QuantiBact A/S, Hvidovre, Denmark.
PLoS One. 2012;7(6):e38451. doi: 10.1371/journal.pone.0038451. Epub 2012 Jun 6.
We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.
我们介绍了定量聚合酶链反应（qPCR）引物和经过改良的多重终点 PCR 引物，即在 5'端添加单个邻位扭曲嵌入核酸（o-TINA）分子。在 qPCR 中，5'-o-TINA 修饰的引物在显著的应激反应条件下允许 qPCR 效率达到 100%，与未修饰的引物相比，提高了 qPCR 检测的稳健性。在掺入基因组 DNA 的样品中，与未修饰的 DNA 引物相比，5'-o-TINA 修饰的引物通过提高灵敏度和特异性来提高稳健性。在未掺入样品中，用 5'-o-TINA 修饰的引物代替未修饰的 DNA 引物可提高 qPCR 的严格性。与未修饰的 DNA 引物相比，这允许在降低引物浓度和增加退火温度的情况下保持 100%的 qPCR 效率，同时保持与单碱基错配引物的交叉反应性不变。在之前发表的针对致泻性大肠杆菌的八重终点 PCR 中，应用 5'-o-TINA 修饰的引物可以进一步减少（>45%或大约一个小时）整个 PCR 程序长度，同时维持所有目标在粗菌裂解物中的扩增和分析灵敏度。对于所有粗菌裂解物，5'-o-TINA 修饰的引物允许在所有八个目标的较低引物浓度和较高退火温度下对 PCR 严格性进行实质性提高。此外，用人类基因组 DNA 掺入的粗菌裂解物显示出较少的非靶标扩增子形成，这意味着稳健性提高。因此，在需要一个或多个引物对在应激反应条件下进行的 PCR 检测中，5'-o-TINA 修饰的引物是有利的。
