Multiplex lateral flow immunoassay for simultaneous detection and specific discrimination of Xoo and Xoc in rice...

Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) are two important rice bacterial pathogens, causing serious losses of global rice yield every year. Effective management of rice bacterial diseases needs timely and accurate detection and quarantine techniques. Herein, a novel Au nanoparticles-based multiplex lateral flow immunoassay (AuNP-mLFIA) was developed for simultaneous detection and specific discrimination of Xoo and Xoc in rice leaf and seed samples. Four anti-Xoo mAbs, four anti-Xoc mAbs, and ten broad-spectrum mAbs towards both Xoo and Xoc were first obtained via hybridoma cell technique. Through checkerboard assays, pairwise mAbs 12H1/19C9 against Xoo and self-paired mAb 18D12 against Xoc were selected to guarantee the highest sensitivity and specificity of the assay. Under the optimal conditions, the AuNP-mLFIA achieved a visual limit of detection (LOD) as low as 7.8 × 10³ CFU mL^-1 for Xoo and 1.6 × 10⁴ CFU mL^-1 for Xoc with no cross-reactivity. In addition, the assay was successfully applied to monitor Xoo and Xoc in 40 field-collected rice leaf and seed samples and the detection results of this assay agreed well with conventional PCR. Therefore, this developed AuNP-mLFIA was sensitive, rapid, and accurate for simultaneous detection of Xoo and Xoc. This study also provides guidelines for the screening of paired mAbs to establish a double-antibody sandwich (DAS)-mLFIA.