Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp
Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp
复制标题
DOI：
10.1111/j.1574-6968.2008.01332.x
复制
发表时间：
2008-11-01
影响因子：
2.1
通讯作者：
Sakai, Masahiro
中科院分区：
文献类型：
--
作者：
Fall, Jean;Chakraborty, Gunimala;Sakai, Masahiro
LAMP是一种在等温条件下高特异性、快速扩增DNA的新方法。本研究利用LAMP方法，建立了对虾中弧菌的快速检测方法。黑盘弧菌（Vibrio nigripulchritudo）是一种与虾病（弧菌病）相关的病原菌，被认为是威胁对虾养殖业的重要病原菌之一。在初始克隆和测序V. nigripulchritudo的16 S和23 S rRNA基因之间的基因间间隔区（ITS）后，设计了一组四个引物（两个内部引物和两个外部引物）用于LAMP反应。反应时间和温度分别优化为63 ℃下60 min。LAMP检测的最低检出量为10（2）CFU mL（-1），而PCR检测的最高检出量为10（3）CFU mL（-1）。LAMP方法可以从实验感染的对虾的心脏、淋巴器官和肌肉中检测到黑脉弧菌的存在。本研究建立了一种高灵敏度、快速检测对虾中黑盘弧菌的方法。本研究所建立的方法可用于常规虾病诊断。
LAMP is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a diagnostic protocol was developed for the detection of Vibrio nigripulchritudo in shrimps. Vibrio nigripulchritudo is associated with distinct shrimp diseases (vibriosis) and is considered one of the threatening pathogens in shrimp industry. After initial cloning and sequencing of the intergenic spacer region (ITS) between 16S and 23S rRNA genes of V. nigripulchritudo, a set of four primers - two inner and two outer - were designed for use in the LAMP reaction. Reaction time and temperature were optimized for 60 min at 63 degrees C, respectively. The detection limit of V. nigripulchritudo by LAMP was 10(2) CFU mL(-1) but PCR could detect up to 10(3) CFU mL(-1). The LAMP method could detect the presence of V. nigripulchritudo from heart, lymphoid organ, and muscle of experimentally infected shrimps with V. nigripulchritudo. This study established a highly sensitive and a rapid diagnostic procedure for detection of V. nigripulchritudo in shrimps. The method developed in this study could be very useful for routine shrimp disease diagnostics.