LightCycler qPCR optimisation for low copy number target DNA
LightCycler qPCR optimisation for low copy number target DNA
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DOI：
10.1016/s0022-1759(02)00218-1
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发表时间：
2002-12-01
影响因子：
2.2
通讯作者：
Shaunak, S
中科院分区：
文献类型：
--
作者：
Teo, IA;Choi, JW;Shaunak, S
LightCycler是一种快速空气加热热循环仪，它包含一个荧光仪，用于检测和定量聚合酶链式反应(PCR)扩增产物。它提供对产品生成的实时逐个周期分析。放大发生在玻璃毛细管中。使用荧光双链DNA结合染料或荧光探针检测产物。然而，在传统的PCR反应中工作良好的条件并不容易转化为LightCycler。在使用这项新技术研究人类组织样本中的一种传染性病原体时，发现了几个参数，这些参数可能会对低拷贝数目标DNA的可靠和可重复性定量产生不利影响。这些问题包括聚合酶链式反应试剂在玻璃上的提取、引物-二聚体的形成、非特异性产物的产生，以及当低拷贝数靶存在于人类染色体DNA的高背景时未能扩增。对于确定的每个问题，都描述了几种解决方案。还描述了新的方法，以确保目标DNA的扩增和定量标准的扩增以相同的效率发生。对目前使用的方案进行适当修改后，LightCycler定量聚合酶链式反应(LC-qPCR)可用于实现超过酶免疫分析的准确度水平。当将这项技术应用于组织样本中病原体的研究时，所描述的LC-qPCR优化策略特别相关。这项技术为可靠和可重复性的低拷贝数目标DNA的定量PCR提供了巨大的潜力。(C)2002 Elsevier Science B.V.保留所有权利。
The LightCycler is a rapid air-heated thermal cycler which incorporates a fluorimeter for the detection and quantification of Polymerase Chain Reaction (PCR) amplified products. It provides real-time cycle-by-cycle analysis of product generation. Amplification occurs in glass capillary tubes. The products are detected using a fluorescent double stranded DNA binding dye or fluorescent probes. However, conditions that work well in conventional PCR reactions do not readily translate to the LightCycler. Whilst using this new technology to study an infectious pathogen in human tissue samples, several parameters were identified which can have an adverse effect on the reliable and reproducible quantification of low copy number target DNA. They included abstraction of PCR reagents on glass, primer-dimer formation, non-specific product generation, and a failure to amplify low copy number target when it is present in a high background of human chromosomal DNA. For each problem identified, several solutions are described. Novel approaches are also described to ensure that amplification of target DNA and of the quantification standards occurs with the same efficiency. With appropriate changes to the protocols currently in use, LightCycler quantitative Polymerase Chain Reaction (LC-qPCR) can be used to achieve a level of accuracy that exceeds that of an enzyme immunoassay. The LC-qPCR optimisation strategies described are of particular relevance when applying this technology to the study of pathogens in tissue samples. The technique offers the enormous potential for reliable and reproducible quantitative PCR of low copy number target DNA. (C) 2002 Elsevier Science B.V. All rights reserved.