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

Clavibacteris an agriculturally important bacterial genus comprising nine host-specific species/subspecies includingC. nebraskensis(Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detectCnin infected plants and distinguish it from otherClavibacterspecies 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 ofClavibacterandCndirectly 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 genusClavibacterandCn,respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies ofClavibacter,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 forClavibacter- andCn-specific primers/probes, respectively. The detection limit forCn-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 onClavibacter-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.

 
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NSF-PAR ID:
10246612
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Scientific Reports
Volume:
11
Issue:
1
ISSN:
2045-2322
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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