Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of Papaya Leaf Curl Guangdong Virus in Passiflora edulis

Passionfruit leaf curl Guangdong virus (PaLCuGdV) is an emerging begomovirus threatening passionfruit production. We established a rapid and simple diagnostic platform based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology for reliable detection of PaLCuGdV. Initial polymerase chain reaction (PCR) and reverse transcription-PCR analyses of symptomatic passionfruit leaves identified double infections with PaLCuGdV and euphorbia leaf curl virus (EuLCV), or triple infections with PaLCuGdV, EuLCV, and cucumber mosaic virus (CMV), which were confirmed by Sanger sequencing. Optimization experiments defined 38°C and 15 min as the optimal RPA conditions, yielding consistent amplification between 30°C and 46°C. Sensitivity assays showed that RPA detected PaLCuGdV DNA at concentrations as low as 100 pg total DNA concentration, representing a 10-fold improvement compared with PCR, while maintaining strict specificity for PaLCuGdV in mixed infections without cross-reactivity to EuLCV or CMV. Incorporation of LFS into the assay enabled simple, equipment-free detection, producing results consistent with PCR and suitable for on-site diagnostics by non-specialists. Tissue distribution analyses revealed the presence of PaLCuGdV in all fruit tissues, including exocarp, mesocarp, endocarp, and seeds. PaLCuGdV was consistently detected in both seed coats and cotyledons, indicating a high potential for mode of seed transmission. Together, these results establish RPA-LFS as a sensitive, specific, and portable diagnostic platform for PaLCuGdV, with broad applicability for field-scale surveillance and seed health testing in passionfruit production systems.