哈维氏弧菌可视化LAMP快速检测方法的建立及应用

Establishment and Application of Rapid Visual LAMPMethod for Detection of Vibrio harveyi

【目的】为加强对哈维氏弧菌(Vibrio harveyi)的早期快速诊断和筛查,探究一种快速检测方法。【方法】以哈维氏弧菌外膜通道蛋白基因Toic为靶基因,以钙黄绿素为检测结果指示剂,通过对反应体系、反应时间和温度进行优化,并检测其特异性和灵敏度,建立一种针对哈维氏弧菌的可视化环介导等温基因扩增(LAMP)检测方法。【结果】该方法的检测时间最快为35 min,灵敏度为100 fg/mL,特异性强,与8种弧菌及金黄色葡萄球菌均无交叉反应。实用性检测结果显示,该方法可准确并特异性地检测到鱼体组织中感染的哈维氏弧菌。【结论】建立一种设备简单、操作简单、反应快速、特异性强、灵敏度高、结果可视化的哈维氏弧菌LAMP快速检测方法,且适用于石斑鱼(Epinephelus)感染哈维氏弧菌的早期快速诊断,为进一步推广该技术在石斑鱼及其他水生动物哈维氏弧菌感染监测中的现场大规模应用提供技术支撑。

【Objective】In order to strengthen the early and rapid diagnosis and screening of Vibrio harveyi,a rapid detection method was explored.【Method】We established a rapid visual loop-mediated isothermal amplification(LAMP)method for detection of V.harveyi by choosing the outer membrane channel protein gene Toic of V.harveyi as the target gene,designating the calcein as the indicator of detection results,optimizing the reaction system,reaction time and reaction temperature,and detecting its specificity and sensitivity.【Results】The shortest detection time was 35 min.The detection sensitivity was 100 fg/mL.The specificity of the LAMP method was strong and no cross-reaction with other 8 kinds of Vibrio and Staphylococcus aureus.The practical test results showed that this LAMP method could accurately and specifically detect V.harveyi in fish tissues infected with V.harveyi.【Conclusion】This study successfully established a visual LAMP method for rapid detection of V.harveyi with simple equipment,simple operation,rapid reaction,strong specificity,high sensitivity,and visual results.This method is suitable for the early and rapid diagnosis of V.harveyi infection in Epinephelus,which provides technical support for further popularizing this technology in the field of disease monitoring of grouper and other aquatic animals.