United States Department of Agriculture-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA 93648.
Contained Research Facility, University of California at Davis, Davis, CA 95616.
Plant Dis. 2022 Dec;106(12):3091-3099. doi: 10.1094/PDIS-09-21-2098-RE. Epub 2022 Nov 17.
Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ' Liberibacter asiaticus' (Las), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of Las is real-time quantitative polymerase chain reaction (qPCR) using either the Las 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of Las-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect Las in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the Las RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to Las, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting Las in fresh citrus crude extracts from leaf midribs and roots from five California strains of Las tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing Las-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.
黄龙病（HLB）是一种破坏性的柑橘病害，影响着全球的柑橘生产。“亚洲韧皮杆菌”（Las），一种局限在韧皮部的细菌，是 HLB 的相关致病因子。目前，Las 的检测标准是使用 Las 16S rRNA 基因或核昔酸还原酶（RNR）基因特异性引物/探针的实时定量聚合酶链反应（qPCR）。qPCR 需要配备精良的实验室和经过培训的人员，这对于快速现场检测 Las 感染的树木并不方便。重组酶聚合酶扩增（RPA）检测是一种基于 PCR 的诊断方法的快速、便携替代方法。在本研究中，开发了一种利用等温扩增在粗提柑橘提取物中检测 Las 的 RPA 检测方法，无需进行 DNA 纯化。引物设计用于扩增 Las RNR 基因的一个区域，荧光标记的探针可在 39°C 下实时检测扩增子，时间在 8 分钟内。该检测方法对 Las 具有特异性，灵敏度与 qPCR 相当，检测限循环阈值为 34。此外，RPA 检测法与侧流装置相结合，可用于现场使用的即时检测。这两种检测方法在加利福尼亚州戴维斯的受控研究设施中测试的来自五个加利福尼亚州 Las 菌株的新鲜柑橘粗提取物的叶片中脉和根部中，对 Las 的检测准确率均为 100%。该检测方法将有助于区分加利福尼亚州感染 Las 的树木与感染其他引起类似疾病症状的病原体的树木，并有助于控制 HLB 的传播。
