Establishment of Real-Time Fluorescence Detection Method for Carnation Etched Ring Virus

<i>Carnation etched ring virus</i> (CERV; genus <i>Caulimovirus</i>, family Caulimoviridae) is a destructive plant pathogen subject to stringent global phytosanitary regulations due to its severe impact on carnation production. As a quarantine organism in multiple countries, including Madagascar and Peru, CERV poses significant risks to international horticultural trade, particularly through asymptomatic infections in propagated planting materials. Current diagnostic challenges, such as the limitations of conventional PCR in balancing speed and sensitivity, underscore the urgent need for robust detection tools to prevent transboundary spread. This study aimed to develop a rapid, sensitive, and specific TaqMan-based real-time PCR assay to enhance phytosanitary screening during port inspections. Targeting a conserved region within the CERV genome (GenBank AJ853858.1, positions 1500–1808), two primer pairs and four probes were systematically evaluated to optimize detection efficiency. The finalized assay demonstrated a sensitivity threshold of 3×10³ copies/μL, comparable to conventional end-point PCR, while significantly reducing processing time. Specificity testing confirmed no cross-reactivity with taxonomically related viruses, including <i>Carnation ringspot virus</i>, <i>Cowpea mosaic virus</i>, <i>Cauliflower mosaic virus</i>, and <i>Carnation latent virus</i>, ensuring reliable discrimination. Thermal cycling conditions were streamlined to a 40-cycle protocol with denaturation at 95°C (10 s), annealing at 56°C (15 s), and extension at 60°C (20 s), enabling completion within 90 minutes. This advancement provides a high-throughput solution for regulatory agencies to intercept contaminated consignments efficiently, addressing critical gaps in existing phytosanitary frameworks. By combining rapid turnaround with robust accuracy, the assay strengthens global efforts to safeguard carnation cultivation from CERV-induced losses. Its implementation in trade inspections is particularly vital for detecting latent infections in asymptomatic plant tissues, a major route of pathogen dissemination. The study underscores the importance of molecular innovation in supporting sustainable agriculture and international biosecurity networks, advocating for the integration of such tools into standardized phytosanitary protocols.