Freeze-drying method for fluorescent PCR (Polymerase Chain Reaction) amplification reagent and application of fluorescent PCR amplification reagent

Abstract

The invention provides a freeze-drying method for a fluorescent PCR (Polymerase Chain Reaction) amplification reagent and application of the fluorescent PCR amplification reagent. The fluorescent PCRamplification reagent is prepared from a fluorescent PCR amplification buffering solution, a primer, a probe, a hot start Taq enzyme, a UNG enzyme, dATP, dGTP, dCTP, dTTP, dUTP and a freeze-drying protection agent. All components of the fluorescent PCR amplification reagent are uniformly mixed; after a mixture is pre-frozen at -35 DEG C for 3h, air pressure of a freeze-drying machine is reduced tobe 10Pa or lower, and then the mixture subjected to vacuum treatment for 2h at -35 DEG C, vacuum treatment for 2h at 10 DEG C and vacuum treatment for 2h at 30 DEG C, so as to obtain fluorescent PCRamplification reagent freeze-dried powder. According to the freeze-drying method provided by the invention, all the components needed by fluorescent PCR are prepared into the same freeze-dried powder,and room-temperature transportation and preservation can be realized; the freeze-dried powder can be dissolved by only utilizing a freeze-dried powder dissolving solution in a utilization process andthen is added into a sample. The freeze-dried powder has the characteristics of convenience for utilization, stability and reliability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a freeze-drying method for fluorescent PCR amplification reagents and an application thereof. Background technique [0002] Fluorescent quantitative PCR technology is a simple, fast, highly sensitive, and specific gene detection method. The necessary conditions for its reaction include: fluorescent PCR reaction buffer system, primers, probes, Taq enzyme, and dNTP. [0003] Among them, the components of the fluorescent PCR reaction buffer system are usually chemical reagents and water, which are relatively stable during storage; primers, probes, and dNTPs are chemically synthesized reagents that need to be stored at low temperature to avoid changes in their properties Thus affecting the use effect; Taq enzyme is an active protein obtained by gene expression method, which needs to be stored at low temperature to maintain its activity; if the enzyme, dNTP, primer probe are m...

AI Technical Summary

Problems solved by technology

The use of this form of product is complicated. The reaction buffer and enzyme must be mixed evenly according to the proportion, then distributed into PCR amplification tubes, and then added to the sample.

Due to the small amount of enzyme (≤0.5 μL), the enzyme solution is relatively viscous, and the preparation process is cumbersome, the preparation error is large, and the reaction repeatability is not good.

Moreover, reagents need to be stored at low temperature (below -20°C), and the cost of product storage and transportation is relatively high

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