[1]植晓玲,李菁菁,顾纪生,等.Cy5光学分子信标的制备及乳腺癌miRNA-21的检测研究[J].徐州医科大学学报,2022,42(05):375-379.[doi:10.3969/j.issn.2096-3882.2022.05.012]
　Preparation of Cy5 optical molecular beacon and detection of miRNA-21 in breast cancer[J].Journal of Xuzhou Medical University,2022,42(05):375-379.[doi:10.3969/j.issn.2096-3882.2022.05.012]
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Cy5光学分子信标的制备及乳腺癌miRNA-21的检测研究()
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《徐州医科大学学报》[ISSN:2096-3882/CN:32-1875/R]

卷:
42
期数:
2022年05期
页码:
375-379
栏目:
出版日期:
2022-05-25

文章信息/Info

Title:
Preparation of Cy5 optical molecular beacon and detection of miRNA-21 in breast cancer
作者:
植晓玲; 李菁菁; 顾纪生; 戴家杰; 孙春鸣; 曹旭; 赵欣玥; 陈明慧*
徐州医科大学医学影像学院，江苏徐州221004
关键词:
miRNA-21; 乳腺癌; 等温循环链置换反应; hDNA
分类号:
R737.9
DOI:
10.3969/j.issn.2096-3882.2022.05.012
文献标志码:
A
摘要:
目的设计一种检测早期乳腺癌筛查肿瘤标志物microRNA-21(miRNA-21)的Cy5光学茎环DNA分子信标探针，构建引物堆叠的等温循环链置换荧光定量分析方法，实现对miRNA-21的高灵敏检测。方法设计Cy5光学标记的茎环hairpinDNA (hDNA)探针，在靶标miRNA-21的配对引发下，激活等温循环链置换扩增反应，采用荧光定量分析方法，优化实验用量，对miRNA-21进行灵敏度、特异度检测，并与反转录荧光定量PCR（qRT-PCR）方法检测结果进行比较。结果本研究设计的Cy5光学hDNA探针构建的等温循环链置换扩增反应，可对miRNA-21 进行高灵敏度检测，其检测限可低至1 fmol/L；检测miRNA-21特异度良好，且不与其他miRNA反应产生荧光，能有效实现单碱基突变miRNA的区分；检测miRNA-21结果与金标准qRT-PCR检测结果的差异无统计学意义（P＞0.05），证明本探针检测结果具有可信性。结论本研究设计的Cy5光学探针检测miRNA-21的灵敏度和特异度高，可实现对早期乳腺癌患者的筛查及诊断。
Abstract:
ob＜x＞jective: MicroRNA-21 (miRNA-21) can be used as a tumor marker for early breast cancer screening.This study aims to study and design a Cy5 optical stem loop DNA molecular beacon probe to detect microRNA-21, construct a primers stack isothermal cyclic chain replacement fluorescence quantitative analysis method, and achieve highly sensitive detection of miRNA-21. Methods: Stem loop hDNA probe with Cy5 optical label was designed, and isothermal cyclic chain replacement amplification reaction was activated under the pairing initiation of target mirNA-21. Fluorescence quantitative analysis method was used to optimize the experimental dosage, and sensitive and specific detection of miRNA-21 was performed. This study will prove the reliability of this study as a detection method for miRNA-21 by comparing the detection results with qRT-PCR method. Results: The chain replacement isothermal amplification reaction constructed by Cy5 optical hDNA probe designed in this study could detect miRNA-21 with high sensitivity, and the detection limit was as low as 1fM. MiRNA-21 has good specificity and does not react with other miRNAs to produce fluorescence, which can effectively distinguish single ba＜x＞se mutated microRNA(miRNA). There was no significant difference between the detection results of miRNA-21 by the probe and that of miRNA-21 by gold standard QRT-PCR (P > 0.05), which proved the reliability of the detection results of the probe. Conclusions: The Cy5 optical probe used in this study can achieve highly sensitive detection of miRNA-21 with high specificity, in order to achieve screening and diagnosis of early breast cancer patients

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备注/Memo

备注/Memo:
基金项目：大学生创新创业训练项目（20201031309Z）*通信作者,E-mail：minghuiai@xzhmu.edu.cn
更新日期/Last Update: 2022-06-07