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. ' Candidatus Liberibacter asiaticus' ( C Las), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of C Las is real-time quantitative polymerase chain reaction (qPCR) using either the C 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 C 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 C Las in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the C 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 C 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 C Las in fresh citrus crude extracts from leaf midribs and roots from five California strains of C Las tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing C Las-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.