Rapid and sensitive isothermal detection of Clavibacter michiganensis subsp. michiganensis using AmplifyRP®
Commercial field detection kits for Clavibacter michiganensis subsp. michiganensis (Cmm) have historically been limited to immunoassay-based formats such as ELISA and lateral flow-based technologies. While effective for high-throughput screening purposes, they are known to cross-react with other Clavibacter michiganensis subspecies, as well as some non-pathogenic bacteria that persist in soil. The cross-reactivity of these methods mandates confirmation testing of positive results by a laboratory equipped for PCR, media plating, and microscopy detection. These confirmatory laboratory techniques may delay critical management decisions to stop the spread of the pathogen. We have developed a rapid isothermal DNA amplification kit using the AmplifyRP® XRT platform, based on the recombinase polymerase amplification (RPA) technique, for Cmm detection. The kit uses specific primers and a probe for real-time fluorescence detection on crude extract in a 20‑minute test, which successfully identifies the presence of Cmm in bacterial culture, tomato fruit, stem, and petiole. Data will be presented on an external validation of the Cmm AmplifyRP® XRT kit collected by an outside seed company. The kit provides a fast and convenient detection method that retains the sensitivity and specificity of PCR while reducing the complexity of molecular diagnostics for inexperienced users.
Davenport, B., Schuetz, K. and Kurowski, C. (2018). Rapid and sensitive isothermal detection of Clavibacter michiganensis subsp. michiganensis using AmplifyRP®. Acta Hortic. 1207, 185-192
recombinase polymerase amplification (RPA), tomato, bacterial canker, pathogen detection methods