Tracing Phosphate Ions Generated during Loop-Mediated Isothermal Amplification for Electrochemical Detection of Nosema bombycis Genomic DNA PTP1.
Tracing Phosphate Ions Generated during Loop-Mediated Isothermal Amplification for Electrochemical Detection of Nosema bombycis Genomic DNA PTP1.
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DOI：
10.1021/acs.analchem.5b01858
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发表时间：
2015-10
影响因子：
7.4
通讯作者：
Shunbi Xie;Yali Yuan;Y. Chai;R. Yuan
中科院分区：
文献类型：
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作者：
Shunbi Xie;Yali Yuan;Y. Chai;R. Yuan
传统上，环介导等温扩增（LAMP）中的扩增DNA检测在复杂的凝胶电泳中或使用昂贵的基于荧光的方法进行。在此，代替依赖于扩增的DNA的直接检测，已经提出了基于通过使用电化学方法追踪在LAMP期间产生的磷酸根离子（Pi）的间接检测用于灵敏的核酸检测。通过预先加入耐热无机焦磷酸酶（PPase），将LAMP中核酸聚合反应的副产物焦磷酸（PPi）水解为Pi。因此，LAMP扩增样品中Pi的总量与起始DNA模板的量成比例。然后，所获得的Pi可以与酸性磷酸盐反应，在电极表面形成磷酸盐沉淀，其充当氧化还原介体，以给出容易测量的电化学信号。通过对家蚕微孢子虫基因组DNA PTP 1的灵敏、准确定量，进一步证明了该方法的实用性。电化学方法可用于靶基因组DNA的定量分析，检测限为17 fg/μL。因此，我们认为，在这项工作中提出的新方法具有优越的上级灵敏度和特异性，以及简单的特点，可以很容易地建立在许多其他种类的核酸的定量分析的辅助LAMP。
Traditionally, amplified DNA detection in a loop-mediated isothermal amplification (LAMP) was carried out in a complicated gel electrophoresis or with expensive fluorescence-based methods. Here, instead of direct detection that relies on amplified DNA, the indirect detection based on tracing phosphate ions (Pi) generated during LAMP by using an electrochemical method has been proposed for sensitive nucleic acid detection. Pyrophosphate (PPi) as the byproduct of nucleic acid polymerization reaction in LAMP was hydrolyzed into Pi by the preaddition of thermostable inorganic pyrophosphatase (PPase). Thus, the total amount of Pi in the LAMP-amplified sample was proportional to the amount of starting DNA templates. The obtained Pi could then react with acidic molybdate to form the molybdophosphate precipitates on the electrode surface, which serve as redox mediators to give a readily measurable electrochemical signal. The practicality of this strategy has been further demonstrated by employing it for sensitive and accurate quantification of Nosema bombycis genomic DNA PTP1. The electrochemical method allowed the quantitative analysis for target genomic DNA with a detection limit of 17 fg/μL. Thus, we suppose that the novel method proposed in this work with superior sensitivity and specificity, as well as the simple feature, can be easily established for quantitative analysis of many other kinds of nucleic acids in the assistance of LAMP.