关键词: 根据脱氧核糖核酸与Gelred结合形成的荧光络合物在紫外灯照射下发出荧光的原理, 建立一种更适用实际应用的快速、简单高通量的非特异性核酸酶酶活测定方法。采用脱氧核糖核酸为底物, 利用凝胶成像仪测定底物减少引起光密度的变化值, 从而计算出非特异性核酸酶活力。研究了影响酶活测定的关键因素, 并对该方法的线性、准确度、精密度、检测限与定量限进行了验证。结果表明, 方法最佳反应时间为3 min, 待测酶活线性范围在4.17～9.01 U/μL, 线性关系、精密度、准确度和灵敏度均符合方法学开发评估要求。相较于紫外吸收法, 此方法检测限更低, 反应时间更短, 且能用于多个样品的分析。

Abstract: A rapid, simple and high-throughput activity assay method of non-specific nuclease has been established, according to the fact that under the irradiation of ultraviolet light and through the combination of nucleic acids with DNA stain Gelred.The fluorescent complex was generated and then emited fluorescence. In the study, the change of optical density caused by the decrease of substrate DNA measured by a gel imager, therefore the enzyme activity was obtained. Key factors involved in the activity assay experiment were studied, as well as the linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) of the method were all verified. The results suggested that the optimal reaction time was 3 min, the linear range of enzyme activity was 4.17-9.01 U/μL, and the linear relationship, precision, accuracy and sensitivity met the evaluation requirements of methodology development. In conclusion, the method exhibited lower LOD and shorter reaction time compared with the UV absorption assay method, and it could be applied for multiple samples analysis.

Key words: non-specific nuclease, enzyme activity, high throughput, optical density, gel imaging