Rapid and visual detection of Verticillium dahliae using recombinase polymerase amplification combined with lateral flow dipstick

Abstract

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes verticillium wilt disease in cotton, resulting in reduced fiber quality and serious economic losses. However, there is still no efficient management strategies that have been developed to control the disease. Therefore, rapid and accurate detection of V. dahliae would be invaluable in disease control programs. In this study, an assay consisting of recombinase polymerase amplification combined with lateral-flow dipstick technology (RPA-LFD) was developed for the rapid and sensitive detection of V. dahliae. The RPA-LFD assay was completed at the isothermal temperature of 37 °C within 25 min. The RPA primers and LF probe exhibited high specificity to V. dahliae. The detection limit of RPA-LFD was 103 fg fungal genomic DNA in a 50 μL reaction volume and 103 spores per gram using artificially inoculated soil samples, whereas conventional PCR detected as low as 103 fg DNA and 104 spores. Additionally, the RPA-LFD assay detected V. dahliae in field soil samples, showing no significant differences compared to conventional PCR. The simple, rapid, and practical nature of the RPA-LFD assay suggests that it will serve as a promising molecular diagnostic tool for the accurate and rapid detection of V. dahliae.