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引用本文:康晋伟,金晶磊,段丽君,周燕,苗亮,周前进,陈炯.香鱼格留虫(Glugea plecoglossi)环介导等温扩增联合横向流动试纸条检测方法的建立.海洋与湖沼,2020,51(6):1501-1512.
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香鱼格留虫(Glugea plecoglossi)环介导等温扩增联合横向流动试纸条检测方法的建立
康晋伟1,2, 金晶磊1,2, 段丽君1,2,3, 周燕1,2,4, 苗亮2, 周前进1,2, 陈炯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.宁波市海曙区畜牧兽医技术管理服务站 宁波 315153;4.1. 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
摘要:
我国的养殖香鱼正面临着严重的香鱼格留虫(Glugea plecoglossi)感染。本研究联合运用环介导等温扩增技术(LAMP)和横向流动试纸条(LFD)的检测技术,建立了快速便捷地检测G. plecoglossi的LAMP-LFD方法。该方法以G. plecoglossi的β微管蛋白基因为检测靶标,在其保守区域设计并筛得6条特异性引物(其中上游内引物用生物素标记),进行LAMP反应,产物再与异硫氰酸荧光素(FITC)标记的特异性探针杂交,在LFD上进行结果判断。结果表明,LAMP-LFD方法能够特异性地检出G. plecoglossi,对梅氏新贝尼登虫、刺激隐核虫、肝肠胞虫阳性的虾组织、派氏异尖线虫、内弯宫脂线虫、鳗弧菌香鱼分离株、杀香鱼假单胞菌,以及香鱼组织等的检测均呈阴性。优化后,LAMP的反应条件为65℃反应45min,与探针杂交的条件为65℃反应5min,加之5min的显色时间,整个检测时程为55min。利用该方法能够检测到2.0fg/μL的含β微管蛋白的质粒DNA,针对G. plecoglossi基因组DNA的检测灵敏度为14.0pg/μL;能够从感染强度达到100个虫体/克的香鱼肝组织中稳定地检测到虫体。该方法可在简单的恒温加热设备(如水浴锅)中完成核酸扩增和探针杂交,无需昂贵的仪器装置。综上,香鱼格留虫LAMP-LFD方法操作便捷、灵敏度高、特异性好、检测快速,而且设备依赖性低,完全适合于基层检测的需求。
关键词:  香鱼格留虫(Glugea plecoglossi)  β微管蛋白  环介导等温扩增技术(LAMP)  横向流动试纸条(LFD)  检测
DOI:10.11693/hyhz20200300076
分类号:S941.42
基金项目:浙江省公益技术研究计划农村农业项目,LGN18C180002号;宁波市科技创新团队项目,2015C110018号;宁波市自然科学基金项目,2018A610342号;宁波市江北区农业和社会发展科技计划项目,2017B02号;农产品质量安全危害因子与风险防控国家重点实验室2020开放基金项目,KF20200105号;浙江省动物预防医学重点实验室开放课题基金项目,ZPKLPVM2017KFKT005号;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,J2018004号;2018年浙江省大学生科技创新活动计划暨新苗人才计划,2018R405078号。
A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION TECHNIQUE COMBINED WITH A LATERAL FLOW DIPSTICK FOR THE DETECTION OF GLUGEA PLECOGLOSSI
KANG Jin-Wei1,2, JIN Jing-Lei1,2, DUAN Li-Jun1,2,3, ZHOU Yan1,2,4, MIAO Liang2, ZHOU Qian-Jin1,2, CHEN Jiong1,2
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.Ningbo Haishu District Animal Husbandry and Veterinary Medicine Technical Management Service Station, Ningbo 315153, China;4.1. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China
Abstract:
Recently, the farming of fish Plecoglossus altivelis in China is facing severe infection of Glugea plecoglossi. Therefore, we developed a novel LAMP-LFD method for rapid detection of G. plecoglossi infection based on loop-mediated isothermal amplification (LAMP) integrated with the visualization on a lateral flow dipstick assay (LFD). With this method, the β-tubulin gene of G. plecoglossi was targeted, and six primers designed in its conserved regions were used among them the forward inner primer was biotinylated. A biotinylated LAMP assay was carried out and the amplicon was hybridized exclusively with fluorescein isothiocyanate (FITC) labeled probe, and then the hybrid product was visualized on a lateral flow dipstick (LFD). Results demonstrate that this LAMP-LFD method could specifically detect G. plecoglossi, and no characteristic amplification was observed when using genomic DNA of Neobenedenia melleni, Cryptocaryon irritans, shrimp tissue with Enterocytozoon hepatopenaei, Anisakis pegreffii, Hysterothylacium aduncum, Vibrio anguillarum ayu-H080701, Pseudomonas plecoglossicida, and P. altivelis tissue. The optimal conditions of LAMP assays were 65℃ for 45min, followed by 5min hybridization at 65℃ and 5min visualization on LFD. Thus, the whole detection of G. plecoglossi could be completed within 55min. The detection limit of LAMP-LFD was 2.0fg/μL for plasmid DNA with β-tubulin gene and 14.0pg/μL for G. plecoglossi genome DNA; and it could detect the spores stably from P. altivelis liver tissue contaminated by G. plecoglossi with an infection intensity of no more than 100 spores/g. In addition, LAMP-LFD could be used in a simple heating equipment such as water bath to complete nucleic acid amplification and probe hybridization without expensive instruments. Therefore, the LAMP-LFD method is easy to operate, sensitive, specific, rapid, and instrument-free, which shows great potential in the routine detection and monitoring of G. plecoglossi.
Key words:  Glugea plecoglossi  β-tubulin gene  loop-mediated isothermal amplification  lateral flow dipstick  detection
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