海豚链球菌LAMP方法的建立及初步应用

doi:10.14088/j.cnki.issn0439-8114.2018.15.025

湖北农业科学 ›› 2018, Vol. 57 ›› Issue (15): 99-104.doi: 10.14088/j.cnki.issn0439-8114.2018.15.025

海豚链球菌LAMP方法的建立及初步应用

张永德¹, 冯世文², 李军², 彭昊², 梁万文¹, 余艳玲¹, 黎铭¹, 杨学明¹, 朱佳杰¹, 罗洪林¹, 陈福艳¹, 雷爱莹¹

1.广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室,南宁 530021;
2.广西兽医研究所/广西兽医生物技术重点实验室,南宁 530001

收稿日期:2018-03-06 出版日期:2018-08-10 发布日期:2019-12-19
通讯作者: 李军（1971-）,广东梅县人,副研究员,博士,主要从事动物疫病防控与病原分子生物学研究,（电子信箱）jlee9981@163.com。
作者简介:张永德（1977-）,男,山东潍坊人,副研究员,博士,主要从事水产动物遗传育种研究,（电话）18577800233（电子信箱）yondar@126.com;
基金资助:
广西自然科学基金项目（2015GXNSFAA139068）; 国家自然科学基金项目（31372553; 3176075）

Development and Preliminary Application of Loop-mediated Isothermal Amplification （LAMP） Method for Rapid Detection of Streptococcus iniae

ZHANG Yong-de¹, FENG Shi-wen², LI Jun², PENG Hao², LIANG Wan-wen¹, YU Yan-ling¹, LI Ming¹, YANG Xue-ming¹, ZHU Jia-jie¹, LUO Hong-lin¹, CHEN Fu-yan¹, LEI Ai-ying¹

1. Guangxi Key Laboratory for Aquatic Genetic Breeding and Healthy Aquaculture/Guangxi Academy of Fishery Sciences, Nanning 530021, China;
2. Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology, Nanning 530001, China

Received:2018-03-06 Online:2018-08-10 Published:2019-12-19

摘要/Abstract

摘要： 为快速准确地检测鱼类海豚链球菌（Streptococcus iniae）,减少由其造成的经济损失,建立了一种基于Sim基因高效、敏感的海豚链球菌环介导等温扩增（LAMP）检测技术,并对739个样品进行了检测。结果表明,该方法可以特异性地检测海豚链球菌,而不能检测无乳链球菌（Streptococcus agalactiae）、副乳房链球菌（Streptococcus parauberis）、停乳链球菌（Streptococcus dysgalactiae）等8种参考菌株。对海豚链球菌DNA最低检测限为5.32拷贝/μL;对739个样品的检测结果显示,8种鱼类海豚链球菌的平均检出率为17.67%,池塘水和海水的检出率分别为13.33%、16.67%;该菌主要发现于罗非鱼脑组织中,其平均检出率为27.04%（43/159）,而其他鱼类则主要发现于皮肤,平均检出率为24.14%（42/174）;阳性样品主要来自于2014年6月至9月期间,占总检出率的65.00%。

关键词: 海豚链球菌（Streptococcus iniae）, 链球菌病, 环介导等温扩增技术, 罗非鱼

Abstract: To develop a rapid and accurate method for detecting Streptococcus iniae and limit the losses because of this disease, a loop-mediated isothermal amplification （LAMP） assay was established with four specific primers designed according to the Sim gene of Streptococcus iniae in GenBank, and totally 739 samples were collected and detected by LAMP. The results showed that the LAMP had a high sensitivity for S. iniae with detection limits of 5.32 copies/μL S. iniae DNA. The LAMP method had a strong specificity, which can be specifically amplified S. iniae, and no amplifications were observed in the 8 reference strains. The detection results of 739 samples showed that the average detection rate of S. iniae in the samples from 8 species of cultured fish was 17.67%, while 13.33% and 16.67% were from pond water and seawater, respectively. The bacteria were found mainly in tilapia brain tissues, and the average positive detection rate was 27.04%, while in other fishes, the average positive detection rate was 24.14%. Higher S. iniae positive carrying rate during Jun to Sep, 2014 was found comparing to Jan to May and Oct to Dec, 2014, with the detection rate reached to 65.00%.

Key words: Streptococcus iniae, streptococcosis, loop-mediated isothermal amplification （LAMP）, tilapia

中图分类号:

S852.61

张永德, 冯世文, 李军, 彭昊, 梁万文, 余艳玲, 黎铭, 杨学明, 朱佳杰, 罗洪林, 陈福艳, 雷爱莹. 海豚链球菌LAMP方法的建立及初步应用[J]. 湖北农业科学, 2018, 57(15): 99-104.

ZHANG Yong-de, FENG Shi-wen, LI Jun, PENG Hao, LIANG Wan-wen, YU Yan-ling, LI Ming, YANG Xue-ming, ZHU Jia-jie, LUO Hong-lin, CHEN Fu-yan, LEI Ai-ying. Development and Preliminary Application of Loop-mediated Isothermal Amplification （LAMP） Method for Rapid Detection of Streptococcus iniae[J]. HUBEI AGRICULTURAL SCIENCES, 2018, 57(15): 99-104.

参考文献

[1] SUANYUK N,SUKKASAME N,TANMARK N,et al. Streptococcus iniae infection in cultured Asian sea bass(Lates calcarifer) and red tilapia(Oreochromis sp.) in southern Thailand[J].Songklanakarin Journal of Science & Technology,2010,32(4):341-348.
[2] DUREMDEZ R,AL-MARZOUK A,QASEM J A,et al.Isolation of Streptococcus agalactiae from cultured silver pomfret,Pampus argenteus(Euphrasen),in Kuwait[J].Journal of fish Diseases, 2004,27(5):307-310.
[3] ANSHARY H,KURNIAWAN R A,SRIWULAN S,et al.Isolation and molecular identification of the etiological agents of streptococcosis in Nile tilapia(Oreochromis niloticus) cultured in net cages in Lake Sentani,Papua,Indonesia[J].Springer Plus,2014,3(1):627.
[4] ABDELSALAM M,EISSA A E,CHEN S C.Genetic diversity of geographically distinct Streptococcus dysgalactiae isolates from fish[J].Journal of Advanced Research,2015,6(2):233-238.
[5] ALI A,HASSAN D,SALEHA A A,et al.Streptococcus agalactiae the etiological agent of mass mortality in farmed red tilapia (Oreochromis sp.)[J].Journal of Animal and Veterinary Advances,2010,9(20):2640-2646.
[6] AGNEW W,BARNES A C.Streptococcus iniae:An aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination[J].Veterinary microbiology, 2007,122(1):1-15.
[7] CHEN M,LI L P,WANG R,et al.PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China[J].Veterinary Microbiology,2012,159(3-4):526-530.
[8] LOW D E,LIU E,FULLER J,et al.Streptococcus iniae: An emerging pathogen in the aquaculture industry[M].Washington. DC:ASM Press;1999.
[9] BAIANO J C F,BARNES A C. Towards control of Streptococcus iniae[J].Emerging Infectious Diseases,2009,15(12):1891-1896.
[10] DODSON S V,MAURER J J,SHOTTS E B.Biochemical and molecular typing of Streptococcus iniae isolated from fish and human cases[J]. Journal of Fish Diseases,1999,22(5):331-336.
[11] GOH S H, DRIEDGER D, GILLETT S, et al.Streptococcus iniae,a human and animal pathogen: Specific identification by the chaperonin 60 gene identification method[J]. Journal of Clinical Microbiology,1998,36(7):2164-2166.
[12] MATA A I,BLANCO M M,DOMNGUEZ L,et al.Development of a PCR assay for Streptococcus iniae based on the lactate oxidase (lctO) gene with potential diagnostic value[J]. Veterinary Microbiology,2004,101(2):109-116.
[13] TORANZO A E,MAGARINOS B,ROMALDE J L.A review of the main bacterial fish diseases in mariculture systems[J]. Aquaculture,2005,246(1):37-61.
[14] TOMITA N,MORI Y,KANDA H,et al.Loop-mediated isothermal amplification(LAMP) of gene sequences and simple visual detection of products[J].Nature Protocols,2008,3(5):877-882.
[15] CAI S H,WANG B,LU Y S,et al.Development of Loop-mediated isothermal amplification method for rapid detection of Streptococcus iniae, the causative agent of streptococcicosis in fish[J]. Journal of Basic Microbiology,2012,52(2):116-122.
[16] HAN H J,JUNG S J,OH M J,et al.Rapid and sensitive detection of Streptococcus iniae by loop-mediated isothermal amplification(LAMP)[J].Journal of Fish Diseases,2011,34(5): 395-398.
[17] SUEBSING R,KAMPEERA J,TOOKDEE B,et al.Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia[J].Letters in Applied Microbiology,2013,57(4):317-324.
[18] BAIANO J C F,TUMBOL R A,UMAPATHY A,et al. Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae[J]. BMC Microbiology,2008, 8(1):1-16.
[19] BUCHANAN J T,STANNARD J A,LAUTH X,et al.Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development[J].Infection and Immunity,2005, 73(10):6935-6944.
[20] MILLER J D,NEELY M N.Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae[J]. Infection and Immunity,2005,73(2): 921-934.
[21] BARNES A C,YOUNG F M,HORNE M T,et al.Streptococcus iniae: Serological differences, presence of capsule and resistance to immune serum killing[J]. Diseases of Aquatic Organisms,2003,53(3):241-247.
[22] LOCKE J B,COLVIN K M,DATTA A K,et al.Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish[J]. Journal of Bacteriology,2007,189(4): 1279-1287.
[23] NGUYEN H T,KANAI K,YOSHIKOSHI K.Ecological investigation of Streptococcus iniae in cultured Japanese flounder(Paralichthys olivaceus) using selective isolation procedures[J]. Aquaculture,2002,205(1):7-17.

海豚链球菌LAMP方法的建立及初步应用

Development and Preliminary Application of Loop-mediated Isothermal Amplification （LAMP） Method for Rapid Detection of Streptococcus iniae

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 4

编辑推荐

Metrics

本文评价

[1]	胡基雄, 王席, 李国攀, 荣俊. 副猪嗜血杆菌sodA基因的表达及其理化性质[J]. 湖北农业科学, 2021, 60(10): 142-147.
[2]	谭仕旦. 藏獒犬链球菌的分离与鉴定[J]. 湖北农业科学, 2018, 57(17): 79-81.
[3]	吴利军, 周丹娜, 邵志勇, 金尔光, 姚艳丰, 王富国, 陈夏冰, 何斌, 杨文海, 刘武, 童伟文, 夏瑜, 陈洁. 仔猪腹泻大肠杆菌分离鉴定及血清型与耐药性分析[J]. 湖北农业科学, 2018, 57(6): 94-97.
[4]	汪最, 孔令严, 邵华斌, 卢琴, 罗青平, 杨玉莹. 体外诱导禽巴氏杆菌链霉素耐药株及耐药机制的初步分析[J]. 湖北农业科学, 2018, 57(8): 96-99.