Development and evaluation of an isothermal recombinase polymerase amplification–lateral flow assay for rapid detection of strawberry vein banding virus in the field. (August 2022) - Zou, Xiaohua
Abstract: The disease caused by strawberry vein banding virus (SVBV) is one of the main virus diseases affecting the growth and development of strawberries, and the development of an on-site method for rapid detection of SVBV would be useful for breeding virus-free strawberry seedlings. In this study, we used isothermal recombinant polymerase amplification (RPA) combined with lateral flow strip (LF strip) testing to achieve rapid diagnosis of SVBV following on-site rapid DNA extraction. The DNA target for the SVBV-RPA amplification was the specific nucleotide region, which is conserved between different SVBV isolates in China. This assay allowed detection of SVBV within 30 min, comprising rapid N3 DNA extraction for 10 min, DNA amplification with RPA for 15 min at 40 °C, and visualization of the amplicons by LF strip for 5 min. The conservative detection limit was as low as 10 pg DNA under optimized conditions. Furthermore, the diagnostic results obtained by SVBV-LF-RPA on-site for detection of suspected samples were consistent with those obtained by polymerase chain reaction (PCR), achieving the aim of highly sensitive on-site rapid diagnosis of most isolated SVBV strains. The system was successfully applied and validated for on-site detection of SVBV in the production of strawberry virus-free seedlings in Shanghai, demonstrating its suitability for on-site diagnosis of SVBV and high potential for application to SVBV detection nationwide. Highlights: The conserved specific primers and probe screened out among most of SVBV isolates from different regions in China. The on-site diagnosis of SVBV-LF-RPA assay established with stable repeatability and optimized reaction conditions. The detection limit of SVBV-LF-RPA assay more sensitive than the conventional PCR assay. The practicability of SVBV-LF-RPA assay effectively evaluated by the key suspected samples in field.