虾类十足目虹彩病毒1(DIV1)LAMP-HNB快检方法的建立

Establishment of LAMP-HNB Rapid Detection Method for Decapod Iridescent Virus 1(DIV1)of Shrimp

[目的]建立一种操作简便、快速、可视化的虾类病原检测方法,实现基层养殖户自检。[方法]针对十足目虹彩病毒1(DIV1),设计环介导等温扩增(LAMP)特异性引物,对反应试剂用量、温度、反应时间等条件进行优化,建立LAMP与羟基萘酚蓝(HNB)结合的LAMP-HNB可视化快速检测方法。[结果]在LAMP反应体系中添加12 mmol/L Mg^(2+)和250μmol/L HNB,65℃恒温条件下反应60 min最佳;DIV1病原质粒最低检出限为102 copies/μL,稳定性和特异性较好,通过带有靶标基因的阳性质粒进行16次重复性试验结果均较为稳定,多种模板扩增试验均未发生假阳性情况,符合试验要求。[结论]该研究建立的LAMP-HNB可视化快速检测方法,适用于上述对虾DIV1的现场快速检测。

[Objective]To establish a simple,rapid and visual pathogen detection method for shrimp,and to achieve self detection by fishermen.[Method]The specific primers of LAMP(loop-mediated isothermal amplification technique)were designed for decapod iridescent virus 1(DIV1).After optimizing the conditions of reagent dosage,temperature and reaction time,a visual and rapid detection method of LAMP combined with hydroxynaphthol blue(HNB)was established.[Result]12 mmol/L Mg^(2+)and 250μmol/L HNB solution in the LAMP reaction system was the most suitable condition at 65℃for 60 min.The minimum detection limits of LAMP-HNB of DIV1 was 102 copies/μL plasmid.The stability and specificity of the detection of DIV1 had also been verified using positive plasmids with target genes,and the results of 16 repeated experiments were relatively stable,multiple template amplification experiments did not show false positives,which meets the experimental requirements.[Conclusion]The LAMP-HNB visualization rapid detection method established in this study is suitable for the on-site rapid detection of shrimp DIV1 mentioned above.