One Simple DNA Extraction Device and Its Combination with Modified Visual Loop-Mediated Isothermal Amplification for Rapid On-Field Detection of Genetically Modified Organisms
One Simple DNA Extraction Device and Its Combination with Modified Visual Loop-Mediated Isothermal Amplification for Rapid On-Field Detection of Genetically Modified Organisms
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DOI：
10.1021/ac301640p
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发表时间：
2013-01-01
影响因子：
7.4
通讯作者：
Yang, Litao
中科院分区：
文献类型：
--
作者：
Zhang, Miao;Liu, Yinan;Yang, Litao
快速、简单、有效是许多领域建立良好分子诊断方法的三大标准。在此，我们报告了一种新型转基因生物（GMO）检测系统，可用于进行现场快速筛查和常规实验室诊断。在该系统中，新设计的廉价 DNA 提取装置与改进的视觉环介导等温扩增 (vLAMP) 测定相结合。 DNA提取装置的主要部分包括硅胶膜过滤柱和改进的注射器。该DNA提取装置无需使用其他实验室仪器即可轻松操作，适用于现场转基因生物测试。使用该设备可以在 15 分钟内从植物组织中快速分离出适合聚合酶链式反应 (PCR) 和等温扩增的高质量基因组 DNA (gDNA)。在改进的 vLAMP 测定中，引入了含有 SYBR 绿色荧光染料的微晶蜡封装检测珠，以避免 LAMP 操作后的染料抑制和交叉污染。该系统已成功应用于筛选和鉴定从现场测试和谷物检验、包装和牲畜饲养场管理局 (GIPSA) 能力测试计划中收集的转基因水稻、大豆和玉米样品，并得到验证，这表明该系统非常适合转基因生物的现场测试和/或常规实验室分析。
Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polyrnerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.