灵敏度均为103拷贝/μL,临床标本检测结果均为阳性。结论建立的脑炎脑膜炎病毒多重PCR结合核酸侵入反应及纳米金显色技术的检测方法,具有较高的检测特异性及灵敏度,检测通量高,肉眼即可观察结果,在传染病病原体检测方面具有广阔的应用前景。
[Abstract]:Objective to establish a method for the detection of viral encephalitis meningitis pathogens with multiple PCR binding nucleic acid invasion and gold nanoparticles. Methods several important encephalitis meningitis viruses (oriental equine encephalitis virus, western equine encephalitis virus) were used. The conserved region of epidemic encephalitis B virus, West Nile virus and Nipavirus) was designed with primers designed for multiplex PCR reaction, nucleic acid invasion reaction and gold nanoparticles reaction, and simultaneous detection of various encephalitis meningitis viruses. The detection specificity was evaluated by four kinds of pathogenic nucleic acids, including Forest Encephalitis virus, St. Louis Encephalitis virus, Kikungunya virus and dengue virus, and its sensitivity was evaluated by in vitro transcription of virus RNA or amplified PCR fragments. The clinical specimens of patients with epidemic encephalitis B were detected. Results A method for detecting the multiple PCR binding nucleic acid invasion of encephalitis meningitis virus and gold nanoparticles was established successfully. The established method can be used specifically for the detection of Japanese encephalitis virus. Detection of the target pathogen, And with the Forest Encephalitis virus, the St. Louis Encephalitis virus, There was no cross reaction between Kikungunya virus and dengue virus. The sensitivity of this method for detecting different targets was 103copies / 渭 L, and the results of clinical specimens were positive. Conclusion the multiplex PCR binding nucleic acid invasion of encephalitis meningitis virus is established. The detection method of the entry reaction and the nanometer gold color development technology, It has high detection specificity and sensitivity, high detection flux, and can be observed with the naked eye, so it has a broad application prospect in the detection of infectious disease pathogens.
【作者单位】：卫生部肠道病原微生物重点实验室江苏省疾病预防控制中心病原微生物研究所;
【基金】：艾滋病和病毒性肝炎等重大传染病防治科技重大专项(No.2013ZX10004103)资助~~
【分类号】：R440;R512.3

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