Department of Clinical Laboratory Medicine, Chinese PLA General Hospital & Chinese PLA Medical School, Beijing 100853, People's Republic of China; College of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325000, People's Republic of China.
Beijing Tag Array Molecular Test Co., Ltd, Beijing 100085, People's Republic of China.
J Virol Methods. 2018 Apr;254:25-30. doi: 10.1016/j.jviromet.2018.01.008.
Quantitative PCR (qPCR) assay using TaqMan™ probe was widely used in the detection of different nucleic acids. However, this technology has several drawbacks, including false negative results caused by primer-dimer (PD) and false positive issues due to primer-probe aggregations. Here, we designed a modified TaqMan™-Molecular Beacon probe by adding an antisense base and a new type of primer pair named central-homo primer pairs bearing 5-10 bases homologous sequence on the 3' end. Using the HBV qPCR assay as a proof of concept, the new design significantly improved the accuracy of the TaqMan™ qPCR assay for HBV detection. Application of the central-homo primer pair led to significantly delayed Ct values by 5-10 cycles compared with conventional primer design. The modified probe containing an antisense base did not produce any detectable signal in repeating primer-probe aggregation experiments. Furthermore, the use of the central-homo primer pair and the non-competitive internal control could solve the false negative problem caused by PD formation. We validated this customized duplex qPCR system using 208 clinical samples collected from patients in clinic showing accuracy was higher than that of the conventional qPCR method.
定量聚合酶链反应 (qPCR) 检测法使用 TaqManTM 探针在不同核酸的检测中被广泛应用。然而，该技术存在几个缺点，包括由引物二聚体 (PD) 引起的假阴性结果和由于引物-探针聚集而产生的假阳性问题。在这里，我们通过添加反义碱基和一种新的引物对设计了一种改良的 TaqManTM-分子信标探针，这种新的引物对称为中心同源引物对，在 3'端具有 5-10 个碱基的同源序列。我们使用 HBV qPCR 检测法作为概念验证，新设计显著提高了 TaqManTM qPCR 检测 HBV 的准确性。与传统的引物设计相比，使用带有 5-10 个循环同源序列的中心同源引物对显著延迟了 Ct 值。含有反义碱基的改良探针在重复的引物-探针聚集实验中不会产生任何可检测的信号。此外，使用中心同源引物对和非竞争性内部对照可以解决由 PD 形成引起的假阴性问题。我们使用从临床收集的 208 个临床样本验证了这个定制的双 qPCR 系统，其准确性高于传统 qPCR 方法。
