A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5' end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR setup. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluoresceinlabeled UT probe for the target gene and a 5-hexachloro-fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT-PCR analysis is relatively flexible, and it could be as short as 56bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT-PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT-PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.