基于 5'UTR 区的快速、灵敏、特异检测肠道病毒 A-D 的温启动比色 RT-LAMP 方法。
WarmStart colorimetric RT-LAMP for the rapid, sensitive and specific detection of Enteroviruses A-D targeting the 5'UTR region.
机构信息
Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, School of Health Sciences, University of Thessaly, Biopolis, Larissa, Greece.
Department of Biochemistry & Biotechnology, Bioinformatics Laboratory, School of Health Sciences, University of Thessaly, Biopolis, Larissa, Greece.
出版信息
J Appl Microbiol. 2021 Jan;130(1):292-301. doi: 10.1111/jam.14770. Epub 2020 Jul 21.
AIMS
The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species A-D targeting the 5' untranslated region (5' UTR) of enteroviruses genome.
METHODS AND RESULTS
The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStart®Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50 min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0·30 CCID assay while the sensitivity for Enterovirus A was 3·00 CCID assay . The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses A-D and validated on 20 clinical isolates.
CONCLUSIONS
This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps.
SIGNIFICANCE AND IMPACT OF THE STUDY
We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5' UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV A-D reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses.
目的
本研究旨在开发一种针对肠道病毒属 A-D 的 5' 非翻译区（5'UTR）的比色环介导等温扩增（LAMP）检测方法。
方法和结果
通过逆转录酶将 RNA 转化为 cDNA，然后通过 WarmStart®Bst DNA 聚合酶在单个反应管中进行 LAMP 扩增，从而将反应时间缩短至 50 分钟。该方法对肠道病毒 B、C 和 D 的检测灵敏度为 0.30 CCID 测定，而对肠道病毒 A 的检测灵敏度为 3.00 CCID 测定。该检测方法对肠道病毒 A-D 的 45 个参考株具有高度特异性和灵敏度，并在 20 个临床分离株上得到验证。
结论
该方法可作为一种快速、敏感和特异的肠道病毒检测诊断工具，由于 LAMP 扩增产物通过颜色变化可视化，无需进行扩增后处理步骤，因此易于在小型临床和研究实验室中实施。
研究的意义和影响
我们开发了一种针对肠道病毒 5'UTR 的比色 LAMP 检测方法，该方法针对肠道病毒 5'UTR，具有高度的特异性和敏感性，基于对 45 株 EV A-D 参考株、20 株 EV B 临床分离株和 3 株非肠道病毒 RNA 病毒的检测性能。
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