Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in Real Time (BART).
Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in Real Time (BART).
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优化的环介导等温扩增 (LAMP) 允许使用实时生物发光测定 (BART) 进行单拷贝检测。
DOI：
10.1007/978-1-0716-2453-1_8
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Hardinge P
中科院分区：
文献类型：
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作者：
Hardinge P
实时生物发光分析（BART）是一种通过记录光输出来检测核酸扩增的分子诊断工具。BART的关键成分是一种耐热荧光素酶，来源于萤火虫pyralis。将BART检测与等温扩增方法相结合，环路介导等温扩增（LAMP）可以使用简单的设备快速获得分子诊断结果。LAMP-BART提供封闭管的定量结果，适用于微升标准测试和纳升微流体分析。在本章中，我们介绍了一种使用LAMP和BART扩增和检测遗传标记的方案。此外，我们提供了优化LAMP检测的建议，以获得高灵敏度和特异性，并消除检测组成部分可能出现的“假阳性”结果的发生率。介绍了利用野碱合成酶终止子（NOSt）和35S启动子（35Sp）序列对转基因玉米进行优化的方法。
The bioluminescent assay in real time (BART) is a molecular diagnostic tool for the detection of nucleic acid amplification by recording light output. The key component for BART is a thermostable luciferase derived from the fireflyPhotinus pyralis. Coupling BART detection with the isothermal amplification method loop-mediated isothermal amplification (LAMP) enables rapid molecular diagnostic results using simple equipment. LAMP-BART provides quantitative results from a closed tube and is appropriate to microliter standard tests and nanoliter microfluidic assays. In this chapter, we introduce a protocol to amplify and detect genetic markers using LAMP with BART. Furthermore, we provide advice to optimize LAMP assays for high sensitivity and specificity and to eliminate the incidence of “false positive” results which can occur from the components of the assay. The optimization of genetically modified (GM) maize by targeting the nopaline synthase terminator (NOSt) and 35S promoter (35Sp) sequences is described.