恒温扩增检测miRNA-21-5p 系统的构建和初步评价

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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 GreenⅠ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（PGⅠ/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、PGⅠ/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。

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关键词 ： RCA 扩增, miRNA-21-5p, Cas9 酶, 胃癌, 胃蛋白酶原Ⅰ/胃蛋白酶原Ⅱ

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 Ⅰfluorescent 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 (PGⅠ/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 constructed 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 PGⅠ/Ⅱalone in the diagnosis of gastric cancer was 0.72 and 0.80,respectively,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.

Key words： RCA amplification MiRNA-21-5p Cas9 enzyme Gastric cancer Pepsinogen Ⅰ/pepsinogen Ⅱ

基金资助:广东省珠海市医学科研项目（2220009000157）。

引用本文:

曾元清；邓宁波▲. 恒温扩增检测miRNA-21-5p 系统的构建和初步评价[J]. 中国当代医药, 2024, 31(13): 4-09.
ZENG Yuanqing DENG Ningbo▲. Construction and preliminary evaluation of a thermostatic amplification detection system for miRNA-21-5p. 中国当代医药, 2024, 31(13): 4-09.

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https://www.dangdaiyiyao.com/CN/ 或 https://www.dangdaiyiyao.com/CN/Y2024/V31/I13/4

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