Rapid detection of the golden potato cyst nematode, Globodera rostochiensis, based on microsatellite markers using Recombinase Polymerase Amplification assay with lateral flow dipsticks

Summary Recombinase polymerase amplification (RPA) is a single tube, isothermal amplification technique, operating at constant temperature with minimal sample preparation. The RPA assay, combined with lateral flow dipsticks (LF) and existing species-specific primers designed previously, has been developed targeting a gene fragment with unique sequence for the golden cyst nematode, Globodera rostochiensis . The assay provided highly specific, sensitive, and rapid detection of this nematode species from crude nematode extracts without a DNA extraction step with a sensitivity of 0.03 second-stage juvenile specimens during approximately 24 min. Specificity of the LF-RPA assay was validated with a wide range of non-target cyst nematodes. The developed LT-RPA assay provides a valuable diagnostic tool for G. rostochiensis detection and has many advantages that may make it a useful alternative to conventional and real-time PCR diagnostics for this pest. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment, and could be exploited for portable laboratory-in-a-suitcase analysis.