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

【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。

[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 reac-tion 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 sam-ples. [Results] The method of a loop-mediated isothermal amplification （LAMP） for detection of Saprolegnia was established. The sensitivity was 10^3 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 Sapro- legnia and its spores.