黄颡鱼实时荧光RPA及侧向流RPA试纸条快速现场鉴定方法的建立与应用  

DEVELOPMENT AND APPLICATION OF REAL-TIME AND LATERAL FLOW DIPSTICK RECOMBINASE POLYMERASE AMPLIFICATION ASSAYS FOR RAPID SPECIES IDENTIFICATION OF PELTEOBAGRUS FULVIDRACO

作　　者：林玉 张旻[1,3] 王娜 何舜平[4] 张辉 唐永凯[6] 方成池[4] 景宏丽[1,3] 张浪 沙航[4] 段志强 王宽 吕继洲 吴绍强 LIN Yu;ZHANG Min;WANG Na;HE Shun-Ping;ZHANG Hui;TANG Yong-Kai;FANG Cheng-Chi;JING Hong-Li;ZHANG Lang;SHA Hang;DUAN Zhi-Qiang;WANG Kuan;LYU Ji-Zhou;and WU Shao-Qiang(Chinese Academy of Inspection and Quarantine,Beijing 100176,China;CAIQ Center for Biosafety,Sanya 572025,China;Technology Innovation Center of Animal and Plant Product Quality,Safety and Control,State Administration for Market Regulation,Beijing 100176,China;Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China;Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China;Freshwater Fisheries Research Center,CAFS,Wuxi 214081,China;Bellagen Biotechnology Co.Ltd,Jinan 250014,China)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]三亚中国检科院生物安全中心,三亚572025 [3]国家市场监督管理总局技术创新中心(动植物产品质量与安全),北京100176 [4]中国科学院水生生物研究所,武汉430072 [5]中国水产科学研究院长江水产研究所,武汉430223 [6]中国水产科学研究院淡水渔业研究中心,无锡214081 [7]山东舜丰生物科技有限公司,济南250014

出　　处：《水生生物学报》2025年第4期120-128,共9页Acta Hydrobiologica Sinica

基　　金：“十四五”国家重点研发计划(2022YFF0608201);国家市场监督管理总局科技创新人才计划(QNBJ202324);中国检验检疫科学研究院基本科研业务费项目(2022JK43)资助。

摘　　要：研究以黄颡鱼为目标对象,基于重组酶聚合酶扩增(Recombinase polymerase amplification,RPA)等温扩增技术,开发了实时荧光型RPA和侧向流RPA试纸条两种现场快速检测方法。这些方法能够用于快速、准确和灵敏地实时检测出黄颡鱼DNA。首先,通过对黄颡鱼及其近缘物种的鱼类线粒体基因组序列进行对比分析,针对COI基因保守区域,设计了特异性探针和引物。测试结果显示,在37℃条件下,实时荧光型RPA和侧向流RPA试纸条都具有良好的特异性。实时荧光型RPA方法能够在20min内完成黄颡鱼DNA的检测,且最低检测限可以达到102拷贝数/反应;而侧向流RPA试纸条的操作更加便捷,能够在10min内可完成对102低拷贝数的黄颡鱼DNA的扩增和鉴定。应用这两种RPA检测技术对149份长江流域常见鱼类进行现场检测,两种方法均显示出较高的准确性(准确率100%)。因此,研究开发的两种用于检测黄颡鱼的实时荧光型RPA和侧向流RPA试纸条方法,拥有操作简便、结果直观、准确率高、不依赖实验室仪器设备等优势,具有良好的现场快速检测应用前景。此外,其克服了形态学物种鉴定专业门槛高、难以鉴定残破组织样品的问题,检测靶标可跨越卵、鱼苗、成鱼等全生命周期,为水产品监管执法提供技术手段。In order to effectively prevent illegal fishing of wild fishery resources in the Yangtze River,it is crucial to provide technical support for the“ten-year ban on fishing in the Yangtze River”and develop methods that can quickly and accurately identify fish species on site.This study focuses on Pelteobagrus fulvidraco and utilizes isothermal amplification technology,specifically Recombinase Polymerase Amplification(RPA),to develop two rapid on-site detection methods:Real-time fluorescent RPA and lateral flow RPA test strips,which are ideally suited for the swift infield detection of P.fulvidraco DNA.We developed an RPA assay based on either Real-time fluorescent detection(Real-time RPA assay)or lateral flow dipstick(RPA-LFD assay)for rapid,accurate,and sensitive detection of P.fulvidraco DNA,facilitating effective management of the fishing ban in the Yangtze River basin.Primers and probes for both assays were designed to target conserved regions of the mitochondrial cytochrome c oxidase subunitⅠ(COⅠ)gene in P.fulvidraco.Tests demonstrated that both the Real-time RPA and RPA-LFD assays exhibited good specificity at 37℃.The Real-time RPA assay achieved a detection limit of 102 copies of P.fulvidraco DNA per reaction,with amplification completed within 20min,though it requires a constant temperature amplification instrument.In contrast,the RPA-LFD assay offers a more convenient operation with a shorter amplification time of only 10min for 102 copies of P.fulvidraco DNA.Additionally,both methods were tested on 149 samples for known fish species,yielding results consistent with actual species identification.The Real-time fluorescence RPA and lateral flow dipstick RPA methods developed in this study for detecting P.fulvidraco DNA have the advantages of easy operation,intuitive results,high accuracy,and no dependence on laboratory instruments and equipment.They have good prospects for rapid on-site detection applications.In addition,it overcomes the problems of high professional threshold for morphological spec

关 键 词：快速物种鉴定 重组酶聚合酶扩增 实时荧光型RPA 侧向流RPA试纸条 黄颡鱼 

分 类 号：Q33[生物学—遗传学] S932.4[农业科学—渔业资源]