Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)
Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)
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DOI：
10.1038/s41598-018-36207-4
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发表时间：
2018-12-04
影响因子：
4.6
通讯作者：
Murray, James A. H.
中科院分区：
文献类型：
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作者：
Hardinge, Patrick;Kiddle, Guy;Murray, James A. H.
环介导扩增 (LAMP) 已广泛用于扩增并检测来自各种病原体、病毒和基因修饰的核酸靶序列。 LAMP 需要两种不同类型的引物；发夹形成LAMP和位移。使用多个引物可产生高特异性，但如果没有最佳的 LAMP 条件，灵敏度可能会很差。我们在此确认了 LAMP 引物设计、浓度和比例对于有效 LAMP 扩增的重要性。我们进一步表明，只要存在加速环引物，置换引物在某些浓度下对于 LAMP 反应不是必需的。我们研究了多种方法来定量来自转基因玉米认证参考材料的 DNA 提取物，以计算呈现给 LAMP 反应的模板的目标拷贝数，并表明，LAMP 可以使用设计用于靶向 35S 启动子 (35Sp) 或 NOS 终止子 (NOSt) 序列的引物来扩增从各种玉米转基因事件中提取的 DNA 中的转基因启动子/终止子序列，并通过实时生物发光 (BART) 和荧光方法进行检测。通过先前的变性和 HPLC 级 LAMP 引物，实现了单拷贝检测，这表明优化的 LAMP 条件可以与 BART 结合用于单拷贝目标，并且与荧光替代品相比，具有简单且经济高效的光检测电子设备。
Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives.