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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。
Using calcein as an indicator, a rapid visualization method of loop-mediated isothermal amplification (LAMP) of Botryosphaeria rhodina was established. In this method, four primers (two inner primers and two outer primers) were designed based on the internal transcribed spacer (ITS) sequence of Pyrococcus furcifera, and the LAMP reaction was optimized Conditions and reaction system, and specificity and sensitivity verification. The optimal reaction temperature was 64 ℃ and the reaction time was 1 h. The results of specificity test showed that LAMP products of 15 strains of Pyrococcus furostrob from different geographical origins and 10 pieces of Guafu disease were positive (green ). The LAMP products of 26 other pathogens and 10 healthy guava fruits were negative (orange). The sensitivity test showed that the detection sensitivity of this method could reach 10 pg at the DNA level. The experimental results show that the rapid, accurate and sensitive LAMP detection method is suitable for the grass-roots departments and fields to detect the use of Guava gorgren.