China and Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
Pest Manag Sci. 2022 Feb;78(2):821-830. doi: 10.1002/ps.6697. Epub 2021 Nov 15.
Benzimidazole resistance in Botrytis cinerea is related to point mutations in the target β-tubulin gene (TUB2). Three mutations (E198A, E198K, E198V) at codon 198 account for most of the resistant strains. A rapid on-site diagnostic assay would be useful to detect the presence and monitor further spread of this resistance mechanism.
A recombinase polymerase amplification combined with lateral flow detection (RPA-LFD) method was established for the rapid detection of methyl benzimidazole carbamate (MBC) resistance in B. cinerea. Based on the three mutations at TUB2 codon 198, three sets of RPA-LFD primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation; no amplification was detected with other mutated or wild-type DNA. The assay was optimized for specificity and sensitivity and was shown to detect the presence of 2 × 10 copies μl of target DNA per reaction within 10 min. DNA from eight other common fungal species of small fruit did not yield a signal. The system worked well over a wide range of temperatures from 25 to 45°C. Crude DNA obtained from boiled mycelium and conidia of symptomatic fruit could be used as templates, which simplified the assay process.
This study developed a novel assay based on RPA-LFD for the rapid and equipment-free detection of MBC-resistant isolates. In combination with a simple DNA extraction method, the assay could detect B. cinerea MBC-resistant isolates even without specialized equipment within 30 min. Considering its specificity, stability and simplicity, the RPA-LFD assay could be a promising tool for rapid on-site diagnosis of fungicide-resistant isolates. © 2021 Society of Chemical Industry.
灰葡萄孢菌对苯并咪唑类杀菌剂的抗性与靶标β-微管蛋白基因（TUB2）的点突变有关。密码子 198 处的三个突变（E198A、E198K、E198V）导致了大多数抗性菌株的出现。一种快速现场诊断检测方法将有助于检测这种抗性机制的存在并监测其进一步传播。
建立了一种重组酶聚合酶扩增结合侧流检测（RPA-LFD）方法，用于快速检测灰葡萄孢菌中甲基苯并咪唑氨基甲酸酯（MBC）的抗性。基于 TUB2 密码子 198 处的三个突变，设计了三组 RPA-LFD 引物，每组引物都能够特异性扩增含有其相应突变的 DNA；而其他突变或野生型 DNA 则没有扩增。该检测方法经过优化，具有特异性和灵敏度，在 10 分钟内可检测到每个反应中存在 2×10 拷贝μl 的目标 DNA。来自其他 8 种小型水果常见真菌物种的 DNA 没有产生信号。该系统在 25 至 45°C 的宽温度范围内运行良好。来自煮沸的菌丝和有症状果实的孢子的粗提 DNA 可以用作模板，简化了检测过程。
本研究开发了一种基于 RPA-LFD 的新型检测方法，用于快速、无需设备检测 MBC 抗性分离株。结合简单的 DNA 提取方法，该检测方法甚至可以在没有专用设备的情况下，在 30 分钟内检测到灰葡萄孢菌 MBC 抗性分离株。考虑到其特异性、稳定性和简单性，RPA-LFD 检测方法可能成为快速现场诊断杀菌剂抗性分离株的有前途的工具。
