香蕉条斑病毒LAMP快速检测方法的建立

Development of a LAMP method for rapid detection of Banana streak virus

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 highly sensitive and specific nucleic acid amplification method that can synthesize rapidly large amounts of DNA under isothermal conditions. The LAMP is successfully used to detect plant viruses and other pathogens. Banana streak virus （BSV） is a badna- virus that causes a viral leaf streak disease of banana and plantain （Musa spp. ）. Combined the sequences of ORF3 published in GenBank with specific sequence isolated from South China, two pairs of primers （B3/F3, BIP/FIP） were designed targeting the conserved region of BSV ORF3. A rapid detection of BSV was estab- lished by a loop-mediated isothermal amplification assay （LAMP） through optimizing the LAMP primers and reaction conditions such as Betaine concentration and MgSO4. BSV-LAMP reaction was carded out at 63℃ for 90 min, the amplified products were colored with SYBR Green I after completion of the reaction, so that the amplification could be detected with naked eyes. The limit of LAMP detection of BSV-infected banana geno- mic DNA was about 3.2 ng·μL-1, which was 25 times more sensitive than PCR method. The LAMP is a further extension of BSV detection and it is a simple and quick method that can detect BSV accurately for field samples. The LAMP is more sensitive than PCR method for BSV detection and mass propagation of culture materials.