水稻细菌性条斑病菌重组酶聚合酶扩增快速检测方法的建立

Recombinase polymerase amplification assay for rapid detection of Xanthomonas oryzae pv. oryzicola in rice

摘要

为便于水稻细菌性条斑病菌（Xanthomonas oryzae pv. oryzicola，Xoc）的早期快速检测，本研究根据GenBank公开报道的水稻细菌性条斑病菌特异性基因序列（序列号：XOCgx_RS03090）设计了重组酶聚合酶扩增（RPA）引物、Exo荧光探针组合及nfo探针组合，并构建含有Xoc基因序列的重组质粒作为阳性对照；建立了水稻细菌性条斑病菌的重组酶聚合酶扩增法（Exo-RPA）和侧流层析试纸条检测法（LFD-RPA）的技术体系，并评估了上述两种不同终端RPA检测方法的灵敏度和特异性，通过优化反应温度、反应时间明确了其对RPA方法扩增效果的影响。结果表明，Exo-RPA和LFD-RPA检测法对水稻细菌性条斑病菌检测的灵敏度均为4.96 copies·μL⁻¹，反应的最佳温度均为39 ℃；优化后的Exo-RPA方法在12 min内检测出水稻细菌性条斑病菌，优化后的LFD-RPA可以在5 min内检测出水稻细菌性条斑病菌；与其他植物病原菌菌株一起进行检测，RPA检测方法可特异地检测出目标病原菌。该方法灵敏度高，特异性强，检测速度快，有望在水稻细菌性条斑病病原监测与病害的早期诊断上得到应用。

Abstract

For early and rapid detection of Xanthomonas oryzae pv. oryzicola (Xoc), the cause of bacterial leaf streak in rice, specific recombinase polymerase amplification (RPA) primers, and Exo fluorescent and nfo probes were designed and screened, respectively, according to the publicly available gene sequence XOCgx_RS03090 of Xoc genome sequence in GenBank. The recombinant plasmid containing Xoc gene was constructed as a positive control. One fluorescent Exo-RPA assay and the other lateral flow strip detection (LFD-RPA) assay were established for rapid detection of Xoc. The sensitivity and specificity of the RPA methods were assessed, and the amplification effects of the RPA methods were explored by optimizing the reaction temperature and reaction time. The results showed that the sensitivity of both the Exo-RPA and LFD-RPA assays for the detection of Xoc were both 4.96 copies·μL⁻¹, and that the optimal temperatures for the reaction were both 39 ℃. Xoc was detected within 12 min by the optimized Exo-RPA method, and within 5 min by the optimized LFD-RPA. The Exo-RPA and LFD-RPA assays developed were highly specific and could specifically and uniquely identify Xoc among plant pathogens in test. Both the RPA methods are highly sensitive, specific and fast, which pose a high potential to the monitoring of the target pathogen and early diagnosis of the disease.

关键词

水稻 / 细菌性条斑病 / 重组酶聚合酶扩增 / 侧流层析试纸条 / 快速检测

Key words

rice / bacterial leaf streak / recombinase polymerase amplification / lateral flow dipstick / rapid detection

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基金资助

海南省科技专项(ZDYF2021XDNY142)

海南省崖州湾种子实验室揭榜挂帅项目(B21HJ0905)