Establishment and application of a real-time fluorescence quantitative PCR assay for detecting EAPV in Passiflora edulis
TL;DR
The SYBR Green-based qPCR method established in this study demonstrated high specificity and sensitivity, providing a reliable tool for rapid EAPV diagnosis and epidemiological investigations.
Abstract
East Asian Passiflora virus (EAPV) is a significant viral pathogen prevalent across passionfruit cultivation regions in China and causes substantial economic losses to the passionfruit industry. The development of a sensitive, rapid, and accurate diagnostic method is essential for virus identification and epidemiological surveillance to support effective disease management strategies. In this study, a SYBR Green-based real-time quantitative PCR (qPCR) assay was developed for EAPV detection using a specific primer set targeting the viral coat protein (CP) gene. The assay was optimized with a primer concentration of 0.2 μmol/L and an annealing temperature of 60°C. The primers exhibited high specificity, generating a standard curve with an amplification efficiency of 90.9% and a coefficient of determination (R2) of 0.992. The limit of detection was 11.41 × 102 copies/μL, representing a 1000-fold greater sensitivity than conventional PCR. Moreover, viral accumulation was successfully detected in both inoculated and systemic leaves of passionfruit plants. In 2025, a total of 120 suspected virus-infected passionfruit samples were collected from Yunnan Province, China, and all samples tested positive for EAPV using the developed qPCR assay. Overall, the SYBR Green-based qPCR method established in this study demonstrated high specificity and sensitivity, providing a reliable tool for rapid EAPV diagnosis and epidemiological investigations.
