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环介导等温扩增(loop-mediated isothermal amplification,LAMP)是一种特异、灵敏、快速的新型基因检测技术。本研究以香蕉条斑病毒(Banana streak virus,BSV)ORF3保守区域为基础针对6个特定区域设计并筛选了4条LAMP扩增引物,通过对LAMP反应中MgSO4、dNTPs、Betaine等主要试剂浓度进行优化,建立了香蕉BSV的LAMP检测方法,63℃反应90 min后通过在反应产物中添加SYBR GreenⅠ染料后颜色的变化,肉眼即可判断检测结果。LAMP具有极高的检测特异性和灵敏性,其检测下限约为3.2 ng·μL-1,是PCR检测灵敏度的25倍,能快速、准确地对疑似样品进行检测,本研究对华南地区部分疑似样品的检测结果显示LAMP阳性检出率比PCR检出率高。本文建立的BSV LAMP检测方法是对BSV检测方法的拓展和延伸,为香蕉病毒的快速检测提供技术保障。
Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for gene detection. In this study, four LAMP amplification primers were designed and screened for 6 specific regions based on the conserved region of ORF3 of Banana streak virus (BSV). The concentration of major reagents such as MgSO4, dNTPs, and Betaine in the LAMP reaction The detection method of LAMP in banana BSV was established. After the reaction at 63 ℃ for 90 min, the color change of SYBR Green Ⅰ dye in the reaction product can be judged visually. LAMP has a high detection specificity and sensitivity. The detection limit of LAMP is about 3.2 ng · μL-1, which is 25 times of the sensitivity of PCR. It can detect suspected samples quickly and accurately. In this study, The test results showed that the positive detection rate of LAMP was higher than the detection rate of PCR. The BSV LAMP detection method established in this paper is an extension and extension of BSV detection methods, providing technical support for the rapid detection of banana virus.