Development and validation of a real-time RT-qPCR assay for the detection of citrus yellow vein clearing virus

), is an emerging pathogen that poses a threat to citrus production worldwide. To address this threat, three reverse transcription quantitative PCR (RT-qPCR) detection assays were developed and validated using all currently available public sequences and in-house high-throughput sequencing data from recent CYVCV detections in California (CA), following guidelines for quantitative and qualitative real-time PCR experiments. Among the RT-qPCR assays tested, the CYVCV-4 outperformed the others in analytical sensitivity, specificity, and diagnostic accuracy, demonstrating high amplification efficiency. The CYVCV-4 assay targets the RNA-dependent RNA polymerase gene of CYVCV and demonstrated high specificity by avoiding cross-reactivity and accurately identifying infected, noninfected, and nontarget citrus pathogens. Its superior performance is attributed to optimized primer and probe design, enabling specific hybridization to the target sequence while minimizing nonspecific amplification. The assay's robustness, interassay and intra-assay variation, and reproducibility were thoroughly validated across multiple laboratories, under varying reaction conditions, and different qPCR instruments. Recent detections of CYVCV in CA underscore the need for rapid and reliable diagnostics to protect citrus production and germplasm. The development and validation of the CYVCV-4 assay demonstrate its effectiveness through comprehensive testing, supporting its use in citrus diagnostic laboratories, quarantine programs, and field surveys, ultimately enhancing CYVCV management efforts. This development highlights the opportunity for plant diagnostics to adopt harmonized validation practices through frameworks like the Diagnostic Assay Validation Network, crucial for national programs, such as the National Clean Plant Network, which depend on validated assays to ensure clean plant systems and agricultural biosecurity.