恒温扩增检测miRNA-21-5p 系统的构建和初步评价

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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

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