登革病毒Ⅰ型拷贝数标准质粒和检测体系的建立及其应用  

Establishment of and application of standard system for detection of copy number of dengue virus type Ⅰ

作　　者：林垚 洪珊 文送娇 席珏敏[1] 王晓丹[1] 陈俊英[1] 潘玥[1] 叶超[1] 管娇琼 孙强明[1] LIN Yao;HONG Shah;WEN Song-jiao;XI Jue-min;WANG Xiao-dan;CHEN Jun-ying;PAN Yue;YE Chao;GUAN Jiao-qiong;SUN Qiang-ming(Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development.on Severe Infectious Disease,Kunming 650118,Ynnnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2019年第1期76-79,共4页Chinese Journal of Biologicals

基　　金：国家十二五重大新药创新科技重大专项(2012ZX09104302);云南省科技厅应用基础重点项目(2016FA029);云南省科技厅联合支持国家科技计划项目(2013GA018);国家自然科学基金面上项目(81171946);中国医学科学院创新工程项目(2016-I2M-1-19);云南自然科学基金项目(2012FB188)

摘　　要：目的建立登革病毒Ⅰ型(dengue virus-Ⅰ,DENV-Ⅰ)拷贝数标准质粒及其检测体系。方法将DENV-Ⅰ的前膜蛋白基因克隆至pMD19-T载体,建立DENV-Ⅰ标准质粒,采用实时荧光定量PCR法进行扩增,获得Ct值与病毒拷贝数的关系,绘制标准曲线,得到相应的标准曲线方程。采用建立的标准质粒及检测体系对3份2017年云南西双版纳DENV-Ⅰ患者的血清进行检测。结果经双酶切及测序鉴定,DENV-Ⅰ拷贝数标准质粒构建正确。实时荧光定量PCR得到的标准曲线方程为y=-0. 300 5 x+12. 133,R^2=0. 991 9。3份血清样品原液的病毒拷贝数分别为9 417 703. 12、21 949 591. 01及19 027 096. 91 copies/μL。结论成功建立了DENV-Ⅰ拷贝数标准质粒及其检测体系,且准确性较好,灵敏度较高,可用于血清样品中DENV-Ⅰ拷贝数的检测。Objective To establish a standard system for detection of copy number of dengue virus-Ⅰ(DENV-Ⅰ).Methods Partial prM gene sequence of DENV-Ⅰwas cloned into vector pMD19T,and the constructed standard plasmid was detected for Ct value of copy number by real-time fluorescence quantitative PCR,based on which a standard curve of virus copy number against Ct was plotted,and the corresponding equation was obtained.Three serum samples of patients with DENV-Ⅰ infection in Xishuangbanna Region,Yunnan Province were detected by the established detection system using the standard plasmid.Results Restriction analysis and sequencing proved that the standard plasmid for detection of copy number of DENV-1 was constructed correctly.The equation of standard curve for real-time fluorescence quantitative PCR was as follows:y =-0.300 5 x + 12.133,R^2 = 0.991 9.The viral copy numbers in three serum samples were 9 417 703.12,21 949 591.01 and 19 027 096.91 copies/μL respectively.Conclusion Viral copy number standard of DENV-Ⅰ was established,which showed high accuracy and sensitivity and might be used for determination of DENV-Ⅰin serum samples.

关 键 词：登革病毒Ⅰ型 病毒拷贝数 标准质粒 

分 类 号：R373.23[医药卫生—病原生物学] Q75[医药卫生—基础医学]