粪便标本中诺如病毒GⅡ拷贝数定量检测方法的建立和应用  

Construction and application of quantitative detection method for Norovirus GⅡ copy number in fecal specimens

作　　者：赵跃丽 饶清 孙强明[2,3] 张兰胜 ZHAO Yue-li;RAO Qing;SUN Qiang-ming;ZHANG Lan-sheng(School of Pharmacy and Chemistry,Dali University,Dali 671000,China;Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China;Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases of Yunnan Province,Kunming 650118,China)

机构地区：[1]大理大学药学与化学学院,云南大理671000 [2]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118 [3]云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国感染控制杂志》2020年第10期864-870,共7页Chinese Journal of Infection Control

基　　金：国家自然科学基金面上项目(81171946);国家十二五重大新药创新科技重大专项(2012ZX09104302)。

摘　　要：目的构建诺如病毒GⅡ(Norovirus GenogroupⅡ,NoV GⅡ)拷贝数标准质粒及其检测体系。方法将合成高度保守的NoV GⅡ基因序列片段克隆至pUC57载体上,构建NoV GⅡ标准质粒,采用聚合酶链反应(PCR)进行验证。通过实时荧光定量PCR扩增曲线结果绘制C t值与病毒拷贝数的标准曲线,求得其相应的标准曲线方程。采用建立的标准质粒及其检测体系对2018年12月昆明市儿童医院4份临床粪便标本进行检测。结果经PCR验证,NoV GⅡ拷贝数的标准质粒构建正确。采用实时荧光定量PCR的扩增曲线获得C t值与病毒拷贝数相对应的标准曲线方程为Y=-3.972X+39.03,R^2=0.991,4份粪便标本原液NoV GⅡ病毒拷贝数分别为30443.45、9468.40、53176.69、4493.12 copies/μL。结论成功建立用于检测粪便标本中NoV GⅡ病毒拷贝数的标准质粒及其检测体系,可为疾病的预防控制和相关试验研究提供有效的定量检测方法。Objective To construct the standard plasmid and detection system for copy number of Norovirus GⅡ(NoV GⅡ).Methods The synthesized highly conserved NoV GⅡgene sequence was cloned into pUC57 vector for constructing NoV GⅡstandard plasmid,then plasmid was verified by polymerase chain reaction(PCR).The standard curve of C t value and copy number of NoV GⅡwas drawn according to real-time fluorescence quantitative PCR amplification curve,and corresponding standard curve equation was obtained.Four clinical fecal specimens obtained from the Children’s Hospital of Kunming in December 2018 were detected by the constructed NoV GⅡstandard plasmid and detection system.Results PCR proved that the standard plasmid for detecting copy number of NoV GⅡwas constructed correctly.Standard curve equation of C t value and viral copy number based on amplification curve of real-time fluorescence quantitative PCR was obtained as follows:Y=-3.972X+39.03,R^2=0.991.Viral copy numbers in 4 fecal specimens were 30443.45,9468.40,53176.69,and 4493.12 copies/μL respectively.Conclusion The standard plasmid and its detection system for detecting the copy number of NoV GⅡvirus in fecal specimens are successfully established,which can provide an effective quantitative detection method for disease prevention and control and related experimental research.

关 键 词：诺如病毒GⅡ 病毒拷贝数 实时荧光定量PCR 标准曲线 

分 类 号：R373[医药卫生—病原生物学]