Tani Hidenori, Kanagawa Takahiro, Kurata Shinya, Teramura Tatsuya, Nakamura Kazunori, Tsuneda Satoshi, Noda Naohiro
Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Anal Chem. 2007 Feb 1;79(3):974-9. doi: 10.1021/ac061506o.
We have developed a simple, cost-effective, and accurate method for the quantification of specific nucleic acid sequences by the combined use of competitive PCR and a sequence-specific fluorescent probe that binds to either the gene of interest (target) or internal standard (competitor), referred to as alternately binding probe (ABProbe). In this method, the target and competitor were coamplified with the ABProbe, and then the fluorescence intensity was measured. The ratio of the target to the competitor can be calculated from the fluorescence intensity of the ABProbe using fluorescence quenching and fluorescence resonance energy transfer, that is, the starting quantity of the target is successfully calculated by end-point fluorescence measurement. Therefore, this method eliminates the complex post-PCR steps and expensive devices for real-time fluorescence measurement. We called this method alternately binding probe competitive PCR (ABC-PCR). We quantified amoA as a model target by ABC-PCR and real-time PCR. By comparison, the sensitivity, accuracy, and precision of ABC-PCR were similar to those of real-time PCR. Moreover, ABC-PCR was able to correctly quantify DNA even when PCR was inhibited by humic acid; therefore, this method will enable accurate DNA quantification for biological samples that contain PCR inhibitors.
我们开发了一种简单、经济高效且准确的方法,通过联合使用竞争性聚合酶链反应(PCR)和一种序列特异性荧光探针来定量特定核酸序列。该荧光探针可与目标基因(靶标)或内标(竞争物)结合,称为交替结合探针(ABProbe)。在这种方法中,靶标和竞争物与ABProbe共同扩增,然后测量荧光强度。利用荧光猝灭和荧光共振能量转移,根据ABProbe的荧光强度可以计算出靶标与竞争物的比例,即通过终点荧光测量成功计算出靶标的起始量。因此,该方法省去了复杂的PCR后处理步骤以及用于实时荧光测量的昂贵设备。我们将此方法称为交替结合探针竞争性PCR(ABC-PCR)。我们通过ABC-PCR和实时PCR对amoA作为模型靶标进行了定量。相比之下,ABC-PCR的灵敏度、准确性和精密度与实时PCR相似。此外,即使PCR受到腐殖酸抑制,ABC-PCR仍能够正确定量DNA;因此,该方法将能够对含有PCR抑制剂的生物样品进行准确的DNA定量。