APS Homepage

Oral: Isothermal Pathogen Detection


Detection of the select agent Rathayibacter toxicus using recombinase polymerase amplification coupled with a lateral flow device
M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) (1) Department of Plant Pathology, Kansas State University, U.S.A.; (2) Department of Plant Pathology, Kansas State University, Australia; (3) Department of Economic Development, Jobs, Tr

Rathayibacter toxicus is a gram-positive plant pathogen responsible for a lethal disease of livestock in Australia. An easy, sensitive in-field detection method would enhance capabilities for port of entry inspections and biosecurity surveillance surveys of annual ryegrass (Lolium rigidum) hay and seed. Recombinase polymerase amplification (RPA) is a fast and sensitive isothermal DNA amplification and detection technology. Long forward and reverse primers (32 bp) and a long probe (49 bp) labelled with FAM were designed using gene involved in toxin production. TwistDX kit was used to amplify the target DNA (at ~37°C) and a lateral flow device used to visualize the amplified products. RPA primers and probes targeting the ITS region of the host genomes were designed for use as internal controls, to enhance the reliability and accuracy of the assays. RPA assays were tested against inclusivity and exclusivity panels for high specificity and accuracy. In-field validation in South Australia confirmed the efficiency and sensitivity of the RPA assays; detected the R. toxicus from infected samples. The proposed RPA method will enhance diagnostics, surveillance, disease management, and in-field biosecurity decisions.