Measurement of microRNA with isothermal DNA amplification on fully automated immunoassay analyzers
Measurement of microRNA with isothermal DNA amplification on fully automated immunoassay analyzers
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DOI：
10.1007/s00216-019-01878-z
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发表时间：
2019-06
影响因子：
4.3
通讯作者：
Makoto Komori;K. Komiya;Takuma Shirakawa;Takamitsu J. Morikawa;T. Yoshimura
中科院分区：
文献类型：
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作者：
Makoto Komori;K. Komiya;Takuma Shirakawa;Takamitsu J. Morikawa;T. Yoshimura
血液样本中的微小RNA（miRNA）通常通过定量逆转录PCR（qRT-PCR）、微阵列和下一代测序（NGS）来测量，其需要耗时的预处理、手动操作和独立仪器。为了克服这些缺点，已经开发了使用临床实验室中常规使用的自动化分析仪进行miRNA检测。等温DNA扩增反应适用于在37 °C下在44分钟内进行提取、扩增和检测过程的全自动免疫测定分析仪。在反应容器中，使用DNA模板、DNA聚合酶和切口核酸内切酶在miRNA存在下扩增预先设计的单链信号DNA。然后，将扩增的信号DNA与附着于磁性颗粒的一种DNA探针和用吖啶酯标记的另一种DNA探针杂交。在化学发光反应之后，自动测量发光强度。癌症相关miRNA的自动化测定在分析仪上进行，通量为每小时66次测试。在一步扩增的测定中，自动检测到浓度低于100 fM的三种miRNA（miR-21- 5 p、miR-18 a-5 p和miR-500 a-3 p），并且miR-21- 5 p与15种相似miRNA的交叉反应性不高于0.02%。在两步扩增的测定中，miR-21- 5 p的检测灵敏度和扩增率分别为3 fM和103倍。在5 fM至1000 pM的目标浓度下，测量的变异系数（CV）小于8%。此外，我们还实现了人血清中核酸的自动检测。所提出的全自动miRNA检测方法显示出高灵敏度、低交叉反应性和适用于临床的可重复性。
MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use.Graphical abstract