番石榴焦腐病菌LAMP检测方法的建立及应用

Development of a loop-mediated isothermal amplification assay for detection of Botryosphaeria rhodina

以钙黄绿素(calcein)为指示剂,建立了番石榴焦腐病菌(Botryosphaeria rhodina)的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)可视化快速检测方法。在该方法中,以番石榴焦腐病菌特异的核糖体转录间隔区(Internal transcribed spacer,ITS)序列为目的 DNA片段,设计4条引物(2条内引物、2条外引物),优化LAMP反应条件和反应体系,并进行特异性和灵敏度验证。研究确定最佳反应温度为64℃,反应时间1 h;特异性检测结果显示,15株不同地理来源的番石榴焦腐病菌和10份番石榴焦腐病病果组织LAMP产物均为阳性(绿色),而26株其它病原菌及10份健康番石榴果实组织LAMP产物均为阴性(橘黄色);灵敏度验证结果显示,该方法的检测灵敏度在DNA水平上可达到10 pg。实验结果表明,快速、准确、灵敏的LAMP检测方法适合基层部门及田间检测番石榴焦腐病菌使用。

A visual LAMP （Loop-mediated isothermal amplification） detection of Botryosphaeria rhodina was developed with calcein as a fluorescence indicator in this study. Based on the ITS （Internal transcribed spacer） sequence of B. rhodina, a set of four LAMP primers was designed. The reaction conditions were optimized and the specificity and sensitivity of LAMP were assayed. The results showed that the constant temperature of 64℃ for h was the optimum reaction condition of LAMP. Specificity assays showed that the amplification results of 15 isolates of B. rhodina from different background and 10 samples of Psidium guajava fruits infected by B. rhodina were positive, and that of the other 26 pathogens and 10 healthy fruits were negative. The detection limit of LAMP was 10 pg pure genomic DNA per 25μL reaction. The LAMP assay developed in this study was sim- ple, fast, sensitive and specific, and can be used to detect B. rhodina infected plant tissues in the field.