基于环介导等温扩增的抗虫基因超灵敏比色生物传感器构建

Construction of an Ultra-sensitive Colorimetric Biosensor for Insect Resistance Genes Based on Loop-mediated Isothermal Amplification

随着转基因技术的发展,快速、稳定和简单的转基因作物检测技术的开发一直是热点。开发了以溴百里酚蓝为显色剂的基于环介导等温扩增的抗虫基因超灵敏比色生物传感器,该方法可以通用检测Cry1Ac和Cry1Ab/Ac基因,具有较好的特异性。新型pH指示剂溴百里酚蓝对于LAMP影响小,通过一步法闭管可视检测,解决了LAMP容易污染的问题,实现了自然光下裸眼可视化半定量检测。最终优化的体系为Tris终浓度3.25 mmol/L,0.1%溴百里酚蓝添加量1.5μL,在此条件下1 h内可检测到单拷贝靶标基因。该方法兼具灵敏性、特异性和稳定性,操作简单,适合现场检测,提供了Cry1Ac和Cry1Ab/Ac基因通用检测的具体操作方法和新的LAMP比色信号输出方式,不仅完善了转基因产品的检测体系,也丰富了LAMP生物传感器信号输出方式。

With the development of genetically modified technology,the development of rapid,stable and simple detection technology for genetically modified crop has always been a hot spot.In this paper,a super-sensitive colorimetric biosensor based on loop-mediated isothermal amplification(LAMP)was developed,using bromophenol thyme blue as the chromogenic reagent.This method can detect the Cry1Ac and Cry1Ab/Ac genes in general,and has good specificity.The new pH indicator bromophenol thyme blue has little effect on LAMP,can achieve one-step closed-tube visual detection,solve the problem of easy pollution of LAMP,and realize semi-quantitative detection with naked eyes under natural light.The final optimized system is the final concentration of Tris 3.25 mmol/L,and the addition amount of 0.1%bromophenol thyme blue is 1.5μL.Under these conditions,a single copy of the target gene can be detected within 1 h.This method combines sensitivity,specificity and stability,is of simple operation,and suitable for on-site detection.It provides specific operation methods for Cry1Ac and Cry1Ab/Ac gene universal detection and a new LAMP colorimetric signal output method,which not only improves the detection system for genetically modified products,also enriches the signal output methods of the LAMP biosensor.