Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye
Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye
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DOI：
10.1038/srep11479
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发表时间：
2015-06-19
影响因子：
4.6
通讯作者：
Neuzil, Pavel
中科院分区：
文献类型：
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作者：
Ahberg, Christian D.;Manz, Andreas;Neuzil, Pavel
自1985年发明以来，聚合酶链式反应(PCR)已经成为一种成熟的双链DNA片段扩增和检测方法。将荧光探针或DNA嵌入染料(如SYBR Green)加入到聚合酶链式反应混合物中，可实现实时反应监测和定量信息提取(QPCR)。具有不同激发光谱的探针可以使用多通道光学检测系统对几个DNA片段进行多重qPCR。在这里，我们展示了使用经济的基于EvaGreen的单光通道检测系统的多路qPCR。以前报道的基于插层染料的非定量多重实时PCR技术是在通过熔融曲线分析(MCA)完成聚合酶链式反应(PCR)后进行的。本文介绍的技术既是定性的，也是定量的，因为它提供了关于测试样本中存在多条DNA链以及起始拷贝数的信息。除了重要的内部控制外，多重定量聚合酶链式反应还允许检测同一样本中不止一条DNA链的浓度。用聚合酶链式反应检测禽流感病毒H7N9是一种成熟的方法。多重定量聚合酶链式反应能够同时区分血凝素(HA)和神经氨酸酶(NA)基因及其比例，从而大大提高了其特异性。
Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex realtime PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.