Fall Jean, Chakraborty Gunimala, Kono Tomoya, Maeda Minoru, Itami Toshiaki, Sakai Masahiro
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan.
FEMS Microbiol Lett. 2008 Nov;288(2):171-7. doi: 10.1111/j.1574-6968.2008.01332.x.
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.
环介导等温扩增（LAMP）是一种在等温条件下以高特异性和快速性扩增DNA的新方法。在本研究中，使用LAMP方法开发了一种用于检测虾中黑美人弧菌的诊断方案。黑美人弧菌与明显的虾病（弧菌病）有关，被认为是虾类养殖业中的威胁性病原体之一。在对黑美人弧菌16S和23S rRNA基因之间的基因间隔区（ITS）进行初步克隆和测序后，设计了一组四个引物——两个内引物和两个外引物——用于LAMP反应。反应时间和温度分别在63℃下优化为60分钟。LAMP对黑美人弧菌的检测限为10² CFU mL⁻¹，但聚合酶链反应（PCR）可检测到高达10³ CFU mL⁻¹。LAMP方法可以从实验感染黑美人弧菌的虾的心脏、淋巴器官和肌肉中检测到黑美人弧菌的存在。本研究建立了一种用于检测虾中黑美人弧菌的高度灵敏且快速的诊断程序。本研究中开发的方法对于虾病的常规诊断可能非常有用。
