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A Recombinase Aided Amplification-Lateral Flow Dipstick Detection Technique for Early On-Site Diagnosis of Bacterial Blight Caused by Xanthomonas oryzae pv. oryzae in Rice

  1. State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Hangzhou 311400, China; Wenzhou Plant Protection and Soil Fertiliser Management Station, Wenzhou 325000, China; Taizhou Academy of Agricultural Sciences, Taizhou 318014, China; #These authors contributed equally to this work
  • Contact: HOU Yuxuan; GONG Junyi
  • Supported by:

    This study was supported by the Zhejiang Provincial Natural Science Foundation of China (Grant Nos. LY23C130004 and LZ24C130004), the National Natural Science Foundation of China (Grant No. 32472115), the National Key Research and Development Program of China (Grant No. 2022YFF1003301), and the Agricultural Sciences and Technologies Innovation Program of Chinese Academy of Agricultural Sciences. We would like to thank Dr. Wang Aiying from China National Rice Research Institute for providing the valuable suggestions on primer design and experimental manipulation.

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Abstract:

Bacterial blight (BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effectiveness once BB has spread. Consequently, it is imperative to develop a rapid, highly sensitive, specific, and easy-to-use detection technique for early on-site diagnosis of BB. We first developed a recombinase-aided amplification-lateral flow dipstick (RAA-LFD) technique for the on-site detection of Xoo. The optimized reaction temperature and time were 37 ºC and 20 min, indicating that the reaction system can be initiated by body temperature independently of any precision instruments. Evaluation of the RAA-LFD technique using the primers (RAAF2/R2) and probe (RAA2-nfo-probe) derived from the XooORF0080 locus exhibited high specificity and eliminated cross-reactivity with other bacterial species. The sensitivity of RAA-LFD is up to 1 pg/μL for Xoo genomic DNA and 1 × 102 CFU/mL for Xoo cells. Significantly, this technique accurately detected Xoo from both artificially inoculated and naturally infected rice leaves at the early stage of infection, directly deploying plant tissue fluid as the template without DNA extraction. These attributes make the developed RAA-LFD system a viable technique for the early diagnosis of BB in the field, providing technical support for early-warning systems and disease control.

Key words: rice bacterial disease, recombinase aided amplification, lateral flow dipstick, Point-of-Care Testing