双链特异性核酸切割酶等温循环扩增结合高效液相色谱高灵敏检测双目标microRNA  

Duplex Specific Nuclease-Enabled Isothermal Signal Amplification Combined with High-Performance Liquid Chromatography for Highly Sensitive Detection of Dual-Target MicroRNAs as Disease Biomarkers

作　　者：祁桐 宋畅 陈文慧 唐良秀 鞠嘉和 沈薇 孔德昭 唐盛 QI Tong;SONG Chang;CHEN Wen-Hui;TANG Liang-Xiu;JU Jia-He;SHEN Wei;KONG De-Zhao;TANG Sheng(School of Environmental and Chemical Engineering,Jiangsu University of Science and Technology,Zhenjiang 212003,China;School of Grain Science and Technology,Jiangsu University of Science and Technology,Zhenjiang 212003,China)

机构地区：[1]江苏科技大学环境与化学工程学院,镇江212003 [2]江苏科技大学粮食学院,镇江212003

出　　处：《分析化学》2021年第2期216-225,共10页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21605105,21705060);江苏省自然科学基金项目(No.BK20170570);江苏省六大人才高峰项目(No.SWYY-021)资助。

摘　　要：通过双链特异性核酸切割酶(DSN)循环扩增策略结合高效液相色谱(HPLC)法,实现了对两种疾病标志物microRNA(miRNA)的选择性分离和高灵敏检测。采用酶循环等温扩增策略增强了目标miRNA的信号,提高了HPLC法检测核酸的灵敏度。利用固定在磁珠上的不同长度和碱基序列的DNA探针实现了不同miRNA信号的色谱分离,磁性分离使背景噪音最小化,动态范围扩大。对目标miRNA-155的检出限为0.30 fmol/L,对miRNA-21的检出限为0.24 fmol/L。将本方法用于检测红斑狼疮、宫颈癌和卵巢癌血清样本中的miRNA-155和miRNA-21,结果与实时荧光定量聚合酶链式反应(qRT-PCR)结果相当。A method for selective separation and highly sensitive quantitation of dual-target miRNAs as disease biomarkers by duplex specific nuclease(DSN)-enabled signal amplification strategy combined with high-performance liquid chromatography(HPLC)was developed.The enzyme-based isothermal amplification strategy enhanced the signals of the target miRNAs and solved the problem of low sensitivity during the detection of nucleic acids utilizing conventional HPLC.To obtain an effective separation of the signals of different miRNAs,DNA probes which were immobilized on magnetic beads were designed with different lengths and base sequences.Effective magnetic separation minimized the background noise and expanded the dynamic range.Consequently,the assay achieved a detection limit of 0.33 fmol/L for miRNA-155 and 0.24 fmol/L for miRNA-21,respectively.The proposed assay was successfully applied to detection of miRNA-155 and miRNA-21 in serum samples from lupus erythematosus,cervical cancer and ovarian cancer patients.The results obtained here were comparable to those obtained by quantitative real-time polymerase chain reaction.

关 键 词：MICRORNA 双目标测定 荧光 双链特异性核酸切割酶 酶循环等温扩增 高效液相色谱 

分 类 号：O657.72[理学—分析化学] R440[理学—化学]