An Improved Recombinase Polymerase Amplification Coupled with Lateral Flow Assay for Rapid Field Detection of ' Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ' Liberibacter asiaticus' (Las), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of Las is real-time quantitative polymerase chain reaction (qPCR) using either the Las 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 Las-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 Las in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the Las RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to Las, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting Las in fresh citrus crude extracts from leaf midribs and roots from five California strains of Las tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing Las-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.