诺如病毒GⅡ.4型荧光RT-RPA检测方法的建立  

Establishment of a fluorescent reverse transcription-recombinase polymerase amplification assay for norovirus type GⅡ.4

作　　者：郑志坤 徐翮飞 丁伟 张娟 杜建森 侯琳[1] 张瑾 ZHENG Zhikun;XU Hefei;DING Wei;ZHANG Juan;DU Jiansen;HOU Lin;ZHANG Jin(Department of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学基础医学院生物化学与分子生物学系,山东青岛266071 [2]青岛国际旅行卫生保健中心 [3]北京大学人民医院青岛医院

出　　处：《精准医学杂志》2025年第2期127-130,共4页Journal of Precision Medicine

基　　金：国家“十四五”科技部研发计划(2022YFC2302800)。

摘　　要：目的建立一种针对诺如病毒(NoV)GⅡ.4型的荧光逆转录-重组酶聚合酶扩增(RT-RPA)检测方法。方法从NCBI获取NoV GⅡ.4型基因序列,进行序列比对后选择保守区域设计引物和探针。以NoV GⅡ.4型标准品作为检测对象,通过优化荧光RT-RPA反应体系的反应温度、引物浓度以及探针浓度等参数,建立针对NoV GⅡ.4型的荧光RT-RPA检测方法。通过检测不同浓度的NoV GⅡ.4型标准品和NoV GⅠ型、NoV GⅡ其他亚型(2型、3型、7型、8型和17型)、轮状病毒、腺病毒、副肠孤病毒、星状病毒、札如病毒核酸,对该方法进行灵敏度检验、特异度检验以及临床样本验证。结果荧光RT-RPA方法检测NoV GⅡ.4型的最佳反应温度为41℃,最佳引物浓度为630 nmol/L,最佳探针浓度为240 nmol/L。NoV GⅡ.4型检出限为1×10^(1)拷贝/μL,其他核酸均未产生仪器可检测到的荧光信号。结论本研究建立了一种针对NoV GⅡ.4型的荧光RT-RPA检测方法,该方法具有灵敏度高、特异度好、反应速度快等特点,有望为口岸现场针对NoV GⅡ.4型的快速检测提供新方法。Objective To establish a fluorescent reverse transcription-recombinase polymerase amplification(RT-RPA)assay for norovirus(NoV)type GⅡ.4.Methods The NoV GⅡ.4 gene sequence was obtained from NCBI,and the primers and probes were designed by selecting the conserved regions after sequence alignment.With NoV GⅡ.4 standards as the detection object,the temperature,primer concentration,and probe concentration of the fluorescent RT-RPA reaction system were optimized to establish a fluorescent RT-RPA assay for NoV GⅡ.4.This method was assessed in terms of sensitivity,specificity,and clinical validation by detecting different concentrations of NoV GⅡ.4 standards and the nucleic acids of NoV GⅠ,other subtypes of NoV GⅡ(NoV GⅡ.2,NoV GⅡ.3,NoV GⅡ.7,NoV GⅡ.8,and NoV GⅡ.17),rotavirus,adenovirus,parechovirus,astrovirus,and sapovirus.Results The fluorescent RT-RPA assay had an optimal reaction temperature of 41℃,an optimal primer concentration of 630 nmol/L,and an optimal probe concentration of 240 nmol/L for detecting NoV GⅡ.4.And the limit of detection was 1×10^(1) copies/μL for NoV GⅡ.4,the other nucleic acids did not produce the fluorescent signal that could be detected by the instrument.Conclusion This study establishes a fluorescent RT-RPA assay for NoV GⅡ.4,which is characterized by high sensitivity,good specificity,and rapid reaction speed,and it is expected to provide a new method for the rapid detection of NoV GⅡ.4 at ports.

关 键 词：诺如病毒 重组酶聚合酶扩增 核酸扩增技术 敏感性与特异性 

分 类 号：R183[医药卫生—流行病学] R373.2[医药卫生—公共卫生与预防医学]