EXODUS系统联合变构DNA纳米开关在帕金森病患者血浆外泌体miR-107检测中的应用  

Application of EXODUS system combined with allosteric DNA nanoswitches in the detection of miR-107 among plasma exosomes of Parkinson′s disease patients

作　　者：王雄[1] 程黎明[1] Wang Xiong;Cheng Liming(Department of Laboratory Medicine,Tongji Hospital,Tongji Medical College of Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院检验科,武汉430030

出　　处：《中华预防医学杂志》2024年第8期1191-1196,共6页Chinese Journal of Preventive Medicine

摘　　要：探索帕金森病患者血浆外泌体miR-107快速检测方法。采用病例对照设计,收集华中科技大学同济医学院附属同济医院检验科2023年12月至2024年1月帕金森病患者组和健康对照组血浆标本各10例。EXODUS系统分离血浆外泌体,纳米颗粒跟踪分析技术和透射电镜鉴定外泌体粒径和形态,Qiagen miRNeasy Micro Kit提取外泌体RNA,变构DNA纳米开关miRacles检测miR-107,琼脂糖凝胶电泳分析miR-107含量。采用Thermo Fisher RevertAid RT Reverse Transcription Kit对外泌体RNA进行逆转录,实时定量PCR检测miR-107。组间比较采用独立样本t检验。EXODUS系统1.5 h完成500μl血浆外泌体的分离,外泌体浓度健康对照组为(4.82±2.02)×10^(10)个/ml,帕金森病患者组为(5.08±2.34)×10^(10)个/ml,两组间差异无统计学意义(t=-0.168,P=0.872),透射电镜证实外泌体形态。变构DNA纳米开关miRacles能检测fM级miR-107,可有效区分miR-107及其家族成员miR-15a和miR-16。琼脂糖凝胶电泳显示,灰度值相对含量健康对照组为(1.00±0.26),帕金森病患者组为(1.86±0.21),帕金森病患者组miR-107显著高于健康对照(t=-8.143,P<0.001),与逆转录实时定量PCR结果类似。EXODUS系统联合变构DNA纳米开关miRacles能实现帕金森病患者血浆外泌体miR-107快速、非酶促、廉价的检测。This study aimed to achieve rapid detection of Parkinson′s disease(PD)plasma exosome miR-107.A case-control design was used to collect ten Parkinson′s disease and ten healthy control plasma samples from the Department of Laboratory Medicine,Tongji Hospital,Tongji Medical College of Huazhong University of Science and Technology from December 2023 to January 2024.Exosome detection via the ultrafast-isolation system(EXODUS)was used to isolate plasma exosomes.The nanoparticle tracking analysis technology and electron microscopy were used to identify exosome particle size and morphology.The Qiagen miRNeasy Micro Kit was used to extract RNA.The microRNA-activated conditional looping of engineered switches(miRacles)was used to detect miR-107,and the relative expression was analyzed by agarose gel electrophoresis.Thermo Fisher RevertAid RT Reverse Transcription Kit was used to perform reverse transcription of RNA,and real-time PCR was used to detect miR-107.The independent samples t-test was used for comparison between groups.EXODUS system completed the isolation of exosomes from 500μl plasma within 1.5 hours.The exosome concentration(mean±SD)was(4.82±2.02)×10^(10)particles/ml in the control group and(5.08±2.34)×10^(10)particles/ml in the PD group.There was no significant difference in exosome concentration between PD patients and healthy controls(t=-0.168,P=0.872).The morphology of exosomes was confirmed by electron microscopy.The miRacles nanoswitch could detect fM-level miR-107 and also effectively distinguish miR-107 from its family members,including miR-15a and miR-16.Agarose gel electrophoresis showed that the mean±SD of relative grey value content was 1.00±0.26 in the control group and 1.86±0.21 in the PD group.The miR-107 in the PD group was significantly higher than that in the control(t=-8.143,P<0.001),which was consistent with the result of real-time PCR.EXODUS combined with miRacles could achieve rapid,non-enzymatic and cheap detection of plasma exosomal miR-107 in PD patients.

关 键 词：外泌体 MicroRNA EXODUS 变构DNA纳米开关 标志物 病例对照研究 

分 类 号：R742.5[医药卫生—神经病学与精神病学]