同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立

doi:10.16431/j.cnki.1671-7236.2015.08.003

›› 2015, Vol. 42 ›› Issue (8): 1935-1942.doi: 10.16431/j.cnki.1671-7236.2015.08.003

同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立

王彩霞¹, 冯春燕¹, 王巧黎², 袁向芬¹, 邓俊花¹, 吕继洲¹, 吴绍强¹

1. 中国检验检疫科学研究院动物检疫研究所, 北京 100029;
2. 青岛市中心医疗集团, 青岛 266005

收稿日期:2015-01-19 出版日期:2015-08-20 发布日期:2015-08-27
通讯作者: 吴绍强 E-mail:sqwu@sina.com
作者简介:王彩霞(1982-),女,山东潍坊人,硕士,助理研究员,研究方向:动物检疫,E-mail:friday128@sina.com
基金资助:
国家质检公益项目(201210018);国家科技支撑计划课题(2012BAK11B04)

Establishment of Duplex Loop-mediated Isothermal Amplification Method for Simultaneous Detection of Shellfish Infected with Perkinsus and Bonamia

WANG Cai-xia¹, FENG Chun-yan¹, WANG Qiao-li², YUAN Xiang-fen¹, DENG Jun-hua¹, LV Ji-zhou¹, WU Shao-qiang¹

1. Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China;
2. Qingdao Center Medical Group, Qingdao 266005, China

Received:2015-01-19 Online:2015-08-20 Published:2015-08-27

摘要/Abstract

摘要： 为满足贝类寄生虫快速检测需求,本研究根据GenBank中发布的派琴虫ITS序列和包纳米虫18S rRNA序列的保守区域分别设计LAMP扩增引物,分别建立了两种寄生虫的单重LAMP检测方法,进一步对反应体系进行优化,组装成两种寄生虫的双重LAMP方法,同时还进行了特异性检测和敏感性检测,并通过对扩增产物进行限制性内切酶酶切检验双重LAMP方法的准确性。结果表明本研究建立的贝类寄生虫双重LAMP方法具有较好的特异性,检测派琴虫时的最低检测限为10拷贝/μL质粒DNA,检测包纳米虫时的最低检测限为100拷贝/μL质粒DNA,同时酶切试验也验证了双重LAMP检测结果的准确性。对随机采自东海和黄海的20份菲律宾蛤仔和10份美国进口牡蛎的检测结果显示,该双重LAMP方法在口岸检疫工作中可以作为派琴虫和包纳米虫的快速检测方法。

关键词: 贝类寄生虫病; 环介导等温扩增反应(LAMP); 检测; 派琴虫; 包纳米虫

Abstract: In order to establish a duplex loop-mediated isothermal amplification (LAMP) method for the detection of shellfish parasitic infection with Perkinsus and Bonamia, primers were designed according to the conserved region of ITS and 18S rRNA gene sequences of Perkinsus and Bonamia deposited in GenBank using the online Primer Explorer 4.0.Firstly, the respective single LAMP detection assay of two parasites were developed, and then the reaction conditions were optimized by combining the two reaction systems into duplex LAMP method.The specificity and sensitivity of duplex LAMP method were examined and the confidence was evaluated by digesting the amplicons with respective endonuclease.The results showed that the established duplex LAMP method was specific enough to distinguish the two parasites from other parasites, and the detection limit of Perkinsus was 10 copies/μL plasmid DNA, the detection limit of Bonamia was 100 copies/μL plasmid DNA.The endonuclease digestion of duplex LAMP products certified the confidence of the established duplex LAMP method.The detection result of 20 Ruditapes philippinarum samples collected randomly from the East China sea and Yellow sea and 10 Ostrea edulis samples imported from USA showed that the duplex LAMP method could be used as a rapid detection method for infection with Perkinsus and Bonamia.

Key words: shellfish parasitic disease; loop-mediated isothermal amplification (LAMP); detection; Perkinsus; Bonamia

中图分类号:

王彩霞, 冯春燕, 王巧黎, 袁向芬, 邓俊花, 吕继洲, 吴绍强. 同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立[J]. , 2015, 42(8): 1935-1942.

WANG Cai-xia, FENG Chun-yan, WANG Qiao-li, YUAN Xiang-fen, DENG Jun-hua, LV Ji-zhou, WU Shao-qiang. Establishment of Duplex Loop-mediated Isothermal Amplification Method for Simultaneous Detection of Shellfish Infected with Perkinsus and Bonamia[J]. , 2015, 42(8): 1935-1942.

参考文献

[1] Wu S Q,Wang C X,Lin X M,et al.Infection prevalence and phylogenetic analysis of Perkinsus olseni in Ruditapes philippinarum from East China[J].Dis Aquat Organ,2011,96:55-60.
[2] 谢丽基,谢芝勋,庞耀珊,等.贝类单孢子虫和折光马尔太虫二重PCR检测方法的建立[J].动物医学进展,2010,31(1):54-57.
[3] 吴绍强,李海燕,林祥梅,等.贝类派琴虫实时荧光定量PCR检测方法的建立和应用[J].渔业科学进展,2009,30(5):58-63.
[4] 谢丽基,谢芝勋,庞耀珊,等.三种贝类原虫多重PCR检测方法的建立[J].中国兽医科学,2009,39(12):1080-1083.
[5] 中华人民共和国国家质量监督检验检疫总局.GB/T 22618—2011 派琴虫病诊断操作规程[S].
[6] 中华人民共和国国家质量监督检验检疫总局.SN/T 2434—2010 贝类包拉米虫病检疫技术规范[S].
[7] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[8] 王彩霞,林祥梅,吴绍强.环媒恒温扩增技术及其在疫病检测中的应用[J].检验检疫学刊,2009,19(4):66-69.
[9] Kasahara K,Ishikawa H,Sato S,et al.Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products[J].FEMS Microbiol Lett,2014,357(2):208-216.
[10] Mahony J,Chong S,Bulir D,et al.Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes[J].J Clin Microbiol,2013,51(8):2696-2701.
[11] Ray S M.A culture technique for the diagnosis of infections with Dermocysti dium marinum.Mackin,Owen,and Collier,in oysters[J].Science,1952,116:360-361.
[12] OIE.Manual of diagnostic tests for aquatic animals[S].2014.
[13] Feng C Y,Lin X M,Wang F,et al.Detection and characterization of Bonamia ostreae in Ostrea edulis imported to China[J].Dis Aquat Organ,2013,106(1):85-91.
[14] 郭书林,陈信忠.重要经济贝类原虫病及其诊断技术研究进展[J].水产科学,2013,32(2):110-116.
[15] Feng C Y,Wang C X,Lin X M,et al.Development of a loop-mediated isothermal amplification method for detection of Perkinsus spp.in mollusks[J].Dis Aquat Organ,2013,104(2):141-148.
[16] Nickens A D,La Peyre J F,Wagner E S,et al.An improved procedure to count Perkinsus marinus in eastern oyster hemolymph[J].J Shellfish Res,2002,21(2):725-732.
[17] 李海艳,吴绍强.牡蛎包纳米虫病的研究进展[J].中国畜牧兽医,2008,35(10):104-107.
[18] Liu X L,Zhao X T,Muhammad I,et al.Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum[J].J Virol Methods,2014,210C:26-31.

同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立

Establishment of Duplex Loop-mediated Isothermal Amplification Method for Simultaneous Detection of Shellfish Infected with Perkinsus and Bonamia

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈吉敏, 李辉煌, 赖连杰, 杨银华, 杨悦, 林瑞意. 鸭坦布苏病毒检测方法及疫苗研究进展[J]. 中国畜牧兽医, 2023, 50(4): 1329-1339.
[2]	和琳, 韩佃刚, 李静, 杜萱, 张家翔, 信吉阁. 牛病毒性腹泻病毒检测方法研究进展[J]. 中国畜牧兽医, 2023, 50(4): 1593-1602.
[3]	张庆勋, 钟震宇, 郭青云, 何宏轩, 白加德. 基于CRISPR-Cas系统的病原体检测研究进展[J]. 中国畜牧兽医, 2022, 49(8): 3190-3199.
[4]	王迁迁, 于永乐, 李月华, 董雅琴, 尼博, 刘拂晓. A型塞尼卡病毒检测方法及疫苗研究进展[J]. 中国畜牧兽医, 2022, 49(6): 2336-2346.
[5]	郭銮英, 李家磊, 金芹芹, 马骏, 刘全. 牛丙型肝炎病毒研究进展[J]. 中国畜牧兽医, 2022, 49(5): 1994-2000.
[6]	梁倩, 包涛涛, 鲜思美, 杨倩, 李鹏飞, 顾庆林, 郑维豪, 杜鹏, 青成欣. 羊口疮病毒PMA-PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(4): 1524-1531.
[7]	李思, 蒋蔚, 程婷婷, 卢春慧, 辛思培, 曹敬政, 王权, 孙卫东. 沙拉沙星间接竞争化学发光酶联免疫检测方法的建立[J]. 中国畜牧兽医, 2021, 48(5): 1772-1783.
[8]	李新苹, 陈晓洁, 赵兵, 刘强德, 丁占强, 张飞, 唐慧芬, 曹剑, 侯凤, 刘洋, 王钢. 气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2021, 48(5): 1876-1882.
[9]	胡瑞瑞, 刘莉, 郭涛, 李雅心, 王小奎, 胡圣伟. 绵羊BMP15基因B2突变可视化检测技术的建立[J]. 中国畜牧兽医, 2021, 48(3): 810-817.
[10]	吴帅斌, 崔泽华, 廖晓萍, 刘雅红, 孙坚. 产碳青霉烯酶肠杆菌科细菌检测方法研究进展[J]. 中国畜牧兽医, 2021, 48(3): 1072-1083.
[11]	杨松鑫, 冯建远, 郭旋, 韦柳源, 梁翼莹, 严昊, 张子仪, 陈海兰. 小分子化合物单克隆抗体的制备及其在动物医学领域的应用[J]. 中国畜牧兽医, 2021, 48(3): 1121-1131.
[12]	石美奕, 杨发龙. RPA技术及其在动物病原检测中的应用[J]. 中国畜牧兽医, 2021, 48(2): 467-476.
[13]	严昊, 冯建远, 张子仪, 杨松鑫, 徐春雨, 韦萌, 黄清萍, 陈海兰. 纳米抗体的制备与临床应用研究进展[J]. 中国畜牧兽医, 2021, 48(2): 685-694.
[14]	尹永康, 王晓亮, 崔艳丽, 梁晓. 禽类产品及饲料中尼卡巴嗪药物检测分析方法研究进展[J]. 中国畜牧兽医, 2021, 48(12): 4725-4734.
[15]	李志强, 陶晨雨, 魏奥龙, 贾青. 猪性控精子纯度实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2021, 48(10): 3682-3690.