Development of Loop-Mediated Isothermal Amplification Assays for Rapid and Sensitive Detection of Three Viruses...

Yellow leaf disease (YLD) is a major threat to the areca palm (Areca catechu L.) industry. Accumulating evidence indicates that areca palm velarivirus 1 (APV1) is a key pathogen associated with YLD. In addition, areca palm latent totivirus 1 (APLTV1) and areca palm yellow leaf-associated ormycovirus (APYLaOMV) are two recently reported viruses that frequently co-infect areca palms. These three viruses can infect betel palms either individually or in combination, and their associated symptoms cannot be reliably distinguished by visual observation alone. In this study, through primer screening and systematic optimization of key factors affecting amplification efficiency, three independent one-step RT-LAMP assays were established for the detection of APV1, APLTV1, and APYLaOMV. The Mg²⁺ and dNTP concentrations, reaction temperatures, and inner-to-outer primer ratios were optimized to give detection limits of 10^-3 for APYLaOMV, 10^-4 for APLTV1, and 10^-5 for APV1. The assays showed no cross-reactivity with the non-target areca palm viruses ANSSV and ANRSV, and were further validated on 26 field samples collected from Hainan Province, where their detection rates were comparable to or higher than those of conventional RT-PCR. Together, these results demonstrate that the developed RT-LAMP assays are sensitive and practical tools for routine molecular diagnosis and epidemiological investigation of APV1, APLTV1, and APYLaOMV in areca palms.