Quantitative method for specific nucleic acid sequences using competitive polymerase chain reaction with an alternately binding probe

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.