【摘要】 目的 建立基于DNA荧光编码微球技术的多重微小核糖核酸(miRNA)检测方法,验证其在肝母细胞瘤(HB)相关miRNA多重检测中的临床应用价值。方法 基于计算机辅助设计(CAD)的四发夹杂交链式反应(qHCR)系统,构建四色荧光(Pacific Bluc、FAM、TAMRA、Cy5)编码微球体系,通过激光共聚焦显微镜对微球的荧光编码性能、形态特征进行表征;以HB相关的7种miRNA(miR-21、miR-26a、miR-27a、miR-122、miR-192、miR-801、miR-223)为靶标,评估方法的检出限、线性范围及特异性;并进一步在血清模拟样本中,采用该方法进行7种miRNA同时检测,验证其多重分析性能。结果 成功构建四色DNA荧光编码微球体系,微球粒径均一,荧光信号稳定性良好。该方法对HB相关miRNA的检出限为1.99 pmol/L(R~2>0.97),且能同时检测HB组中7种miRNA。结论 基于DNA荧光编码微球的多重miRNA检测技术具有高灵敏度、高特异性及多重分析能力,可实现HB相关miRNA的快速多重检测,为HB的早期诊断和病情监测提供新的技术支撑。更多还原

【Abstract】 Objective To establish a multiplex microRNA(miRNA) detection method based on DNA fluorescent-cncoded microsphere technology,and verify its clinical application value in the multiplex detection of hepatoblastoma(HB)-associated miRNAs.Methods A four-color fluorescent(Pacific Blue,FAM,TAMRA,Cy5)-encoded microsphere system was constructed based on a computer-aided design(CAD)-enabled quadhairpin hybridization chain reaction(qHCR) system.The fluorescent encoding performance and morphological characteristics of the microspheres were characterized using a laser confocal microscope.Seven HB-associated miRNAs(miR-21,miR-26a,miR-27a,miR-122,miR-192,miR-801,miR-223) were used as targets to evaluate the method’s limit of detection(LOD),linear range,and specificity.Furthermore,simultaneous detection of the seven miRNAs was performed using this method in serum-mimetic samples to verify its multiplex analysis performance.Results The four-color DNA fluorescent-encoded microsphere system was successfully constructed,with uniform microsphere particle size and excellent fluorescent signal stability.For HB-associated miRNAs,the method exhibited a LOD of 1.99 pmol/L(R~2>0.97) and could simultaneously detect the seven miRNAs in HB-rclated samples.Conclusion The multiplex miRNA detection technology based on DNA fluorescent-encoded microspheres possesses high sensitivity,high specificity,and multiplex analysis capability.It enables rapid multiplex detection of HB-associated miRNAs and provides new technical support for the early diagnosis and disease monitoring of HB.更多还原