Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis

• Published in: Scientific reports 2021-06, Vol.11 (1), p.12017-12017, Article 12017
• Main Authors: Larrea-Sarmiento, Adriana, Stack, James P., Alvarez, Anne M., Arif, Mohammad
• Format: Article
• Language: English
• Subjects: 631/326; 631/326/1320; 631/326/2522; 631/449; ABC transporter; ABC transporters; ATP-binding protein; Blight; Body temperature; Clavibacter michiganensis nebraskensis; Deoxyribonucleic acid; DNA; DNA probes; Genomics; Humanities and Social Sciences; Infections; Laboratories; multidisciplinary; Pathogens; Permease; Plant bacterial diseases; Probes; Protein transport; Quarantine; Recombinase; Science; Science (multidisciplinary); Species; Temperature requirements; Wilt
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-021-91336-7

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis ( Cn ), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter - and Cn -specific primers/probes, respectively. The detection limit for Cn -specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter -specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.