Xie Shunbi, Yuan Yali, Chai Yaqin, Yuan Ruo
State Key Laboratory of Silkworm Genome Biology, College of Chemistry and Chemical Engineering, Southwest University , Chongqing 400715, China.
Anal Chem. 2015 Oct 20;87(20):10268-74. doi: 10.1021/acs.analchem.5b01858. Epub 2015 Oct 5.
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.
传统上，环介导等温扩增（LAMP）中的扩增DNA检测是通过复杂的凝胶电泳或昂贵的基于荧光的方法进行的。在此，提出了一种基于电化学方法追踪LAMP过程中产生的磷酸根离子（Pi）的间接检测方法，而非依赖扩增DNA的直接检测方法，用于灵敏的核酸检测。LAMP中核酸聚合反应的副产物焦磷酸（PPi）通过预先添加耐热无机焦磷酸酶（PPase）水解成Pi。因此，LAMP扩增样品中Pi的总量与起始DNA模板的量成正比。然后，获得的Pi可以与酸性钼酸盐反应，在电极表面形成磷钼酸盐沉淀，作为氧化还原介质产生易于测量的电化学信号。通过将该策略用于家蚕微孢子虫基因组DNA PTP1的灵敏和准确定量，进一步证明了该策略的实用性。电化学方法能够对目标基因组DNA进行定量分析，检测限为17 fg/μL。因此，我们认为本文提出的新方法具有卓越的灵敏度和特异性，且操作简单，在LAMP的辅助下能够轻松建立用于多种其他核酸的定量分析方法。
