Development of a Multiplex RT-PCR Assay for Simultaneous Detection of Velarivirus arecae, Arepavirus arecae and Arepavirus arecamaculatum

Areca Palm Velarivirus 1 (<i>Velarivirus arecae</i>, APV1), Areca palm necrotic ringspot virus (<i>Arepavirus arecae</i>, ANRSV), and Areca palm necrotic spindle-spot virus (<i>Arepavirus arecamaculatum</i>, 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. Specific primers were designed based on the <i>Coat Protein</i> (CP) gene sequences of the three target viruses: APV1. A specific primer pair targeting the <i>coat protein</i> (CP) region was designed for APV1, while primer pairs for ANRSV and ANSSV were designed based on conserved sequences surrounding the Nla-VPg/Nla-Pro protease cleavage sites. A multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR) assay was subsequently developed to simultaneously amplify the target sequences. The multiplex RT-PCR detection system was optimized by adjusting critical parameters, including the annealing temperature, extension time, and number of cycles, to ensure high specificity and sensitivity. The optimized multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR) successfully yielded distinct amplification products for all three target viruses: 938 bp for APV1, 527 bp for ANRSV, and 250 bp for ANSSV. The size differences among the amplicons allowed them to be clearly distinguishable by 2% agarose gel electrophoresis. The optimal reaction conditions were determined to be an annealing temperature of 53.4 °C and 35 cycles. Applying the optimized multiplex RT-PCR method, we analyzed 414 field samples collected from Hainan province. APV1 was identified as the most prevalent virus, detected in 22.71% of the total samples. ANRSV and ANSSV were detected at significantly lower rates, in 3.86% and 0.2% of the samples, respectively. 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%. 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.