Adaptation of CANARY biosensors for rapid detection of plant pathogens.
Z. LIU (1), K. Rappaport (1), L. Levy (1) (1) USDA APHIS PPQ CHPST, Beltsville, MD, USA.
Phytopathology 99:S75 .

Abstract: The CANARY (Cellular Analysis and Notification of Antigen Risk and Yield) technology uses a recombinant B cell line expressing membrane-bound antibodies that are pathogen-specific, and at the same time expresses the calcium sensitive bioluminescent protein, aequorin. Upon crosslinking of antigens of a specific pathogen to the antibodies, B cells produce an elevated level of calcium. The process triggers an aequorin conformation change and leads to light emission through conversion of its prosthetic group coelenterazine into coelenteramide and CO2. The emitted light can be easily detected by a luminometer. We adapted this technology for detection of regulated plant pathogens. In this report, we present the initial results on testing with a Ralstonia-specific B cell line. Two strains of Ralstonia solanacearum race 3 biovar 1 and race 1 biovar 1were used. In the first six days of a 15-day assay period, the limit of detection of 3 CFU/CANARY test can be achieved in spiked geranium extract. We can still detect as low as 300 CFU/test at the end of the 15-day period. Comparable results were obtained with killed bacterial suspension. The procedure requires simple instrumentation and sample testing only takes a few minutes. Testing for cross-activity with related organisms and infected plant tissues are under way.