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Freeze-dried microspheres of LAMP isothermal amplification reagent as well as preparation method and application of freeze-dried microspheres

A technology of isothermal amplification and reagents, which is applied in biochemical equipment and methods, and microbial measurement/inspection. The effect of transportation cost, ease of packaging, and increased stability

Active Publication Date: 2021-03-12
河南中检食安生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The loop-mediated isothermal amplification reagent obtained by the above preparation method is powdery, and the lyophilized powder reagent is not easy to quantify and pack, and cannot meet the needs of instant detection products

Method used

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  • Freeze-dried microspheres of LAMP isothermal amplification reagent as well as preparation method and application of freeze-dried microspheres
  • Freeze-dried microspheres of LAMP isothermal amplification reagent as well as preparation method and application of freeze-dried microspheres
  • Freeze-dried microspheres of LAMP isothermal amplification reagent as well as preparation method and application of freeze-dried microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation method of the freeze-dried microspheres of the LAMP isothermal amplification reagent of the present embodiment comprises the following steps:

[0034] 1) Preparation of lyoprotectant:

[0035] For the preparation of threonine with a mass volume ratio (W / V) of 8%, weigh 8g of threonine with an electronic balance, add 80ml of purified water, put it in a magnetic stirrer for heating and dissolving, and finally set the volume to 100ml , filtered with a 4um filter, placed at 4°C, and set aside.

[0036] For the preparation of bovine serum albumin with a mass volume ratio (W / V) of 10%, weigh 10g of bovine serum albumin with an electronic balance, add 80ml of purified water, put it in a magnetic stirrer for heating and dissolving, and finally set the volume to 100ml , filtered with a 4um filter, placed at -20°C, and set aside.

[0037] For the preparation of PEG20000 with a mass volume ratio (W / V) of 25%, weigh 25g of PEG20000 with an electronic balance, add ...

Embodiment 2

[0056] The preparation method of the freeze-dried microspheres of the LAMP isothermal amplification reagent in this example is the same as that in Example 1, and the difference from Example 1 is that the amount of the lyoprotectant added is: bovine serum albumin 3 μL, Su 3 μL of amino acid and 5 μL of PEG200000; the reaction system is shown in Table 3; the freeze-drying procedure is as follows: first drying at -50°C for 16 hours, followed by secondary sublimation drying at 20°C for 3 hours.

[0057] Table 3 Reaction system for constant temperature amplification of 2019-nCOV virus nucleic acid

[0058]

[0059]

[0060] The freeze-dried microspheres of the LAMP isothermal amplification reagent in this embodiment are the freeze-dried microspheres of the LAMP isothermal amplification reagent prepared by the method for preparing the freeze-dried microspheres of the LAMP isothermal amplification reagent in this embodiment.

Embodiment 3

[0062] The preparation method of the freeze-dried microspheres of the LAMP isothermal amplification reagent in this example is the same as that in Example 1, and the difference from Example 1 is that the amount of the lyoprotectant added is: bovine serum albumin 3 μL, Su Amino acid 5 μL and PEG200007 μL; the freeze-drying procedure is: first drying at -60°C for 10 hours, followed by secondary sublimation drying at 30°C for 3 hours; the reaction system is shown in Table 4.

[0063] Table 4 Reaction system for constant temperature amplification of 2019-nCOV virus nucleic acid

[0064]

[0065]

[0066] The freeze-dried microspheres of the LAMP isothermal amplification reagent in this embodiment are the freeze-dried microspheres of the LAMP isothermal amplification reagent prepared by the method for preparing the freeze-dried microspheres of the LAMP isothermal amplification reagent in this embodiment.

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Abstract

The invention relates to a freeze-dried microsphere of an LAMP reagent as well as a preparation method and application of the freeze-dried microsphere, and the freeze-dried microsphere of the LAMP reagent comprises an LAMP primer group, a freeze-drying protective agent and a reaction solution, wherein the LAMP primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB; the freeze-drying protective agent is prepared from bovine serum albumin, threonine and PEG (Polyethylene Glycol) 20000; the volume parts of theLAMP primer group, the freeze-drying protective agent and the reaction liquid are respectively as follows: 7-14 parts, 9-17 parts and 9.2-17.75 parts. The freeze-dried microsphere of the LAMP isothermal amplification reagent can improve the stability of the detection reagent, can be transported at normal temperature, saves energy and transportation cost, and is convenient for quantitative packaging.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a freeze-dried microsphere of a LAMP isothermal amplification reagent, a preparation method and an application thereof. Background technique [0002] Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification, LAMP) is a nucleic acid in vitro amplification technology invented by Eiken Co., Ltd. in Japan. According to the 6 regions of the target gene, 4 specific primers are designed, and Bst DNA is used to The strand displacement of Polymerase can be completed within 1 hour at 60-65°C for 10 9 ~10 10 double amplification. Since the LAMP amplification process relies on the recognition of 6 independent regions of the target sequence, the reaction specificity is very strong, and the nucleic acid amplification process is carried out under constant temperature conditions, and ordinary water baths or equipment with stable heat ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2527/125
Inventor 王云龙王群智邵俊影王欣林圣博李强王丽王雯丽王昱堃
Owner 河南中检食安生物科技有限公司
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