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

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, LAMP primer sets were designed based on the conserved sequences of the three viruses. Through primer screening and optimization of key factors affecting amplification efficiency, including Mg²⁺ concentration, dNTP concentration, and outer-to-inner primer ratio, independent LAMP assay systems were separately established for each virus. The optimal LAMP conditions for APYLaoMV were 12 mM Mg²⁺, 1.7 mM dNTPs, an outer-to-inner primer ratio of 2:1, and a reaction temperature of 65 °C, with a detection limit of 10-3 dilution of the template cDNA. For APV1, the optimal system contained 10 mM Mg²⁺, 1.0 mM dNTPs, an outer-to-inner primer ratio of 4:1 at 65 °C, yielding a detection limit of 10-5 dilution. For APLTV1, the optimum conditions were 10 mM Mg²⁺, 0.8 mM dNTPs, an outer-to-inner primer ratio of 8:1, and a reaction temperature of 63 °C, giving a detection limit of 10-4 dilution. 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.