Real-time fluorescent quantitative PCR probe detection method and reaction solution and kit thereof

Abstract

The invention discloses a real-time fluorescent quantitative PCR probe detection method and a reaction solution and a kit thereof. The reaction solution comprises a PCR buffer solution, a Taq hot start enzyme, dNTP, betaine with the final concentration of 0.04-0.05M, trehalose with the final concentration being not higher than 0.05M and tetramethylammonium chloride with the final concentration of30-40mM, and in the reaction solution, magnesium chloride is further added till the final concentration of the magnesium chloride is 3.0-5.0mM. The normal activity of the Taq hot start enzyme can be protected at high temperature through addition of the betaine and the trehalose with the specific concentrations into the reaction solution, the specificity of a PCR reaction can be improved through addition of the tetramethylammonium chloride with the specific concentration, and the concentration of the magnesium chloride in the reaction solution is further increased, so that the yield and the efficiency of amplification are significantly improved and the time required for the amplification can be effectively shortened while no influence on the specificity of the amplification is guaranteed.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a real-time fluorescence quantitative PCR probe detection method, a reaction solution and a kit thereof. Background technique [0002] Real-time fluorescent quantitative PCR (Real-time fluorescent quantitative PCR, qPCR) was first launched by Applied Biosystems in 1996, and has been widely used in gene expression differential analysis, nucleic acid copy number detection, single nucleotide polymorphism analysis , tumor gene detection, prenatal diagnosis and other basic scientific research, as well as clinical diagnosis, detection of pathogenic microorganisms, disease research and drug development and other fields. This technology can be divided into non-specific fluorescent dye method and specific fluorescent probe detection method. In terms of specific gene detection and quantification, Taqman and MGB probe detection methods are the most widely used, and there are c...

AI Technical Summary

Problems solved by technology

In applications using real-time fluorescent quantitative PCR, samples are often precious and low in content, and the length of reaction time has a great impact on stability and detection sensitivity. Short reaction time is conducive to ensuring stability and improving detection sensitivity, and vice versa. Test results

However, most of the reaction solution qPCR probe mix on the market currently has a reaction time of about 2 hours, and the reaction time is long; some commercial qPCR probe mixes shorten the reaction time, but the stability will be greatly reduced , it is difficult to achieve high amplification efficiency and detection sensitivity at the same time

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