The invention discloses a PCR amplification detection method, which comprises the following steps: 1) constructing a PCR
reaction system including: a
buffer solution, a dATP solution, a dCTP solution, a dGTP solution and a dTTP solution, an upstream primer, a downstream primer and a probe, that are complementary with a detection sample, and a bis-hot-start
DNA polymerase, which is formed by mixing chemical-modified hot-start Taq
enzyme and nano-
antibody-blocked hot-start Taq
enzyme according to
concentration ratio of 1:1; 2) blending the PCR
reaction system with a detection sample to perform PCR amplification; 3) acquiring circulation threshold value after the PCR amplification, and according to the circulation threshold value, determining the quantity of a to-be-detected substance in the detection sample. In the invention, the nano-
antibody is selected for preparing the hot-start Taq
enzyme to form the bis-hot-start
DNA polymerase, so that the PCR amplification detection method not only can simulate a
polymerase monoclonal antibody for application in hot-start PCR, but also can functionally replace the
monoclonal antibody, which is poor in stability and is greatly reduced in preparation cost.