Abstract Citrus tristeza virus (CTV) and ‘Candidatus Liberibacter asiaticus’ (CLas), are two of the most significant systemic pathogens associated with decline threatening citrus crops globally, including those in tropical and subtropical regions. Effective disease management and the prevention of their spread rely on rapid, sensitive, and reliable diagnostic tools aiming at bud wood certification and clean stock propagation. An assay capable of simultaneous and sensitive detection of both the virus and fastidious prokaryotes (CTV and CLas) is expected to simply the routine indexing and certification procedures in citrus. This study describes the development and validation of a highly sensitive and rapid duplex isothermal RT-RPA/RPA assay for the simultaneous detection of CTV and CLas. The assay utilizes a simplified pulverized tissue extract template prepared in a 50 mM NaOH : 2.5 mM EDTA buffer (1 : 1, pH 8.0), making it suitable for field-based or limited-resource laboratory settings. The developed assay resulted in an optimal performance at 37°C for 30 min. A higher sensitivity was observed with the assay detecting CTV and CLas infections at concentrations up to a 10–9 fold dilution (0.1 pg/μL concentration), a significant improvement over conventional duplex RT-PCR/PCR. Validation results revealed mixed infections of CLas and CTV in 83.3% of samples. The duplex RPA-based assay represents a substantial advancement in diagnostics for these key citrus pathogens, enabling rapid, cost-effective, and accurate detection. This assay holds great promise for large-scale nursery indexing and phytosanitary programs, thereby contributing to sustainable citrus cultivation in tropical and subtropical environments.
Wangkhem et al. (Wed,) studied this question.