Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

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.

本研究介绍了在5'端添加单个邻位扭曲插入核酸（ortho-Twisted Intercalating Nucleic Acid，o-TINA）分子修饰的实时荧光定量聚合酶链式反应（quantitative polymerase chain reaction，qPCR）引物与多重终点聚合酶链式反应（multiplex end-point PCR）引物。在实时荧光定量聚合酶链式反应（qPCR）中，经5'端o-TINA修饰的引物可在显著严苛的反应条件下实现100%的qPCR扩增效率，相较于未修饰引物，可提升qPCR检测的稳健性。在掺入基因组DNA的样本中，相较于未修饰的DNA引物，经5'端o-TINA修饰的引物可通过提升灵敏度与特异性来增强检测稳健性。在未掺入基因组DNA的样本中，用5'端o-TINA修饰的引物替换未修饰的DNA引物，可提升qPCR反应的严谨性。相较于未修饰的DNA引物，该修饰引物可在更低的引物浓度与更高的退火温度下实现100%的qPCR扩增效率，且对存在单个碱基错配的引物仍保持不变的交叉反应性。在此前已发表的针对致泻性大肠埃希菌的八重终点聚合酶链式反应体系中，使用5'端o-TINA修饰的引物可进一步缩短总PCR程序时长（降幅超45%，或缩短约1小时），同时在粗制细菌裂解液样本中仍能维持所有靶标的扩增效果与分析灵敏度。针对所有粗制细菌裂解液样本，5'端o-TINA修饰的引物可显著提升PCR反应严谨性，具体表现为所有8个靶标均可在更低的引物浓度与更高的退火温度下完成扩增。此外，掺入人类基因组DNA的粗制细菌裂解液样本中非靶标扩增产物的形成量更少，这意味着检测稳健性得到提升。综上，在需要一对或多对引物在严苛反应条件下完成扩增的PCR检测体系中，5'端o-TINA修饰的引物具有显著优势。