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Freeze-drying protecting agent of reagent for PCR amplification, product as well as preparation method and application of reagent

A technology for freeze-drying protective agents and reagents, which is applied in the direction of biochemical equipment and methods, and microbial determination/inspection. and other problems, to achieve the effect of improving sensitivity and accuracy, good resolubility, and prolonging the validity period

Pending Publication Date: 2020-10-27
ZHUHAI LIVZON DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, sugars, especially trehalose and sucrose, are more common in the freeze-drying of PCR systems, but sucrose is prone to Medela reaction in the high-temperature denaturation of PCR reactions, which will affect the detection results and generally lead to changes in the sensitivity and uniformity of reagents. Difference
Trehalose has bound water and is easy to absorb moisture. The moisture absorption of the lyophilized powder during storage will directly affect the validity period of the lyophilized reagent, and the stability of the lyophilized reagent is closely related to the residual moisture of the lyophilized powder. More, the worse the stability, so using only trehalose, the stability of the reagent will not be optimal

Method used

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  • Freeze-drying protecting agent of reagent for PCR amplification, product as well as preparation method and application of reagent
  • Freeze-drying protecting agent of reagent for PCR amplification, product as well as preparation method and application of reagent
  • Freeze-drying protecting agent of reagent for PCR amplification, product as well as preparation method and application of reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 trehalose concentration selection

[0073] 1. Prepare 40% trehalose mother liquor: weigh 40g trehalose, dissolve it with ultrapure water to 100mL, fully dissolve and mix, divide into small tubes, and store in a freezer.

[0074] 2. Prepare PCR reaction solutions with different trehalose contents (1%, 2%, 4%, 6% and 8%) according to Table 1-1, and pack 50 μL per hole in each group into eight PCR tubes, and put them into freeze-dried machine.

[0075] Table 1-1: Formulation table of trehalose system with different concentrations Unit: μL

[0076]

[0077] 3. Freeze drying. Freeze-drying conditions are as follows:

[0078] Table 1-2: Freeze-drying conditions

[0079]

[0080]

[0081] 4. After the freeze-drying is completed, press again, take out the reagent and press the cap. Record the appearance results of all groups, pack them into bags, carry out the 0-day test and record the reconstitution results.

[0082] Table 1-3: Appearance and recons...

Embodiment 2

[0093] Concentration selection of embodiment 2 hydroxypropyl-beta-cyclodextrin (hereinafter referred to as HP-beta-CD)

[0094] 1. Prepare 20% HP-β-CD mother solution: weigh 20g of hydroxypropyl-β-cyclodextrin, dissolve it in ultra-pure water to 100mL, fully dissolve and mix, subpackage in small tubes, and store in a freezer:

[0095] 2. Prepare PCR reaction solutions with 4% trehalose and different HP-β-CD contents according to Table 2-1, pack 50 μL per well in each group into eight PCR tubes, and put them into a freeze dryer;

[0096]Table 2-1: Formulation table of HP-β-CD system with different concentrations Unit: μL

[0097]

[0098] 3. Freeze-dry according to the conditions in Table 1-2.

[0099] 4. After the freeze-drying is completed, press again, take out the reagent and press the cap. Record the appearance results of all groups, pack them into bags, carry out the 0-day test and record the reconstitution results.

[0100] Table 2-2: Appearance and reconstitution ...

Embodiment 3

[0111] The selection of embodiment 3 inulin concentrations

[0112] 1. Prepare 5% inulin mother liquor: weigh 5g of inulin, add ultrapure water to 100mL, fully dissolve and mix well, subpackage in small tubes, and store in a freezer:

[0113] 2. Prepare 4% trehalose, 1% hydroxypropyl-β-cyclodextrin and PCR reaction solutions with different contents (0.02%, 0.05%, 0.10%, 0.20% and 0.50%) of inulin according to Table 3-1, Dispense 50 μL per well in each group into eight PCR tubes, and put them into a freeze dryer;

[0114] Table 3-1: Formula table of different concentrations of inulin system

[0115]

[0116]

[0117] 3. Freeze-dry according to the conditions in Table 1-2.

[0118] 4. After the freeze-drying is completed, press again, take out the reagent and press the cap. Record the appearance results of all groups, pack them into bags, carry out the 0-day test and record the reconstitution results.

[0119] Table 3-2: Appearance and reconstitution results of PCR sys...

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Abstract

The invention relates to the technical field of biology, and in particular provides a freeze-drying protective agent of a reagent for PCR amplification, a product as well as a preparation method and application of the reagent. The freeze-drying protecting agent of the reagent for PCR amplification comprises trehalose, hydroxypropyl-beta-cyclodextrin, inulin and carrageenan. The freeze-drying protecting agent can significantly prolong the validity period of the reagent for PCR amplification, and at the same time can effectively reduce the possibility of aerosol formation of the PCR amplification reaction system during use, and improve the sensitivity and accuracy of PCR.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a freeze-drying protectant for a PCR amplification reagent, a product, a preparation method and application thereof. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) is an in vitro control nucleic acid technology, which in a suitable buffer system, in the presence of specific primers, deoxyribonucleic acid, suitable polymerase and cations, through Repeated cycles of denaturation-annealing-extension, a method to obtain a large number of specific gene fragments in a short period of time. [0003] Since many components in the PCR system, especially enzymes, primers, probes, and dNTPs, are sensitive to temperature, fluorescent PCR reagents are very demanding in terms of use, storage, and transportation conditions. At present, most of the reagents used for PCR amplification are in liquid state, with poor stability and easy failure, and the validity...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686
Inventor 欧格邓京何绮婷勾宏娜
Owner ZHUHAI LIVZON DIAGNOSTICS
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