Fraunhofer Institute for Cell Therapy and Immunology - Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany.
Sci Rep. 2022 May 3;12(1):7137. doi: 10.1038/s41598-022-11320-7.
Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.
环介导等温扩增（LAMP）是最知名和最受欢迎的等温扩增方法之一。它的简单性和速度使其特别适合即时诊断。然而，假阳性结果仍然是一个主要的缺点。许多（下游）应用程序都已知用于检测 LAMP 扩增子，如比色测定法、原位 LAMP 或 CRISPR-Cas 系统。通常，需要对 LAMP 产物进行修饰，以用于不同的检测应用，如侧向流动测定法。这通常通过预修饰引物来实现。本研究旨在评估不同修饰核苷酸（如 Cy5-dUTP、生物素-dUTP 和氨基丙基-dUTP）对扩增子的标记作为预标记引物的替代方法。为此，研究了分子大小、核苷酸数量和靶浓度对扩增和标记效率的影响。这项研究表明，在 LAMP 过程中可以使用不同的修饰核苷酸对 LAMP 扩增子进行多样化标记，并且可以通过多种下游应用（如荧光光谱、凝胶电泳、微阵列和侧向流动系统）对这些样品进行分析。此外，基于微阵列的检测以及识别和区分假阳性的能力被证明是概念验证。
