An improved Recombinase polymerase amplification coupled with lateral flow assay for rapid field detection of "Candidatus Liberibacter asiaticus"

Authors: RATTNER, RACHEL - Cooperative Agricultural Support Services; GODFREY, KRIS - University Of California, Davis; HAJERI, SUBHAS - Central California Tristeza Eradication Agency; Yokomi, Raymond - Ray

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2022
Publication Date: 5/20/2022
Citation: Rattner, R., Godfrey, K., Hajeri, S., Yokomi, R.K. 2022. An improved Recombinase polymerase amplification coupled with lateral flow assay for rapid field detection of "Candidatus Liberibacter asiaticus". Plant Disease. https://doi.org/10.1094/PDIS-09-21-2098-RE.
DOI: https://doi.org/10.1094/PDIS-09-21-2098-RE

Interpretive Summary: Huanglongbing (HLB) is a destructive citrus disease which can spread rapidly between citrus trees by feeding of the Asian citrus psyllid (ACP) which is infected with the pathogen, “Candidatus Liberibacter asiaticus” (CLas). Early detection and rouging of HLB-infected trees along with effective ACP control is the best management strategy to limit HLB spread. Isothermal amplification allows rapid amplification of nucleic acids in plants without the need for nucleic acid extraction. An end-point Recombinase Polymerase Assay (RPA) was developed to detect HLB based on the detection of the multicopy RNR gene of CLas. CLas detection by RPA was comparable to real time quantitative PCR (qPCR) which requires purified CLas DNA. The RPA assay was coupled to a lateral flow device for an end-point-of-use assay that is field deployable and user friendly. The RPA/LF assay was validated with crude extracts from CLas-infected citrus trees and CLas-infective ACP. This method can be used by growers, extension agents, PCAs, and commercial diagnostic laboratories to assist the grower make rapid decisions on management tactics to control HLB.

Technical Abstract: Huanglongbing (HLB) is a destructive citrus disease that affects production worldwide. Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium, is the causal agent of Huanglongbing. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either CLas 16S rRNA gene- or the multi-copy ribonucleotide reductase (RNR) gene-specific primers/probe. This diagnostic technique requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas. Recombinase Polymerase Amplification (RPA) assay is a fast, portable alternative to polymerase chain reaction (PCR)-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in 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 fluorescent labeled probe allowed for detection of the amplicon in real-time within eight minutes. The assay was CLas-specific and sensitive to Ct 34 when compared with qPCR. 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 citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility. 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.