Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent

A technology of freeze-drying protective agent and amplification reaction, which is applied in the biological field, can solve the problems of harsh storage and transportation conditions of fluorescent quantitative PCR reagents, the influence of PCR reagent performance sensitivity, and the limitation of freezing and thawing times of solutions, so as to improve convenience, Low cost, reduced storage and transportation effects

Inactive Publication Date: 2017-04-26
ZHUHAI LIVZON DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fluorescent quantitative PCR reagents have strict requirements on storage and transportation conditions. Generally, they need to be stored at -20°C and there are restrictions on the number of freeze-thaw solutions. Dry ice transportation is generally required.
If these conditions are not met, it will have a great impact on the performance of PCR reagents, especially the sensitivity, and even cause the reagents to fail.

Method used

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  • Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent
  • Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent
  • Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0045] Implementation Case 1, HCV Fluorescent Quantitative PCR Freeze-dried Reagent

[0046] 1. Preparation of 5×RT-PCR lyoprotectant

[0047] Weigh 16g of trehalose and 0.4g of Ficoll400, add DEPC water to 40ml, mix the above ingredients in a Corning centrifuge tube bottle, place in a water bath at 60°C for 10 minutes to dissolve, mix evenly, pack in small tubes, and put Store in a 2-8°C refrigerator for later use.

[0048] 2. Preparation of HCV Fluorescent Quantitative PCR Reaction Reagent

[0049] The preparation of HCV reaction reagent is as follows:

[0050] Tth PCR Buffer: 1×;

[0051] Tth enzyme: 7.5U;

[0052] HCV pf: 400nM;

[0053] HCV pr: 400nM;

[0054] HCV pb: 100nM;

[0055] RT-PCR lyoprotectant: 1×; make up 50 μl with water.

[0056] The HCV-PCR lyophilized reagent containing lyoprotectant was prepared as follows:

[0057]

[0058]

[0059] After the above components were mixed upside down, 50 μl per tube was dispensed into eight PCR tubes.

[00...

Embodiment example 2

[0083] Implementation case 2, HIV-1 fluorescent quantitative PCR freeze-dried reagent

[0084] 1. Preparation of 5×RT-PCR lyoprotectant

[0085] Weigh 16g of trehalose and 0.4g of Ficoll400, add DEPC water to 40ml, mix the above ingredients in a Corning centrifuge tube bottle, place in a water bath at 60°C for 10 minutes to dissolve, mix evenly, pack in small tubes, and put Store in a 2-8°C refrigerator for later use.

[0086] 2. Preparation of HIV-1 Fluorescent Quantitative PCR Reaction Reagent

[0087] The preparation of HIV-1 reaction reagent is as follows:

[0088] Tth PCR Buffer: 1×;

[0089] Tth enzyme: 7.5U;

[0090] HIV-1 pf1: 400 nM;

[0091] HIV-1 pr1: 400nM;

[0092] HIV-1 pf2: 400 nM;

[0093] HIV-1 pr2: 400 nM;

[0094] HIV-1 pb1: 100 nM;

[0095] HIV-1 pb2: 100 nM;

[0096] RT-PCR lyoprotectant: 1×; make up 50 μl with water.

[0097] The HIV-1-PCR lyophilized reagent containing lyoprotectant was prepared as follows:

[0098]

[0099] After the abov...

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Abstract

The invention discloses a freeze-drying protective agent and a freeze-drying method for an RNA amplification reaction agent, belonging to the field of biotechnologies. The freeze-drying protective agent contains mycose, polysucrose and water, and is prepared by mixing 6.4-16.0 g of mycose and 0.4-1.0 g of polysucrose and adding with water till the volume is 40ml. The invention further provides the freeze-drying method for the RNA amplification reaction agent. The freeze-drying method comprises the following steps: a, uniformly mixing the freeze-drying protective agent and a real-time fluorescent RT-PCR reaction agent, thus obtaining real-time fluorescent RT-PCR reaction liquid; and b, carrying out freeze drying on the RT-PCR reaction liquid, thus obtaining the RT-PCR freeze-dried reagent. With the adoption of the freeze-drying protective agent and the freeze-drying method provided by the invention, the RNA fluorescent quantitative PCR reaction agent can be stably stored for a long time at 2-8 DEG C or even at the room temperature.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a freeze-drying protectant for RNA amplification reaction reagents and a freeze-drying method. Background technique [0002] Real-time fluorescence quantitative PCR technology was introduced by Applied Biosystems in 1996. This technology refers to adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve. . Real-time fluorescent quantitative PCR is a method of detecting the total amount of products after each cycle of polymerase chain reaction (PCR) with fluorescent chemicals in DNA amplification reactions. Real-time detection of PCR progress through fluorescent signals. Since there is a linear relationship between the Ct value of the template and the initial copy number of the template during the exponential ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邓京欧格
Owner ZHUHAI LIVZON DIAGNOSTICS
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