The title Development of a multiplex RT‑PCR assay for simultaneous detection of Areca palm velarivirus 1, Areca palm necrotic ringspot virus and Areca plam necrotic spindle-spot virus

Areca palm velarivirus 1 (APV1), Areca palm necrotic ringspot virus (ANRSV), and Areca palm necrotic spindle-spot virus (ANSSV) are major viral pathogens that cause significant economic losses in areca palm cultivation. Rapid and reliable detection methods are essential for the early diagnosis and management of these viruses in affected regions. In this study, a one-step multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR) assay was developed for the simultaneous detection of APV1, ANRSV, and ANSSV. Three pairs of specific primers were designed from conserved genomic regions of each virus, generating amplification products of 938 bp for APV1, 527 bp for ANRSV, and 250 bp for ANSSV. The PCR products were clearly distinguishable by 2% agarose gel electrophoresis. Optimal amplification conditions were determined to be 53.4 °C for annealing temperature and 35 cycles. Subsequently, the established multiplex RT-PCR detection method was applied to areca leaf samples collected from the main areca planting areas in Hainan. This method enabled efficient and accurate identification of single and mixed infections in field samples. Virus detection in areca samples from Hainan Island revealed clear regional differences in disease incidence, with higher rates in the eastern and central regions—particularly Baoting, Lingshui, Wanning, and Qionghai—averaging 46.73%. A decreasing trend in severity was observed from east to west, with milder symptoms in areas like Danzhou and Baisha. Together, these results demonstrate that the developed multiplex RT-PCR is a sensitive and practical tool for the routine molecular diagnosis and epidemiological investigation of APV1, ANRSV, and ANSSV in areca palms.