哈维氏弧菌RT-LAMP检测方法建立与应用

哈维氏弧菌(Vibrio harveyi)是引起海南省后水湾深水网箱养殖卵形鲳鲹(Trachinotus ovatus)"烂身病"的主要病原菌。为了能够快速诊断该病原菌,急需建立一种耗时短、准确以及便捷的检测方法。本研究利用分离到的致病菌株QT520的ToxR基因序列设计特异性引物和加入SYTO-9特异荧光染料,建立了一种可以实时、快速检测哈维氏弧菌的LAMP法(RT-LAMP)。该方法对哈维氏弧菌的基因组DNA及菌液灵敏度分别为100 fg/μL和10~3 cfu/mL,与Real-time PCR法检测灵敏度相当,比普通PCR的检测灵敏度要分别高1000倍和10倍,能有效区分哈维氏弧菌与坎氏弧菌(Vibrio campbellii);可以实时观察检测结果,且检测时间只需要40 min;具有耗时短、特异性和灵敏度高、仪器便携、操作简单且能实时观察检测结果等优点,非常适合在生产现场进行哈维氏弧菌的检测。

Development and application of real-time fluorescence loop mediated isothermal amplification for detection of Vibrio harveyi

Vibrio harveyi is considered a major pathogen of the golden pompano (Trachinotus ovatus) causing rot body disease in the deep-sea cage aquaculture area in Houshui Bay, 2016. Building a more rapid, accurate, and convenient technical detection analysis rather than the conventional methods, for example, physiological and biochemical identification, 16S rRNA gene sequence analysis, and immunodiagnostic technique, which are labor-intensive and time-consuming, is of great importance to disease prevention and control. According to the principle of the LAMP method, real-time loop-mediated isothermal amplification (RT-LAMP) techniques were developed for detection of fish pathogenic V. harveyi through designing specific primers based on ToxR genes of V. harveyi QT520 after adding fluorescent dye SYTO-9 to the reaction system. The results showed that this method could effectively distinguish the V. harveyi and V. campbellii, and the detection limit of this method for V. harveyi with both of pure cultures and DNA concentrations were found to be 10³ CFU/reaction and 100 fg/uL, being equivalent to real-time PCR (RT-PCR), which was 1000 and 10 times that of general PCR, respectively. Furthermore, the detection process through this method only took 40 min and the results could be displayed on the LCD panel. Thus, it has high specificity, high sensitivity, and the interpretation of results is simple and objective. Furthermore, it has simple operation characteristics, being suitable for application and promotion in basic levels of fishery technology promotion departments and fishery professional cooperatives.