Xiao Caiyun, Li Rongyu
Institute of Crop Protection, Guizhou University, Guiyang 550025, China.
The Provincial Key Laboratory for Agricultural Pest Management in Mountainous Region, Guiyang 550025, China.
Biology (Basel). 2021 Nov 5;10(11):1136. doi: 10.3390/biology10111136.
Soft rot causing is one of the most destructive diseases of Kimura et Migo in China that reduces yield and quality. A key challenge for an integrated management strategy for this disease is the rapid and accurate detection of on . Therefore, a new loop-mediated isothermal amplification (LAMP) assay was developed for this purpose. In this study, the primers were selected and designed using the translation elongation factor-1α () gene region as the target DNA sequence in order to screen the best system of reaction of LAMP to detect through optimizing different conditions of the LAMP reaction, including time, temperature, concentrations of MgSO, and concentrations of inner and outer primers. The optimized system was able to efficiently amplify the target gene at 62 °C for 60 min with 1.2 μM internal primers, 0.4 μM external primers, 7 mM Mg, and 5 fg/µL minimum detection concentration of DNA for . The amplified products could be detected with the naked eye after completion of the reaction with SYBR green I. We were better able to control the effect of soft rot in using fungicides following a positive test result. Additionally, the control effect of synergism combinations against soft rot was higher than 75%. Thus, LAMP assays could detect in infected tissues of and soils in field, allowing for early diagnosis of the disease.
软腐病是中国荸荠上最具毁灭性的病害之一，会降低荸荠的产量和品质。针对这种病害的综合管理策略的一个关键挑战是在荸荠上快速准确地检测到[病原菌名称未明确]。因此，为此开发了一种新的环介导等温扩增（LAMP）检测方法。在本研究中，选择并设计了以翻译延伸因子-1α（[具体英文名称未明确]）基因区域作为目标DNA序列的引物，通过优化LAMP反应的不同条件，包括时间、温度、MgSO浓度以及内、外引物浓度，来筛选检测[病原菌名称未明确]的最佳LAMP反应体系。优化后的体系能够在62℃下60分钟内高效扩增目标基因，内引物浓度为1.2μM，外引物浓度为0.4μM，Mg浓度为7mM，[病原菌名称未明确]的DNA最低检测浓度为5fg/µL。反应完成后，用SYBR green I可肉眼检测到扩增产物。检测呈阳性结果后，使用杀菌剂能更好地控制荸荠上软腐病的病情。此外，协同组合对软腐病的防治效果高于75%。因此，LAMP检测方法能够检测荸荠感染组织和田间土壤中的[病原菌名称未明确]，实现对该病的早期诊断。
