An empirical approach for quantifying loop-mediated isothermal amplification (LAMP) using Escherichia coli as a model system.
An empirical approach for quantifying loop-mediated isothermal amplification (LAMP) using Escherichia coli as a model system.
复制标题
使用大肠杆菌作为模型系统量化环介导等温扩增 (LAMP) 的经验方法。
DOI：
10.1371/journal.pone.0100596
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Gomez RD
中科院分区：
文献类型：
--
作者：
Subramanian S;Gomez RD
环路介导的等温扩增(LAMP)是一种高效、选择性、快速的DNA扩增技术，用于病原菌的遗传筛选。然而，尽管它很受欢迎，但还没有数学模型来量化结果，也没有明确定义的衡量标准来比较可用的结果。LAMP本质上是复杂的，涉及到基因复制的多条途径，使得基础模型几乎难以处理。为了绕过这一困难，引入了一种替代的经验模型，该模型将允许人们从浓度-时间曲线中提取一组参数。文中还给出了一个推导阳性反应时间的简单配方--Tp--一个类似于聚合酶链式反应(PCR)中的阈值循环时间的参数。这些参数可以作为LAMP放大的客观和明确的指标。以产生Vero-毒素(VT)的两个基因片段为例，对产生VT的菌株(O157和O45)和不产生VT的菌株(DH5α)进行了测试。使用已建立的LAMP引物和程序对适当的靶序列进行选择性扩增，并在特定时间间隔使用Qubit 2.0荧光计测量扩增产物的浓度。数据符合广义Logistic函数。除了提供精确的筛查指标外，用一小组数字表示数据还具有显著的优势。它便于独立于采样技术的LAMP反应的比较。它还消除了解释中的主观性，简化了数据分析，并允许对大样本总体进行轻松的数据归档、检索和统计分析。据我们所知，这项工作是对LAMP进行定量建模的第一次尝试，并提供了一种标准方法，为高通量自动筛选铺平了道路。
Loop mediated isothermal amplification (LAMP) is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp - a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR), is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT) production and tested against VT-producing (O157 and O45) and non-VT producing (DH5 alpha) strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening.
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