Advanced Technology Research Laboratories, Panasonic Corporation, 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan.
Molecules. 2013 Sep 25;18(10):11751-67. doi: 10.3390/molecules181011751.
An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF) cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.
一种基于磁珠（MBs）上不对称聚合酶链反应（A-PCR）和随后应用循环探针技术（CPT）的端粒酶活性测定方法。在该测定中，端粒酶反应产物固定在 MBs 上，然后用 MBs 洗涤以去除通常存在于临床样品中的 PCR 抑制剂。然后通过 A-PCR 优先扩增端粒酶反应产物的富含鸟嘌呤的序列（5'-(TTAGGG)n-3'），并通过 CPT 检测扩增产物，其中具有荧光团的探针 RNA 在存在靶 DNA 的情况下在 RNase H 的作用下水解。催化剂介导的探针 RNA 切割增强了探针 5'端的荧光。该测定方法成功地选择性检测了 HeLa 细胞，而不是正常的人皮肤成纤维细胞（NHDF）细胞。重要的是，这种选择性在不存在和存在过量 NHDF 细胞的情况下对 HeLa 细胞的检测产生了相同的结果；因此，该测定可用于实际的临床应用。HeLa 细胞的检测下限为 50 个细胞，低于常规端粒重复扩增协议测定的检测下限。我们的测定还消除了由 PCR 抑制剂引起的假阴性结果。此外，我们表明该测定适用于筛选 G-四链体配体以寻找抑制端粒酶活性的配体。
