中国兽医科学2024，Vol.54Issue(10)：1334-1344,11.DOI:10.16656/j.issn.1673-4696.2024.0179
基于双重信号放大技术的非洲猪瘟病毒快速现场核酸检测方法的开发与应用
Development and application evaluation of a rapid on-site nucleic acid detection method for African swine fever virus based on enzymes medi-ated duplex exponential amplification
摘要
Abstract
This study introduces a novel nucleic acid detection method based on enzymes mediated duplex exponential amplification,designed for rapid,highly sensitive,and specific on-site testing,which has been scaled for broader application.The method employs specific primers and a recombinase complex to target ASFV genetic sequences,followed by rapid RNA amplification at a constant temperature using T7 RNA polymerase.The amplified RNA hybridizes with a fluorescently labeled DNA probe,and the DSN enzyme-mediated cleavage releases a fluorescent signal,amplifying the probe's detection by more than 1 000 times.By assessing various primer,probe,and reagent combinations,we evaluated sensitivi-ty,specificity,and contamination control on standard plasmids and mock samples,while also streamlin-ing sample processing steps.The results demonstrate that this method can stably and specifically de-tect 5-10 copies of the target gene within 25 minutes,reducing contamination risks compared to qPCR.In a test of 393 clinical samples,all 155 positive and 238 negative samples were correctly identified,achieving a 100％accuracy rate.This research successfully established a nucleic acid detection method based on dual signal amplification,offering reliable technical support for the clinical moni-toring and diagnosis of ASFV.关键词
非洲猪瘟病毒/双重信号放大技术/现场核酸检测/临床应用评估Key words
African swine fever virus/dual signal amplification technology/on-site nucleic acid detection/clinical application evaluation分类
农业科技引用本文复制引用
胡振新,覃鸿妮,吴尧,谢钰珍,吴凡,李秋实,戴东升,张勇,牛志杰..基于双重信号放大技术的非洲猪瘟病毒快速现场核酸检测方法的开发与应用[J].中国兽医科学,2024,54(10):1334-1344,11.基金项目
国家生物药技术创新中心揭榜挂帅项目(NCTIB2022HS03002) （NCTIB2022HS03002）
江苏省科技厅面上项目(BK20221255) （BK20221255）
江苏省高校优秀科技创新团队项目(2023年) （2023年）
江苏省精准诊疗药物创制工程研究中心(苏州大学)开放课题(SDGC2239) （苏州大学）
苏州市关键核心技术攻关项目(2023年度) （2023年度）
苏州工业园区服务外包职业学院科研项目(SISOKY-202309) （SISOKY-202309）
