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2025年8月6日 星期三
农业生物技术学报  2024, Vol. 32 Issue (4): 961-973    DOI: 10.3969/j.issn.1674-7968.2024.04.020
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
重组酶聚合酶扩增技术检测南美白对虾急性肝胰腺坏死病
金东升1,2, 漏懿荣2, 王瑶华3, 段丽君2, 李其昂2, 陈琛3, 闫茂仓3, 陈炯1,2, 周前进1,2,*
1 宁波大学 农产品质量安全危害因子与风险防控国家重点实验室,宁波 315211;
2 宁波大学 海洋学院,宁波 315832;
3 浙江省海洋水产养殖研究所 浙江省近岸水域生物资源开发与保护重点实验室;温州 325005
Detection of Acute Hepatopancreatic Necrosis Disease in Pacific White Shrimp (Penaeus vannamei) by Recombinase Polymerase Amplification
JIN Dong-Sheng1,2, LOU Yi-Rong2, WANG Yao-Hua3, DUAN Li-Jun2, LI Qi-Ang2, CHEN Chen3, YAN Mao-Cang3, CHEN Jiong1,2, ZHOU Qian-Jin1,2,*
1 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China;
2 School of Marine Science, Ningbo University, Ningbo 315832, China;
3 Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-resource, Zhejiang Mariculture Research Institute, Wenzhou 325005, China
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摘要 急性肝胰腺坏死病(acute hepatopancreatic necrosis disease, AHPND)是一种新近出现的对虾(Penaeidae)细菌性疾病,给世界范围内的对虾养殖业造成了巨大的经济损失。AHPND主要由携带毒力基因pirApirB的弧菌(Vibrio spp.)引起。本研究以pirApirB为检测靶标,分别设计和筛选特异性引物和探针,建立重组酶聚合酶扩增(recombinase polymerase amplification, RPA)方法。RPA反应在39 ℃下进行,反应时间设定为20 min。结果显示,针对pirApirB基因分别筛选到1套引物/探针组合。应用该引物/探针组合对RPA方法进行系列测试,发现引物/探针组合均能够特异性检出目标毒力基因,对溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(V. parahaemolyticus),以及虾肝肠孢虫(Enterocytozoon hepatopenaei)等13种微生物的基因组DNA未出现扩增。灵敏度实验结果发现,使用pirA的引物/探针组合最低能够稳定检测出0.01 fg/μL的重组质粒pirAB-pMD19,而使用pirB的引物/探针组合最低能够稳定检测出0.1 fg/μL的重组质粒pirAB-pMD19。经优化,检测pirApirB的RPA反应的最优引物浓度为0.4 µmol/L,探针浓度为0.16 µmol/L。使用优化的引物/探针组合对可疑南美白对虾(Penaeus vannamei)组织样品利用RPA方法进行检测,结果显示,利用建立的RPA方法能够准确地从对虾组织样品中检测到pirApirB,综合检测结果与世界动物卫生组织(World Organisation for Animal Health, WOAH)推荐的PCR方法结果一致。综上,本研究分别针对pirApirB建立的PRA方法能够快速、准确地检出ANPND病原,而且操作简便、耗时短,有望成为AHPND病害日常监测和快速检测的重要补充。
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金东升
漏懿荣
王瑶华
段丽君
李其昂
陈琛
闫茂仓
陈炯
周前进
关键词 重组酶聚合酶技术(RPA)急性肝胰腺坏死病(AHPND)pirApirB检测    
Abstract:Acute hepatopancreatic necrosis disease (AHPND) is a newly emerged bacterial disease in shrimps (Penaeidae), causing significant economic losses in the global shrimp farming industry. AHPND is primarily caused by Vibrio spp. carrying virulent pirA and pirB. In this study, specific primers and probes were designed and screened for pirA and pirB, and a recombinase polymerase amplification (RPA) method was developed. This RPA reaction ran in 39 ℃ for 20 min. The results revealed successful screening of primer/probe combinations for both pirA and pirB genes. Through a series of tests using the RPA method, these primer/probe combinations demonstrated specific detection of the target virulence genes, without amplification of genomic DNA from 13 microorganisms, such as Vibrio alginolyticus, V. parahaemolyticus, and Enterocytozoon hepatomegaly. The detection limit of the RPA method was 0.01 and 0.1 fg/μL of recombinant plasmid pirAB-pMD19, when the primers and probes targeting pirA or pirB were used. The primer concentrations were optimized at 0.4 µmol/L each, while the optimal concentration for the probe was 0.16 µmol/L. The RPA method was used to detect suspicious tissues of Penaeus vannamei using optimized sets of primers and probe. The results showed that the RPA method could accurately detect pirA and pirB from shrimp tissues, which was consistent with the PCR method recommended by the World Organization for Animal Health (WOAH). In summary, this PRA methods targeting pirA and pirB respectively could rapid and accurately detect ANPND-causing bacteria with easy operation and short time-consumption, making it a promising supplementary tool for routine monitoring and rapid detection of AHPND.
Key wordsRecombinase polymerase amplification (RPA)    Acute hepatopancreatic necrosis disease (AHPND)    pirA    pirB    Detection
收稿日期: 2023-05-08     
ZTFLH:  S945.1  
基金资助:浙江省公益技术研究计划/农村农业(LGN22C010001); 浙江省重点研发项目(2021C02059); 国家自然科学基金(42276110); 宁波市科技特派员项目(2022S210); 浙江省“三农九方”农业科技协作计划项目(2023SNJF062); 浙江省近岸水域生物资源开发与保护重点实验室开放基金(J2022001); 宁波大学One health交叉学科研究项目(HZ202201); 温州市基础性科研项目(S2020020)
通讯作者: *zhouqianjin@nbu.edu.cn   
引用本文:   
金东升, 漏懿荣, 王瑶华, 段丽君, 李其昂, 陈琛, 闫茂仓, 陈炯, 周前进. 重组酶聚合酶扩增技术检测南美白对虾急性肝胰腺坏死病[J]. 农业生物技术学报, 2024, 32(4): 961-973.
JIN Dong-Sheng, LOU Yi-Rong, WANG Yao-Hua, DUAN Li-Jun, LI Qi-Ang, CHEN Chen, YAN Mao-Cang, CHEN Jiong, ZHOU Qian-Jin. Detection of Acute Hepatopancreatic Necrosis Disease in Pacific White Shrimp (Penaeus vannamei) by Recombinase Polymerase Amplification. 农业生物技术学报, 2024, 32(4): 961-973.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.04.020     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I4/961
 
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