Bis-hot-start DNA polymerase containing nano-antibody and PCR amplification detection method

Abstract

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.

Description

【Technical Field】 [0001] The invention belongs to the technical field of DNA polymerases for fluorescence quantitative PCR amplification and detection, and particularly relates to a double hot-start DNA polymerase and a PCR amplification detection method. 【Background technique】 [0002] When DNA polymerase performs PCR reaction, it still has residual enzyme activity during the process from low temperature (20℃~37℃) to high temperature (94℃~95℃), and low-level non-specific annealing and extension are at the beginning and This occurs during thermal cycling, which in turn leads to the formation of non-specific amplification products in subsequent PCR cycles, ultimately reducing the sensitivity and yield of the reaction by reducing the required amplification signal or the signal is masked by high background values. Hot-start PCR is to start the PCR enzymatic reaction after the initial denaturation stage. The hot-start reduces the formation of primer dimers, primer mismatches and non-...

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Problems solved by technology

[0004] Chinese patent application No. CN201510707423.1 discloses a quantitative PCR method containing dye EvaGreen and dual hot-start enzymes. The dual hot-start enzymes in this patent include: HotStarTaq DNA polymerase and Anti Taq DNA polymerase. The Anti Taq DNA polymerase is a common monoclonal antibody. Although the hot-start Taq polymerase blocked by monoclonal antibody has strong specificity, its preparation process is complicated, time-consuming and expensive.

[0005] Generally speaking, the stability and sensitivity of the above-mentioned PCR amplification method are still not high, therefore, it is necessary to provide a new dual hot start DNA polymerase to solve the above problems

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