Establishment and Application of LAMP Rapid Detection Assay for Stephylium solani
XIE Xue-Wen1,*, LIU Shi-Cheng1,*, SHI Yan-Xia1, ZHANG Sheng-Feng2, CHEN Li-Da1, CHAI A-Li1, FAN Teng-Fei1, LI Lei1,**, LI Bao-Ju1,**
1 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 Xuzhou Tairui Agricultural Technology Co., Ltd., Xuzhou 221133, China
Abstract：Stephylium solani is an important plant pathogenic fungus with a wide range of hosts. Loop-mediated isothermal amplification (LAMP) technology has been widely used in many fields such as plant protection because of its strong specificity, high sensitivity, rapid response and low cost. To establish a rapid, efficient and accurate detection technology for Lycopersicon esculentum petiole leaf spot, SYBR GreenⅠwas used as the indicator and the glyceraldehyde-2-phosphate dehydrogenase (gpd) gene was used as the target. 6 primers (2 inner primers FIP/BIP, 2 outer primers F3/B3 and loop primer LB25/LF25) were designed for LAMP amplification. At the same time, a set of rapid LAMP detection system for S. solani was established by accelerating the reaction with loop primers. The results of specificity verification showed that only 16 strains of S. solani had positive amplification and had high specificity. The sensitivity results showed that the LAMP detection sensitivity of the system was 2.4×103 fg/μL, significantly higher than the PCR detection technology. The results showed that the addition of ring primers could shorten the reaction time to 27 min, and effectively accelerate the LAMP reaction. Through the color reaction of calcein, under natural light, only the reaction solution with the template of S. solani turned green, and the other reaction solutions were orange. The system was used to detect the dynamic changes of pathogen content in tomato leaves after inoculation with S. solani. 12 h after inoculation, the LAMP detection had a positive color reaction, and the color gradually became darker with time, which was consistent with the severity of tomato leaves after artificial inoculation. The reaction results can be directly observed. The loop primer accelerates the reaction to further shorten the detection time to 27 min, which can quickly and accurately reflect the pathogen content. The LAMP detection system established in this study has strong advantages in the rapid detection of S. solani. It can be used for the rapid detection of latent and non symptomatic stages of S. solani. It is of great significance for the timely and scientific control of tomato petioles leaf spots.
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