Rapid and sensitive colorimetric detection of Xanthomonas axonopodis pv. citri by immunocapture and a nested-polymerase chain reaction assay

We have developed a sensitive and specific assay for Xanthomonas axonopodis pv. citri, the causal agent of citrus bacterial canker. The assay is based on sequential nested amplification by polymerase chain reaction (PCR) of a region of plasmid DNA that is very highly conserved in X. axonopodis pv. citri. Specific amplification products were observed in reactions containing three or fewer target molecules, an improvement of 50- to 100-fold over single-stage PCR, and similar results were observed when beginning with purified DNA or living bacterial cells. Colorimetric detection of amplification products was performed with the DIANA (detection of immobilized amplified nucleic acids) method, which uses labeled primers to allow amplification product capture and detection in a microtiter plate. Predicted amplification products were produced from all strains of X. axonopodis pv. citri and from four of six strains of X. axonopodis pv. aurantifolii but not from other xanthomonads, including citrus epiphytes, except for X. axonopodis pv. vignicola and one strain isolated from Feronia elephantiacum, consistent with previous hybridization results. No amplification products were observed from strains of X. axonopodis pv, citrumelo that incite citrus bacterial spot disease in Florida citrus nurseries. Amplification was completely inhibited by copper hydroxide when present in the reaction mix at 13.6 micrograms/ml. Concentrated leaf extracts from tangelo and mandarin orange, but not similar extracts from other citrus varieties, also inhibited amplification. Immunomagnetic separation of target bacteria prior to amplification was used to concentrate and recover X. axonopodis pv. citri from samples containing compounds that inhibit amplification (i.e., copper and concentrated citrus extracts). Immunocapture, by concentrating target bacteria from dilute plant extracts, improved the sensitivity of the assay by 100-fold over nested-PCR alone. The combination of sensitivity, specificity, and speed of the assay could make this a widely used assay both in plant quarantine and in areas where X. axonopodis pv. citri is endemic and clean planting stock programs are to be initiated.