Freeze-drying protection system required for nucleic acid amplification reagent and preparation method of freeze-drying protection system

A technology of freeze-drying protection and freeze-drying protection agent, which is applied in the field of pharmaceutical preparation, can solve the problems of high temperature and inactivation of reagents, and achieve the effects of less energy consumption, convenient storage, and avoiding repeated freezing and thawing.

Inactive Publication Date: 2019-04-09
百康芯(天津)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: the temperature of vacuum drying or rapid air drying is too high, and there is a risk of reagent inactivation

Method used

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  • Freeze-drying protection system required for nucleic acid amplification reagent and preparation method of freeze-drying protection system
  • Freeze-drying protection system required for nucleic acid amplification reagent and preparation method of freeze-drying protection system
  • Freeze-drying protection system required for nucleic acid amplification reagent and preparation method of freeze-drying protection system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] A kind of LAMP amplification reagent lyophilization protection system and lyophilization treatment steps are as follows:

[0057] (1). Freeze-drying of the whole system: Mix all the reagents required for the Lamp reaction together. The specific components of the reagents are as follows:

[0058] components

volume

content

Bst enzyme (8U / μL)

80μL

640U

UNG enzyme (1U / μL)

0.5μL

0.5U

dNTPS (100mM)

20 μL

2μM

MgSO4(100mM)

40μL

4μM

Buffer(10X)

100μL

/

SYBR GREEN I(250X)

15μL

3.75X

Betaine (5M)

2000μL

15mM

purified water

44.5μL

/

[0059] The lyoprotectant is one of the following formulations:

[0060] components

volume

content

Mannitol (18%, w / v)

1110μL

5％

BSA (10%, w / v)

200μL

0.5％

Glycine (100mM)

200μL

5mM

purified water

290μL

/

[0061] (2). Fractional freeze-dry...

Embodiment 2

[0069] The formula screening of embodiment 2 freeze-drying protection reagent

[0070] The formula of the freeze-drying protective agent is as follows, and the balance is purified water:

[0071] Formula 1: dextran 5%, polyethylene glycol 2%, sucrose 2%;

[0072] Formula 2: mannitol 8%, polyethylene glycol 1%, threonine 1%;

[0073] Formula 3, Mannitol 5%, Bovine Serum Albumin 2%, Glycine 1%;

[0074] Formula 4, polyvinylpyrrolidone 3%, bovine serum albumin 2%, trehalose 1%.

[0075] The above four groups of formulas were mixed with the LAMP reagent (the A solution part in the sub-system) and then lyophilized. The lyophilized reagents were subjected to sensitivity experiments and morphological comparisons, and a control group was set up. The control group was the reaction reagent without lyophilization ( Does not contain lyoprotectant).

[0076] serial number

[0077] After lyophilization, the PCR tube was taken out of the lyophilizer, and the tube was fastened a...

Embodiment 3

[0078] Embodiment 3 thermal stability test

[0079] After screening the lyophilized reagent formula, formula 2 and formula 3 were selected for reagent thermal stability test (37°C constant temperature), and the stability of the reagent was initially evaluated. At the same time, a control group (without adding lyoprotectant reaction system), the test results refer to Figure 2-5 as shown,

[0080] sensitivity

[0081] In the present invention, the response curve Ct≤48 in the sensitivity test can determine the effectiveness of the reagent. From the above results, it can be seen that the freeze-dried protection reagent of the present invention, especially reagent 2, can be stored at 37°C for 4 weeks or even longer, which is greatly improved. The storage stability of the LAMP amplification reagent is improved.

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Abstract

The invention belongs to the technical field of medicine preparation, and particularly relates to a freeze-drying protection system required for a nucleic acid amplification reagent and a preparationmethod of the freeze-drying protection system. The freeze-drying protection system comprises the nucleic acid amplification reagent and a freeze-drying protection additive, wherein the nucleic acid amplification reagent is a reagent used for LAMP reaction amplification, the freeze-drying protection additive is one or a compound of the following reagents: polyethylene glycol, mannitol, polyvinylpyrrolidone, glucan, trehalose, sucrose, bovine serum albumin, collagen, threonine and glycine, and the concentration of the weight-to-volume ratio of the freeze-drying protection additive and the amplification reaction reagent is 3% to 25%. The freeze-drying protection additive used in the invention has the advantages of small volume, short freeze-drying time, high efficiency and low energy consumption, and the freeze-drying protection system can be directly used for gene chip experiments, does not cause repeated freeze-thaw and waste of reagents, and can effectively ensure the activity of active substances in the freeze-drying process.

Description

technical field [0001] The invention belongs to the technical field of medicine preparation, and in particular relates to a freeze-drying protection system required for nucleic acid amplification reagents and a preparation method thereof. Background technique [0002] Loop-mediated isothermal amplification technology (loop-mediated isothermal amplification, LAMP) is a technology that can achieve nucleic acid amplification in a short period of time under constant temperature conditions. It has the advantages of "simple, fast, low-cost, and accurate". This technology is comparable to PCR reaction in terms of sensitivity, specificity and detection range, and can achieve on-site high-throughput rapid detection without any special equipment, and the detection cost is much lower than that of fluorescent quantitative PCR. However, the amplification system of the LAMP reaction contains active ingredients such as polymerase and dNTP, which need to be stored at low temperature. When u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119
Inventor 杨玲玲屈彬彬高亚平张国豪吴国君
Owner 百康芯(天津)生物科技有限公司
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