用于高灵敏可视化检测松材线虫的闭管等温扩增法

建立了一种基于环介导等温核酸扩增技术(LAMP)的松材线虫高灵敏可视化闭管检测方法。针对松材线虫核糖体DNA的序列保守区域设计LAMP引物,通过优化LAMP体系中的Mg2+、甜菜碱浓度和反应温度等因素,建立了环介导等温扩增法;并结合蜡封反应管对产物进行检测,检测结果可直接通过肉眼观察SYBR Green I荧光显色进行判定。结果表明,本方法可检测到低至10拷贝/管的松材线虫核酸片段,可对单条线虫进行检测,并且具有很高的特异性,能区分检测松材线虫与拟松材线虫。由于整个反应恒温进行,无需热循环仪;闭管检测极大地降低了扩增产物交叉污染的风险;检测速度快,整个检测过程只需40 min,为松材线虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。

A Closed-Tube Isothermal Amplification Method for Highly Sensitive & Visualized Detection of Pine Wood Nematode (Bursaphelenchus xylophilus)

A visualized closed-tube loop-mediated isothermal amplification (LAMP) assay with high sensitivity method to detect pine wood nematode (Bursaphelenchus xylophilus) was established. LAMP primers were designed aiming at the conservative domain of ribosomal DNA sequence of B. xylophilus through an optimization of LAMP reaction conditions such as concentration of Mg²⁺, betaine, and reaction temperature etc and established the LAMP method. The results of determination was observed through naked eyes by judging color changes of fluorescent coloration SYBR Green I of the product sealed with wax at the bottom of the detection tube. The results indicated that the sensitivity of the proposed method was as low as 10 copies of genomic DNA fragment of B. xylophilus, and it was sensitive enough to detect a single nematode. The method had a very high specificity, and could easily distinguish B. xylophilus from B. mucronatus. Because the whole reaction was carried out in constant temperature, no thermal cycler was needed. The closed-tube LAMP reaction greatly decreased the risk of cross-contamination from amplified products. The assay was very fast, it needed only 40 minutes for the whole detection process. Therefore, the established method provided a simple, highly sensitive and highly specific tool for rapid screening of pine wood nematode on the site.