Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to 10⁴ cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The Bühlmann-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.

中文翻译：

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开发用于苹果和梨火疫病现场诊断的改进的环介导的等温扩增测定法。

需要快速而准确的诊断来根除和管理经济上重要的侵入性疾病，例如火疫病。环介导的等温扩增（LAMP）被认为是最好的现场诊断方法，因为它快速，对靶标高度特异性，并且对样品中的抑制剂不敏感。在这项研究中，与以前开发的LAMP分析相比，开发出了LAMP分析，可为现场诊断火疫病提供更一致的结果。新LAMP测定（命名为DS-LAMP）引物从一个组氨酸tRNA的设计连接酶基因（EAMY_RS32025）E.火疫病菌CFBP1430基因组中。DS-LAMP仅从解淀粉链球菌菌株的基因组DNA扩增DNA（阳性检测），而不是从E. pyrifoliae（引起黑梢枯萎病）或丁香假单胞菌PV。丁香科（引起苹果树上的枯萎病）。DS-LAMP的检出限为每个LAMP反应10^个细胞，相当于每毫升样品提取物中10 ^4个细胞。DS-LAMP成功地在四个受火疫病感染的苹果和梨果园中诊断出病原体。另外，它可以区分黑梢枯萎病和火枯萎病。该BUHLMANN-LAMP，以前为现场诊断火疫病的开发，没有给特异性一致的结果E.梨火疫病和现场诊断; 它对三株E. pyrifoliae和两株丁香假单胞菌pv产生了积极的反应。丁香科。它还对一些健康的样品提取物产生了积极的反应。在这项研究中开发的DS-LAMP，可以对火疫病进行更准确的现场诊断，尤其是在大火疫病和黑梢病共存的大韩民国。