Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection.
Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection.
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DOI：
10.1093/nar/gkt004
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发表时间：
2013-04
影响因子：
14.9
通讯作者：
Li Q
中科院分区：
文献类型：
--
作者：
Liao Y;Wang X;Sha C;Xia Z;Huang Q;Li Q
允许在一次测定中检测多个核酸靶标的多重分析系统可以提供样品的快速表征，同时仍然节省成本和资源。然而，很少有系统被证明能够提供高通量和成本有效的中重（例如10至50重）分析解决方案。在这里，我们描述了荧光颜色和解链温度（Tm）作为虚拟2D标记的组合使用，其能够在实时聚合酶链反应平台上对比当前策略多一个数量级的靶进行同质检测。靶首先与一对连接寡核苷酸杂交，其中一个含有人工序列，当与报告荧光探针杂交时，该人工序列具有独特的Tm。然后通过通用引物对扩增连接的产物，并通过解链曲线分析程序变性。通过在相应的荧光检测通道中它们各自的Tm值来鉴定靶标。通过对15种高危人乳头瘤病毒和48种人类单核苷酸多态性进行基因分型，验证了这种方法的原理证明。在两种情况下，通过分析大量临床样品证明了该方法的耐用性。多重性、灵活性和简单性的综合优点应使这种方法适合于各种应用。
Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.
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