Crude sap based colorimetric duplex RT-RPA assay for rapid detection of citrus yellow vein clearing virus and citrus yellow mottle-associated virus in citrus.

Citrus yellow vein clearing virus (CYVCV) and citrus yellow mottle-associated virus (CiYMaV) frequently co-infect citrus, posing challenges for reliable diagnosis and certification systems. In this study, a duplex reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for the simultaneous detection of both viruses using crude leaf sap of citrus. The assay operates under isothermal conditions (40 °C, 25 min) and incorporates an endogenous internal control (Elongation factor 1α) to support assay reliability and reduce the likelihood of false-negative results. The duplex RT-RPA assay performed effectively with crude sap, enabling detection without the need for nucleic acid extraction. It specifically amplified CYVCV and CiYMaV without cross-reactivity with other citrus pathogens and showed performance comparable to duplex RT-PCR. A hydroxynaphthol blue (HNB)-based colorimetric format was further evaluated to allow visual identification of positive samples without electrophoresis, although it does not differentiate between the two viruses. Validation using citrus samples confirmed detection of both single and mixed infections, with results consistent with those obtained by duplex RT-PCR. Overall, the combined approach provides a rapid and simplified method for citrus virus detection and may be useful for routine screening and certification programs.