Simultaneous detection of multiple sdhB SNPs in Botrytis cinerea using a universal quenching probe-based LAMP assay.

Succinate dehydrogenase inhibitors (SDHIs) are widely used to control Botrytis cinerea, but resistance caused by multiple point mutations in the sdh genes has become a major concern. To enable rapid and accurate identification of SDHI‑resistant genotypes, we developed a loop‑mediated isothermal amplification (LAMP) assay using a universal quenching probe (UQProbe LAMP) capable of simultaneously detecting multiple single nucleotide polymorphisms (SNPs) within the same target region. Using a single UQProbe and a common LAMP primer set, the method successfully distinguished four sdhB genotypes-wild type, H272R, H272Y, and H272V-based on characteristic melting temperatures (approximately 56 °C, 50 °C, 44.5 °C, and 38.5 °C, respectively). This single‑probe, multi‑SNP detection strategy eliminates the need for mutation‑specific primers or multiple probes, providing a simple and efficient workflow for SNP typing. The UQProbe LAMP system developed in this study offers a rapid, cost‑effective, and versatile platform for multiplex SNP discrimination, applicable not only to fungicide resistance monitoring but also to a wide range of genetic diagnostic applications requiring precise SNP detection.