Yang Wenchao, Mi Lijuan, Cao Xueyan, Zhang Xiaodong, Fan Chunhai, Hu Jun
Nanobiology Laboratory, Bio-X Life Science Research Center, College of Life Science and Biotechnology, Shanghai JiaoTong University, Shanghai 200240, People's Republic of China.
Nanotechnology. 2008 Jun 25;19(25):255101. doi: 10.1088/0957-4484/19/25/255101. Epub 2008 May 14.
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.
已证实，当使用适量的金纳米颗粒（AuNPs）时，其能够提高聚合酶链反应（PCR）的特异性或增加其效率。然而，对于AuNPs在实时荧光定量PCR中的应用仍缺乏系统评估。在本研究中，首先在实时荧光定量PCR中测试了AuNPs的DNA降解和荧光猝灭效应。然后使用两种不同的Taq DNA聚合酶，即天然Taq聚合酶和重组Taq聚合酶，来评估AuNPs对实时荧光定量PCR中熔解曲线、标准曲线和扩增曲线的影响。同时也测试了天然Taq DNA聚合酶与AuNPs不同比例的组合。结果发现，AuNPs可应用于实时荧光定量PCR，相关系数R²>0.989。2.09 nM AuNPs与3.75 U天然Taq DNA聚合酶的组合可使扩增曲线左移，提高实时荧光定量PCR的效率（未添加AuNPs时为0.628 39，添加2.09 nM AuNPs时为0.717 89），从而与对照样品相比能够更快地进行检测。然而，在基于重组rTaq DNA聚合酶的实时荧光定量PCR中未发现AuNPs具有改善作用。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。
