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Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method

A technology for nucleic acid amplification reaction and freeze-drying protection, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA preparation, etc., and can solve the problems of unstable storage of nucleic acid amplification reaction reagents, lack of long-term storage of reagents, and disease detection To achieve the effect of protecting biological activity, keeping sensitivity stable, and prolonging the shelf life

Inactive Publication Date: 2014-07-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, a variety of nucleic acid amplification methods are widely used in the detection of pathogens, but the nucleic acid amplification reaction reagents are unstable when stored at room temperature, and become invalid after only one month. About three months, and the storage period at -20°C is not ideal
This requires that the isothermal amplification reaction reagents be stored, transported and used under low temperature conditions as much as possible, otherwise the diagnostic reagents are prone to failure, so the long-term storage and long-distance transportation of the kit will be greatly restricted, and it is easy to be diagnosed due to Improper storage temperature of the reagent reduces its sensitivity or even completely invalidates it, which eventually leads to the untimely detection of the disease and causes the epidemic
[0010] At present, there is no method for long-term storage of nucleic acid amplification reaction reagents at 4°C or room temperature

Method used

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  • Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method
  • Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method
  • Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method

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Experimental program
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Effect test

Embodiment 1

[0071] Embodiment 1, the preparation of heat-resistant lyoprotectant

[0072] The selection and ratio of heat-resistant lyoprotectant components have a significant impact on the forming and preservation effects of diagnostic reagents. In order to make the appearance and shape of the freeze-dried product of the nucleic acid amplification reaction reagent better, and to prolong its shelf life, long-term analysis and research have been carried out on the components of the heat-resistant freeze-drying protective agent. After a series of tests, the optimized The types and contents of various components are selected from the following components: trehalose, mannitol, and bovine serum albumin.

[0073] 1. Preparation of lyoprotectant

[0074] Weigh 21.55g of trehalose, 5.4g of mannitol, and 5.4g of bovine serum albumin; add the above ingredients into 50ml of water for injection preheated to 37°C, fully dissolve, mix well, and filter out with a 0.22μm filter membrane. Bacteria were ...

Embodiment 2

[0111] Example 2. Preparation process of reverse transcription loop-mediated isothermal amplification reaction reagent freeze-dried product

[0112] 1. Preparation of lyoprotectant

[0113] Weigh 11.9g of trehalose, 2.98g of mannitol, and 2.98g of bovine serum albumin; add the above ingredients in sequence to 50ml of water for injection that has been preheated to 37°C, fully dissolve and mix well, and filter with a 0.22μm filter membrane Sterilize by filtration to obtain a freeze-dried protective agent, which is stored in a refrigerator at 4°C for future use.

[0114] 2. Preparation of reverse transcription loop-mediated isothermal amplification reaction reagents

[0115] 1) Preparation of reaction buffer: weigh KCl0.0423g, MgSO 4 0.1109g, (NH 4 ) 2 SO 4 Add 0.0733g to 20ml of water for injection, measure 1.125ml of 1M Tris-HCl (pH8.8), and add 1.125ml of Tween200.05625ml to the above solution, add water for injection to 25ml, shake well, and store in a refrigerator at 4°...

Embodiment 3

[0152] Example 3 Preparation process of rolling circle amplification reaction reagent freeze-dried product

[0153] 1. Preparation of lyoprotectant

[0154] Weigh 22.22g of trehalose, 5.56g of mannitol, and 5.56g of bovine serum albumin; add the above ingredients in sequence to 50ml of water for injection preheated to 37°C, fully dissolve, mix well, and filter with a 0.22μm filter membrane Sterilize by filtration to obtain a freeze-dried protective agent, which is stored in a refrigerator at 4°C for future use.

[0155] 2. Preparation of reagents for rolling circle amplification reaction

[0156] 1) Preparation of dNTP mixture: take equal volumes of dATP, dTTP, dCTP, and dGTP four nucleotide solutions, mix them, and store them in a 4°C refrigerator for later use.

[0157] 2) Primer solution configuration: Take the designed primer solution RCA1 and store it in a 4°C refrigerator for later use.

[0158] RCA1 (5′-GTGTAGGAACGGCTGACATTCTGG-3′)

[0159] 3) Preparation of rolling...

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Abstract

The invention relates to a freeze-drying protective agent of nucleic acid amplification reaction reagents and a freeze-drying method. The invention provides a composition for freeze-drying protection, which is the following 1) or 2): 1) the composition comprises mycose, mannitol, bovine serum albumin, and water with a ratio of 4.0-25g:1-7g:1.0-7g:49-50ml; 2) the composition comprises mycose, mannitol, bovine serum albumin, Tween, Tris-HCl and water with a ratio of 4.0-25g:1-7g:1.0-7g:0.1-0.25ml:2.5*10<-3>-5.0*10<-3>mol:49-50ml. The freeze-drying protective agent of the invention is low in raw material cost, simple in operation, and suitable for large-scale production, can reduce production cost, and can effectively prolong the storage life of nucleic acid amplification reaction reagents.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a freeze-drying protectant for nucleic acid amplification reaction reagents and a freeze-drying method. Background technique [0002] Loop-mediated isothermal amplification technology (loop-mediated isothermal amplification, LAMP) is a novel constant temperature nucleic acid amplification method developed in 2000. This technology is different from the conventional PCR method, which relies on the design of specific regions on the target sequence. Several sets of specific primers and a Bst DNA polymerase with strand displacement characteristics, under isothermal conditions, through the unique reverse combination of the primers themselves, Bst DNA polymerase performs the classic amplification reaction of strand automatic cycle displacement DNA, which is simple and fast , Strong specificity. At present, a variety of loop-mediated isothermal amplification kits have been launched on the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 吴文学刘旭徐威
Owner CHINA AGRI UNIV
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