基于树枝状微流控芯片的分子诊断平台

病原体的快速准确诊断对于公共卫生具有至关重要的意义，传统诊断方法，如酶联免疫吸附实验（Enzyme-linked Immunosorbent Assay, ELISA）和聚合酶链式反应（Polymerase Chain Reaction, PCR），虽然被广泛应用，但它们存在诸如检测时间长、需要复杂设备等局限性，难以满足快速和现场检测的需求。近年来，微流控技术因其微小尺寸和高度集成化的特点，为快速、高效、灵敏度高的病原体检测提供了新的技术平台。

本研究将重组酶聚合酶扩增（Recombinase Polymerase Amplification, RPA）技术、T7转录技术以及成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）技术结合，研发了一种可用于乙型肝炎病毒（Hepatitis B Virus, HBV）核酸检测的方法。RPA技术使得核酸能在恒温条件下迅速扩增，结合T7转录可以将扩增产物转录成RNA，并被CRISPR/Cas13a技术进一步识别从而进行信号放大。与商用RPA荧光检测试剂盒相比，RPA-T7-Cas13a检测技术的终点荧光信号值可达到商用RPA检测试剂盒的10倍，具有更高的检测灵敏度。

在此基础上，本研究创新性地设计开发了一种基于树枝状结构的离心式微流控数字化检测芯片，采用离心驱动机制，降低了对注射泵等外部液体驱动控制设备的依赖。该平台利用树枝状微阵列结构缩小了反应单元体积，增加了样本反应单元数量。该检测平台能够在30 min内完成HBV病毒的核酸检测，检测灵敏度达到1 aM，并且能够在多种病原体混合物中准确检测到目标病原体，特异性良好。与高内涵成像系统结合还可以对HBV病毒核酸进行高效的定量分析。同现有的商业数字化微流控检测仪器相比，本研究开发的微流控检测平台无需复杂的芯片结构设计和样本液滴化处理过程，仅通过离心即可快速完成检测单元的分散，为病原体数字化检测的发展提供了新的思路和工具。

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