Construction and preliminary evaluation of a thermostatic amplification detection system for miRNA-21-5p
曾元清 1邓宁波1
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作者信息
1. 广东省中医院珠海医院检验科,广东珠海 519015
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摘要
目的 建立一种基于滚环扩增和CRISPR-Cas9系统的荧光检测miRNA-21-5p方法,并对检测性能进行初步评价.方法 设计一种特异性的锁式探针识别靶标miRNA-21-5p,在大肠杆菌DNA连接酶和Phi29 DNA聚合酶的作用下,进行滚环扩增,扩增形成一条长重复单链.在Cas9酶的辅助下进行特异性识别,并形成双链DNA,然后通过加SYBR Green Ⅰ荧光染料与形成双链DNA产物结合,产生荧光信号的荧光强度与双链DNA产物浓度成正比,并对该系统进行特异性和灵敏度进行分析.用构建的方法对胃癌患者血清和健康人血清进行miRNA检测,是否成功检测miRNA,同时检测两组血清中胃蛋白酶原Ⅰ/胃蛋白酶原Ⅱ(PG Ⅰ/Ⅱ)水平,绘制受试者工作特征(ROC)曲线,根据曲线下面积(AUC)分析miRNA-21-5p、胃蛋白酶原对胃癌的诊断价值.结果 构建的扩增系统能检测到血清中miRNA,胃癌血清中miRNA-21-5p荧光强度高于健康对照组,差异有统计学意义(P＜0.05);ROC曲线结果显示,miRNA-21-5p、PG Ⅰ/Ⅱ单独诊断胃癌的AUC分别为0.72、0.80,两者联合检测诊断的AUC为0.85,高于任意单个指标诊断.结论 用该方法直接检测miR-21-5p操作方便快捷,不需要复杂热循环仪器,适用范围广.
Abstract
Objective To establish a fluorescence detection method for miRNA-21-5p based on rolling ring amplification and CRISPR-Cas9 system,and to preliminarily evaluate the detection performance.Methods A specific lock-in probe was designed to identify the target miRNA-21-5p,and rolled loop amplification was performed under the action of Escherichia coli DNA ligase and Phi29 DNA polymerase to form a long repeat single strand.With the help of Cas9 enzyme,the specificity is identified and double-stranded DNA is formed,and then the fluorescence intensity of the fluorescence signal was proportional to the concentration of the double-stranded DNA product by adding SYBR Green Ⅰ fluorescent dye.The constructed method was used to detect miRNA in serum of patients with gastric cancer and serum of healthy people.Whether miRNA was successfully detected and the levels of pepsinogen Ⅰ/pepsinogen Ⅱ(PG Ⅰ/Ⅱ)in serum of the two groups were detected at the same time,and receiver operating characteristic(ROC)curve was drawn.The diagnostic value of miRNA-21-5p and pepsinogen in gastric cancer was analyzed according to area under the curve(AUC).Results The con-structed amplification system could detect miRNA in serum,and the fluorescence intensity of mirNA-21-5P in serum of gastric cancer was higher than that in healthy control group,the difference was statistically significant(P＜0.05).ROC curve results showed that the AUC of miRNA-21-5p and PG Ⅰ/Ⅱ alone in the diagnosis of gastric cancer was 0.72 and 0.80,re-spectively,and the AUC of combined detection was 0.85,higher than that of any single index.Conclusion Using this method to directly detect miR-21-5p has convenient and fast operation,no need for complex thermal cycling instruments,and a wide range of applications.
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