An Ultrasensitive Immunocapture (IC)-RPA-CRISPR/Cas12a Assay with Three Readout Modes for Detecting Xanthomonas oryzae pv. oryzicola of Rice Bacterial Leaf Streak

Abstract

Xanthomonas oryzae pv oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS) and causes enormous losses of rice yields in many countries every year. Development of sensitive diagnostic techniques is crucial for its prevention and control. Here, we developed an ultrasensitive IC-RPA-CRISPR/Cas12a assay with three readout modes [qPCR machine, UV lamp, and lateral flow strip (LFS)] for Xoc detection in rice, which combined advantages of immunocapture, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based cleavage. Especially, the immunocapture step allows to capture and enrich Xoc from samples, which minimizes the interference from rice debris to benefit nucleic acid release and amplification and enhances the specificity and sensitivity of this assay. The detection limits of its three readout modes for Xoc bacterial suspension is 2, 6, and 60 CFU/mL, respectively. Collectively, this study provides a specific, ultrasensitive, practical approach for quarantine and detection of Xoc that will benefit the prevention and control of BLS.