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Loop-mediated isothermal amplification reagent capable of being transported at normal temperature as well as preparation method and application of reagent

A ring-mediated isothermal and reagent technology, applied in the field of molecular biology, can solve the problems of high concentration of reagents, airgel pollution downstream detection, and influence, and achieve the effects of no loss of detection sensitivity, convenient transportation, and simple operation

Active Publication Date: 2019-03-19
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the limitations of the existing technology, the purpose of the present invention is to provide a ring-mediated isothermal amplification reagent that can be transported at room temperature, a preparation method and its application, so as to solve the problem of high concentration of the existing reagents and easy airgel pollution. And the problem that affects the downstream detection

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  • Loop-mediated isothermal amplification reagent capable of being transported at normal temperature as well as preparation method and application of reagent
  • Loop-mediated isothermal amplification reagent capable of being transported at normal temperature as well as preparation method and application of reagent
  • Loop-mediated isothermal amplification reagent capable of being transported at normal temperature as well as preparation method and application of reagent

Examples

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preparation example Construction

[0040] The preparation method of the reagent of the present invention is as follows: mix Bst DNA Polymerase, dNTP, primer mixture, indicator, protective agent and aerosol pollution prevention agent according to the above ratio, freeze-dry at -70~-80°C for 8~16h, namely Loop-mediated isothermal amplification reagents are available.

[0041] The reagent of the invention can be transported under normal temperature conditions and can be used for nucleic acid detection. Generally, the specific detection process is as follows: mix the loop-mediated isothermal amplification reagent and buffer, add 1-2 μl of the template to be tested (ie, nucleic acid), and then place it at a constant temperature of 63-67°C for 30-60 minutes, and then raise the temperature to React at 83-87°C for 5 minutes; finally, place the reaction tube under a blue light for observation. Bright green is positive and brick red is negative. Among them, the buffer is composed of betaine, KCl, Tris-HCl, (NH 4 ) 2 S...

Embodiment 1

[0045] First prepare the indicator mixture, dissolve 62mg HNB in ​​10ml deionized water to obtain 10mM HNB solution; take 100μl 10000×Gelgreen and add 900μl deionized water to obtain 1000×Gelgreen solution. Then take 375 μl of 10 mM HNB, 35 μl of 1000×Gelgreen and 590 μl of water to prepare a 25× indicator mixture.

[0046] To prepare a protective agent mixture, mix 75 μl of 2% bovine serum albumin, 75 μl of 0.05% soybean lecithin, 50 μl of 100 mM mannitol and 50 μl of 0.25% hydroxyethyl starch, and make up 500 μl of deionized water to obtain a 25× protective agent mixture.

[0047] To prepare an aerosol pollution prevention agent, take 100 μl of 0.25% glycyrrhizin, 100 μl of 0.5% sodium alginate and 100 μl of 2.5% tert-butanol, then supplement 1ml with 700 μl of deionized water, and store in a water bath at 50-85°C.

[0048] Then add 0.6 μl water, 1.4 μl 10 mM dNTP, 0.4 μl primer mix, 0.4 μl indicator mix, 1 μl protectant mix, 1 μl aerosol pollution preventive agent, 0.4 μl B...

Embodiment 2

[0060] The difference between this example and Example 1 is that the concentration of tert-butanol is 0.1%, and the addition amount is 100 μl.

[0061] The loop-mediated isothermal amplification reagent prepared in this example was prepared into a buffer, and then mixed with the E. coli DNA template to determine its freeze-drying efficiency, as Image 6 shown.

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Abstract

The invention discloses a loop-mediated isothermal amplification reagent capable of being transported at a normal temperature as well as a preparation method and application of the reagent. The reagent comprises Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive. The preparation method comprises the following steps: mixing the previouscomponents, and performing freeze drying at a temperature of 70-80 DEG C below zero for 8-16 hours, thereby obtaining the loop-mediated isothermal amplification reagent. A loop-mediated isothermal amplification reagent used for nucleic acid test is prepared in the invention, can be transported and preserved at the normal temperature, and is convenient to transport and excellent in stability; meanwhile, the problem that popularization of a LAMP (Loop-mediated Isothermal Amplification) technology is limited by aerosol pollution is solved, and the application and popularization difficulty of theLAMP technology is greatly reduced. The activity of the Bst DNA Polymerase in the reagent is 150%, the reagent can be preserved at the normal temperature of one year or longer, only a buffer solutionneeds to be added during use, the operation is simple, the activity of DNA polymerase is high, the detection sensitivity is loss-free, the interference of the protective agent is low, and the cost islow.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a loop-mediated isothermal amplification reagent that can be transported at room temperature, a preparation method and an application. 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 sta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2537/1376C12Q2527/125
Inventor 闫亚平张鑫
Owner SHAANXI NORMAL UNIV
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