LAUSR

Huanglongbing (HLB) is a destructive citrus disease that affects production worldwide. "Candidatus Liberibacter asiaticus" (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either CLas 16S rRNA gene- or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase Polymerase Amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas from crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within eight minutes at 39°C. The assay was specific to CLas and the sensitivity was comparable to qPCR, with a detection limit of cycle threshold (Cq) 34. Additionally, the RPA assay was combined with a lateral flow device (LFD) for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from other pathogens that cause similar disease symptoms, helping to control HLB spread.