水霉菌环介导等温扩增检测方法的建立

Establishment of a loop-mediated amplification assay for the specific detection of Saprolegnia and its spores

摘要:

【目的】建立一种快速简单检测水霉病病原菌的方法。【方法】针对水霉菌ITS区基因序列设计4条特异性引物, 包括两条外引物和两条内引物, 优化反应条件, 观察检测结果。对该方法的特异性和敏感性进行研究。【结果】建立了环介导等温扩增技术检测水霉菌的方法, 确定了其最适反应条件。该方法能够检测到浓度低至103个/mL的水霉菌孢子, 其灵敏度是普通PCR方法的100倍。【结论】建立的检测水霉菌的LAMP技术, 具有操作简便快速等特点, 可用做特异性水霉及其孢子的快速鉴定。

Abstract:

[Objective] To develop a loop-mediated isothermal amplification (LAMP) method for detection of Saprolegnia. [Methods] Four specific LAMP primers (two inner primers and two outer primers) were designed by targeting ITS gene of Saprolegnia. After optimizing reaction condition, the positive results of LAMP assay were judged through visible green colour. To evaluate the sensitivity and specificity of the assay by detection of Saprolegnia spore samples. [Results] The method of a loop-mediated isothermal amplification (LAMP) for detection of Saprolegnia was established. The sensitivity was 103 spores per mL and was 100 times higher than normal PCR. [Conclusion] LAMP assay is specific and simple to operate without the need of special equipment, our assay presents a promising method for detection of Saprolegnia and its spores.