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 mediated duplex exponential amplification

作　　者：胡振新 覃鸿妮[1,4] 吴尧 谢钰珍 吴凡 李秋实 戴东升 张勇 牛志杰 HU Zhenxin;QIN Hongni;WU Yao;XIE Yuzhen;WU Fan;LI Qiushi;DAI Dongsheng;ZHANG Yong;NIU Zhijie(Suzhou Industrial Park Institute of Services Outsourcing,Suzhou 215000,China;GeneVide Biotechnology Co.,Ltd,Suzhou 215000,China;Suzhou Research Institute of Shandong University,Suzhou 215000,China;Jiangsu Province Engineering Research Center of Precision Diagnostics and Therapeutics Development(Soochow University),Suzhou 215000,China;Suzhou ProRNA Biomedical Co.,Ltd,Suzhou 215000,China)

机构地区：[1]苏州工业园区服务外包职业学院,江苏苏州215000 [2]苏州晶睿生物科技有限公司,江苏苏州215000 [3]山东大学苏州研究院,江苏苏州215000 [4]江苏省精准诊疗药物创制工程研究中心(苏州大学),江苏苏州215000 [5]苏州启环生物医药有限公司,江苏苏州215000

出　　处：《中国兽医科学》2024年第10期1334-1344,共11页Chinese Veterinary Science

基　　金：国家生物药技术创新中心揭榜挂帅项目(NCTIB2022HS03002);江苏省科技厅面上项目(BK20221255);江苏省高校优秀科技创新团队项目(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);苏州市关键核心技术攻关项目(2023年度);苏州工业园区服务外包职业学院科研项目(SISOKY-202309)。

摘　　要：本研究开发了一种新的基于双重信号放大技术的非洲猪瘟病毒(ASFV)核酸检测方法,旨在提供快速、高灵敏度和高特异性的现场检测解决方案,并已进行规模化测试。该方法采用特定引物和重组酶复合体靶向ASFV基因序列,通过T7 RNA聚合酶在恒温下快速扩增RNA。扩增的RNA与荧光标记的DNA探针杂交后,经DSN酶水解释放荧光信号,放大探针检测逾1000倍。采用不同的引物、探针和试剂组合,在标准质粒和模拟样本上进行了灵敏度、特异性和污染防控的评价,并简化了样品处理步骤。结果表明,该方法能够在25 min内稳定且特异地检测到5~10 copies的靶标基因,与qPCR相比降低了污染风险。在393个临床样本的检测中,155个阳性和238个阴性样本的检测结果均符合预期,准确率达100%。本研究成功建立了一种适用于现场检测ASFV的双重信号放大核酸检测方法,为ASFV的临床监测和诊断提供了可靠的技术支持。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 1000 times.By assessing various primer,probe,and reagent combinations,we evaluated sensitivity,specificity,and contamination control on standard plasmids and mock samples,while also streamlining sample processing steps.The results demonstrate that this method can stably and specifically detect 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 monitoring 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 

分 类 号：S852.659.1[农业科学—基础兽医学]