Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye
Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye
复制标题
金纳米颗粒作为 SYBR Green I 染料实时聚合酶链反应添加剂的评价
DOI：
10.1088/0957-4484/19/25/255101
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发表时间：
2008-06-25
期刊：
影响因子：
3.5
通讯作者：
Hu, Jun
中科院分区：
文献类型：
--
作者：
Yang, Wenchao;Mi, Lijuan;Hu, Jun
金纳米粒子(AuNPs)已被证明能够提高聚合酶链式反应(PCR)的特异性或效率，当使用适量的AuNPs时。然而，目前还缺乏对AuNPs进行实时荧光定量检测的系统评价。在这项研究中，首次在实时荧光聚合酶链式反应中检测了DNA的降解和AuNPs的荧光猝灭效应。然后用两种不同类型的Taq DNA聚合酶，即天然Taq聚合酶和重组Taq聚合酶，研究了AuNPs对实时荧光定量PCR的阈值循环(C-T)值、标准曲线和熔化曲线的影响。还检测了不同比例的天然Taq DNA聚合酶和AuNPs的量。结果表明，AuNPs可用于实时荧光定量聚合酶链式反应，相关系数R-2>；为0.989。2.09 nM的AuNPs与3.75U的天然Taq DNA聚合酶的结合可使扩增曲线左移，提高了实时荧光定量聚合酶的检测效率(不含AuNPs的为0.628 39，而含2.09 nM的AuNPs为0.717 89)，从而使检测速度比对照样品更快。然而，在基于重组rTaq DNA聚合酶的实时荧光定量PCR中，没有发现AuNPs的改进能力。此外，结果表明AuNPs与天然Taq DNA聚合酶之间存在复杂的相互作用。
Gold nanoparticles (AuNPs) have been proven to be able to improve the specificity or increase the efficiency of a polymerase chain reaction (PCR) when a suitable amount of AuNPs was used. However, there is still a lack of systematic evaluation of AuNPs in real-time PCR. In this study, DNA degradation and the fluorescence quenching effect of AuNPs were first tested in real-time PCR. Then two different kinds of Taq DNA polymerase, native and recombinant Taq polymerase, were employed to evaluate the AuNPs' effect on the threshold cycle (C-T) values, standard curves and melting curves in real-time PCR. Different ratios of the amount of native Taq DNA polymerase to the amount of AuNPs were also tested. It was found that AuNPs could be applied in real-time PCR with correlation coefficient R-2 > 0.989. The combination of 2.09 nM AuNPs with 3.75 U of native Taq DNA polymerase could make the amplification curves shift to the left and enhance the efficiency of the real-time PCR (0.628 39 without AuNPs compared with 0.717 89 with 2.09 nM AuNPs), thus enabling faster detection in comparison with those of control samples. However, no improvement ability of AuNPs was found in real-time PCR based on recombinant rTaq DNA polymerase. Besides, the results suggest that a complex interaction exists between AuNPs and native Taq DNA polymerase.