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首页> 外文期刊>Forest Pathology >Development of two alternative Loop-mediated isothermal amplification tools for detecting pathogenic pine wood nematodes
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Development of two alternative Loop-mediated isothermal amplification tools for detecting pathogenic pine wood nematodes

机译：开发两种可替代的Loop介导的等温扩增工具，用于检测病原性松木线虫

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摘要

Loop-mediated isothermal amplification (LAMP) detection tools have great potential for diagnosing the causal agents of infectious diseases in clinics and in agriculture. In this work, we developed two alternative LAMP protocols for detecting the pathogenic nematode Bursaphelenchus xylophilus, causal agent of pine wilt disease. We first identified a pectate lyase 3 gene as a biomarker for developing a LAMP Detection Kit, as there was no homologue in non-pathogenic nematodes that live in pine timber or bark and show structural similarities to B.xylophilus. The first LAMP protocol used the Genie II equipment and an isothermal master mix containing Geobacillus sp. M 2.0 large fragment DNA polymerase showed approximately 10 times greater sensitivity with a shorter incubation period compared with that of the second LAMP protocol, which utilized a fluorescence metal indicator, calcein and an engineered Bacillus stearothermophilus DNA polymerase I, large fragment (Bst 2.0 DNA polymerase). However, the LAMP reactions with calcein and Bst 2.0 polymerase were the cost-effective method because the reaction could be performed using a simple isothermal block and relatively inexpensive calcein as a fluorescence indicator visible to the naked eye.
机译：回路介导的等温扩增（LAMP）检测工具在临床和农业中诊断传染病的病因具有巨大潜力。在这项工作中，我们开发了两种替代的LAMP方案，用于检测致病性线虫Bursaphelenchus xylophilus，它是松萎病的病原体。我们首先将果胶酸裂合酶3基因鉴定为开发LAMP检测试剂盒的生物标记物，因为在生活在松木或树皮中且与B.xylophilus结构相似的非致病性线虫中没有同源物。第一个LAMP协议使用Genie II设备和包含Geobacillus sp。的等温预混液。与第二个LAMP方案相比，M 2.0大片段DNA聚合酶显示了大约10倍的灵敏度，并且孵育时间更短，后者使用了荧光金属指示剂，钙黄绿素和工程改造的嗜热脂肪芽孢杆菌DNA聚合酶I，大片段（Bst 2.0 DNA聚合酶）。但是，与钙黄绿素和Bst 2.0聚合酶进行LAMP反应是一种经济有效的方法，因为该反应可以使用简单的等温块和相对便宜的钙黄绿素作为肉眼可见的荧光指示剂进行。

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