CN-115181786-B - Real-time fluorescence PCR detection solid detection reagent and preparation method thereof

Abstract

The invention relates to a real-time fluorescence PCR solid detection reagent and a preparation method thereof, wherein the real-time fluorescence PCR solid detection reagent is prepared by mixing a protection system, a glycerol-free buffer solution, dNTPs premix, a specific primer probe and glycerol-free Taq polymerase and then performing freeze drying treatment. The real-time fluorescence PCR solid detection reagent can keep the original diagnosis effect and sensitivity for at least 2 years at normal temperature, and can detect 2 copy number nucleic acid molecules at the lowest. Solves the problems that the existing real-time PCR detection reagent is required to be prepared on site and requires strict cold chain technology in long-distance transportation, greatly increases transportation and storage expenditure, and causes long transportation time, insufficient inventory of relevant reagents in some undeveloped areas and the like.

Inventors

• YANG SIYU
• WEN WEIJIA

Assignees

• 温维佳
• 温维佳
• 杨斯宇
• 杨斯宇

Dates

Publication Date

20260421

Application Date

20210401

Priority Date

20210401

Claims (4)

1. 1. The real-time fluorescence PCR solid detection reagent is characterized by being prepared by mixing a protection system, a glycerol-free buffer solution, dNTPs premix, a specific primer probe and glycerol-free Taq polymerase and then freeze-drying, wherein the protection system is an aqueous solution of the components with mass concentration of 0.1-0.5 g/mL, 5-25 mg/mL of Type A gelatin, 0.5-2.5 mg/mL of dextrin, 0.1-0.5 mg/mL of chitosan, 0.05-0.25 g/mL of polysucrose and 0.5-1.0 mg/mL of cellulose, the solvent is ultrapure water, the glycerol-free buffer solution and dNTPs premix is composed of the components with the molar concentration of ：0.2 mol/L KCl、0.2 mol/L Tris-HCl、0.2 mol/L (NH 4 ) 2 SO 4、 0.05 mol/L MgCl 2、 0.5mmol/L dNTPs, solvent is ultrapure water, and the cellulose is hydroxyethyl cellulose or methylcellulose.
2. 2. A method for preparing the real-time fluorescent PCR solid detection reagent according to claim 1, comprising the steps of: 1) Premixing the protection system; 2) Uniformly mixing 2-6 mu L of the glycerol-free buffer solution, 5-15 mu L of dNTPs premix and 1 mu mol/L of specific primer probe with 10-30 mu L of the protection system in a vibrating manner; 3) Adding 1-3 units of glycerol-free Taq polymerase, and vibrating and mixing uniformly; 4) Placing the mixed solution prepared in the step 3) at a low temperature of-25 to-30 ℃ for 2-6 hours; 5) Immediately transferring the material obtained in the step 4) into a vacuum dryer, and performing vacuum drying treatment for 1-3 hours at room temperature; 6) And after the drying is finished, the real-time fluorescent PCR solid detection reagent is obtained, and the obtained real-time fluorescent PCR solid detection reagent is packaged in a PP reagent tube, sealed and stored at normal temperature.
3. 3. The method for preparing a real-time fluorescent PCR solid detection reagent according to claim 2, wherein the room temperature of the step 5) is 16-35 ℃.
4. 4. The method for preparing a real-time fluorescent PCR solid detection reagent according to claim 2, wherein the vacuum drying treatment time of the step 5) is 2 hours.

Description

Real-time fluorescence PCR detection solid detection reagent and preparation method thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a solid real-time fluorescence PCR solid detection reagent and a preparation method thereof. Background Polymerase Chain Reaction (PCR) provides a general method to replicate specific DNA sequences for further investigation. Real-time fluorescent PCR was developed on the basis of PCR technology, which can monitor amplification of a target DNA sequence or molecule during PCR by detecting fluorescent signals caused by fluorescent dyes in a reaction system and can achieve extremely high sensitivity, thereby achieving a target DNA with a limit of detection (LOD) of less than 5 copies (in some cases, only 1 copy), and the technical means has rapidly been developed and started to be widely used in biological research, clinical diagnosis, criminal investigation, biomedical research, and the like. Besides the sample extract liquid to be detected during the real-time fluorescence PCR detection, the detection reagent mainly comprises aqueous solution prepared by polymerase, primer probes, dNTPs and buffer solution, the components are sensitive to temperature change and sensitivity of the storage environment, the components are required to be kept at constant low temperature (generally about minus 20 ℃) in the production, transportation and storage processes and are kept in liquid state under the condition of normal temperature (25 ℃) for a period of time not more than 2 days, and the components are required to be independently stored at low temperature and are prepared immediately, so that the transportation and storage cost of the detection reagent is greatly increased, the insufficient detection reagent reserves of a plurality of countries and regions are caused, and the components are more serious in some development and later regions, so that the regions cannot find and cope with some emergent public health events such as novel coronavirus epidemic. Lyophilization is considered one of the ways to solve this problem, however, current lyophilized reagent products still suffer from short shelf life, low sensitivity, and difficulty in stable control of both. In order to ensure the efficacy of the detection reagent, in the storage and transportation processes of most of the existing products, all components of the detection reagent need to be kept under constant low-temperature conditions, and uninterrupted cold chain transportation technology is ensured in the process of cross-regional allocation, so that the products can be kept under enough low-temperature environments at the production end, the transportation end and the use end. The detection mechanism stores the reagent components or the mixed solution of the components which are pretreated and other necessary components in a low-temperature environment respectively, and the reagent components are prepared on site. There are few reports that the freeze-drying technology is used for treating the real-time fluorescence PCR detection reagent to improve the quality guarantee period at normal temperature, but the problems that the quality and the detection sensitivity of the product are difficult to control, the quality guarantee period is relatively short, and the product must be stored in a strictly controlled dry environment still exist. Meanwhile, the existing reagent is difficult to realize in some underdeveloped areas because of the continuous low-temperature storage environment and continuous constant-temperature cold chain transportation in the transregional allocation process, the continuous low-temperature storage environment and the severe cold chain transportation requirement. At normal temperature, most real-time fluorescence PCR detection reagents containing polymerase only maintain the original efficacy for about 24 hours. Meanwhile, temperature fluctuation and storage environment which does not reach low enough temperature can influence the detection effect of polymerase and even other components in the detection reagent, so that misdiagnosis and missed diagnosis are caused. In few existing products for treating real-time fluorescent PCR solid detection reagents by using a freeze drying technology and some protection formulas, the problems that the quality of the products is difficult to control, the detection sensitivity is relatively low, the shelf life is relatively short, the products still need to be always in a strictly controlled dry environment and the like still exist. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a real-time fluorescence PCR solid detection reagent and a preparation method thereof, wherein the solid detection reagent is prepared by mixing a protection system, a glycerol-free buffer solution, dNTPs premix, a specific primer probe and glycerol-free Taq polymerase and then performing freeze drying