摘要
目的 建立一种呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色的检测方法.方法 针对几种重要的呼吸道病原体（甲型流感病毒、乙型流感病毒、SARS冠状病毒、嗜肺军团菌、脑膜炎奈瑟菌以及腺病毒）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,对多种呼吸道病原体同时进行检测,以人偏肺病毒、呼吸道合胞病毒、人鼻病毒、肺炎链球菌4种呼吸道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.结果 成功建立了一种呼吸道病原体多重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,且与人偏肺病毒、呼吸道合胞病毒、人鼻病毒、肺炎链球菌无交叉反应.该方法对不同靶标的检测灵敏度介0.5 ～50拷贝/μL.结论 建立的呼吸道病原体多重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.
Objective To develop a method for detecting respiratory pathogens by using multiplex PCR combined with invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles.Methods Primers were designed based on the conservative regions of respiratory pathogens（Influenza viruses type A and B,Severe Acute Respiratory Syndromes coronaviruses （SARS-CoV）,Legionella pneumophila （LP）,Neisseria Meningitidis （N.Men.） and Adenovirus （ADV）.Multiplex PCR system,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were established to detect different respiratory pathogens in one reaction.Human metapneumovirus （HMPV）,Respiratory Syncytial Virus （RSV）,Human Rhinovirus （HRV）,and Streptococcus pneumoniae （SP） were used to test its specificity.Quantitative RNA transcribed in vitro and PCR fragments were used to assess its sensitivity.Results A method for detecting respiratory pathogens by using multiplex PCR,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were successfully established.This method could detect targeted pathogens specifically,and it had no cross reaction with HMPV,RSV,HRV and SP.The detecting limitation for different targets was between 0.5～ 50 copies/μL.Conclusions The method has characteristics of high specificity,sensitivity and throughput.The results can be observed by visual inspection.This method has broad application prospects in pathogen detection.
作者
樊欢
戚宇华
朱政
葛以跃
崔仑标
吴涛
史智扬
FAN Huan QI Yu-hua ZHU Zheng GE Yi-yue CUI Lun-biao SHI Zhi-yang(Institute of Pathogen Mi- crobiology, Jiangsu Provincial Center for Disease Prevention and Control, Key Laboratories of Enteric Patho- genic Microbiology, Ministry of Health, Nanjing 210009, China)
出处
《国际病毒学杂志》
2016年第5期326-331,共6页
International Journal of Virology
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（2013ZX10004103）
江苏省自然科学基金（BK20131451）
关键词
呼吸道病原体
多重PCR
侵入反应
纳米金显色
Respiratory pathogen
Multiplex PCR
Invasive reaction
Chromogenic Reaction Cata-lyzed by Gold Nanoparticles
