Fast detection of Citrus chlorotic dwarf-associated virus by loop-mediated isothermal amplification

Citrus chlorotic dwarf-associated virus (CCDaV) is among the most damaging pathogens that can cause significant reduction in both the yield and quality of some economically important citrus varieties. The timely and accurate detection of CCDaV is a key factor for development of efficient management strategies. This study describes the development of a reliable diagnostic assay for rapid detection of CCDaV based on loop-mediated isothermal amplification (LAMP). A set of six specific primers targeting the coat protein-encoding regions of CCDaV was designed. The isothermal temperature and incubation duration were optimized for LAMP. No cross-reactivity was observed with other non-target citrus pathogens. The detection sensitivity of the LAMP assay was equal to quantitative PCR after optimization of the reaction conditions. Combined with the leaf crude extraction method, the LAMP assay accomplished the detection of CCDaV isolates in less than 35 min, and the results can be seen by observing color changes in the reaction tubes. The proposed LAMP assay is a powerful tool for the detection of CCDaV and will be particularly useful for large scale indexing of field samples in regional laboratories with only basic equipment.