LAUSR

BACKGROUND Benzimidazole resistance in B. cinerea is related to the 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. RESULTS A recombinase polymerase amplification combined with lateral flow stripes (RPA-LFD) method was established for the rapid detection of benzimidazole 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, while 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 2×102 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 at 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. CONCLUSION This study developed a novel assay based on lateral flow strip-based recombinase polymerase amplification (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 fungicides resistant isolates. This article is protected by copyright. All rights reserved.