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ARS Home » Pacific West Area » Riverside, California » National Clonal Germplasm Repository for Citrus » Research » Publications at this Location » Publication #428236
Research Project: Citrus and Date Genetic Resource Conservation and Utilization

Location: National Clonal Germplasm Repository for Citrus

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

Author
item OSMAN, FATIMA - University Of California, Davis
item BODAGHI, SOHRAB - University Of California, Riverside
item Krueger, Robert
item SMITH, TAYLOR - Florida Department Of Agriculture And Consumer Services
item HODZIC, EMIR - University Of California, Davis
item MAPES, SAMANTHA - University Of California, Davis
item Abrahamian, Peter
item TIAN, TONGYAN - California Department Of Food And Agriculture
item Rivera Rivera, Yazmin
item VIDALAKIS, GEORGIOS - University Of California, Riverside

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/26/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Citrus yellow vein clearing virus (CYVCV, Potexvirus citriflavivenae) is an emerging pathogen of citrus, for which reliable assays are needed. This paper reports development of a real time (quantitative) polymerase chain reaction (qPCR) assay for detection of CYVCV. Four qPCR assays were developed, one of which proved superior to the other three and to a previously published assay, showing high sensitivity and specificity. The robustness, inter and intra assay variation, reproducibility, and transferability of the assay were rigorously assessed and validated across different laboratories, qPCR instruments, and reagent kits, demonstrating its practicality for widespread use in diagnostic settings.

Technical Abstract: Citrus yellow vein clearing virus (CYVCV, Potexvirus citriflavivenae) is an emerging pathogen of citrus. In this study, we developed and validated reverse transcription quantitative polymerase chain reaction (RT-qPCR) detection assays following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Analysis of all available CYVCV GenBank sequences allowed for the design of four RT-qPCR assays targeting distinct regions within the viral genome. The performance of the four RT-qPCR assays for detecting CYVCV was evaluated, comparing their specificity, sensitivity, and viral load detection. The CYVCV4 RT-qPCR assay, targeting the RNA Dependent RNA Polymerase (RdRP) gene, demonstrated superior performance, with the highest specificity, sensitivity, and viral load average among the developed assays. The assay detected CYVCV in 100% of the infected samples tested, with a mean viral load of 3.13E+09 copies/100mg of tissue, outperforming the other three and a previously reported CYVCV RT-qPCR assay. The CYVCV4 RT-qPCR assay showed high sensitivity, detecting CYVCV down to a dilution equivalent of 0.1 pg/µl and a limit of detection of <10 copies/µL. The assay also demonstrated specificity, avoiding cross-reactivity and correctly classifying samples of infected, non-infected, and non-target citrus pathogens. The robustness, inter and intra assay variation, reproducibility, and transferability of the CYVCV4 RT-qPCR assay were rigorously assessed and validated across different laboratories, qPCR instruments, and reagent kits, demonstrating its practicality for widespread use in diagnostic settings. The newly developed CYVCV4 RT-qPCR assay can be used in citrus pathogen detection laboratories, field surveys, and citrus germplasm and quarantine programs.