Agent for loop-mediated isothermal amplification and kit and application thereof

A ring-mediated isothermal and kit technology, applied in the field of nucleic acid amplification, can solve the problems of low detection cost and high cost, and achieve good comparability, ensure sensitivity, and good application prospects

Inactive Publication Date: 2019-01-08
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects in the prior art, to provide a reagent for loop-mediated isothermal amplification, as well as the reagent kit containing the reagent and the application of the reagent kit, the present invention solves the problem of high cost of LAMP reagents While ensuring the detection quality, the kit described in the present invention has the same detection effect as imported reagents, and its sensitivity, specificity and stability can meet the needs of clinical experiments, but the detection cost can be greatly reduced

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  • Agent for loop-mediated isothermal amplification and kit and application thereof
  • Agent for loop-mediated isothermal amplification and kit and application thereof
  • Agent for loop-mediated isothermal amplification and kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 - Reagents for LAMP loop-mediated isothermal amplification

[0036]The reagents for LAMP loop-mediated isothermal amplification involved in this example include the following components: nanoparticles (0.1mg / mL), DTT (2M), BSA (0.25g / mL), TMAC (50mM), seaweed At least one of sugar (80mM); wherein, the nanoparticle is made of gold, silver, silicon dioxide, polystyrene or graphene, its surface is positively charged or uncharged, and its diameter is 0-1000nm.

Embodiment 2

[0037] Example 2 - Kit for LAMP loop-mediated isothermal amplification

[0038] The kit involved in this embodiment includes the following components: 10× constant temperature buffer, dNTP mix (25mM), MgSO 4 (100mM), Bst DNA polymerase (8U / μL), neutral red dye, nanoparticles (0.1mg / mL), DTT (2M), BSA (0.25g / mL), TMAC (50mM), trehalose (80mM ); wherein, the nanoparticle is made of gold, silver, silicon dioxide, polystyrene or graphene, its surface is positively charged or uncharged, and its diameter is 0-1000nm. The kits described above may also optionally comprise an amplification primer composition.

[0039] In the ring-mediated isothermal amplification reaction system prepared by the kit, the final concentration of each component is as follows: nanoparticles 0-10mg / mL; dithiothreitol 0-10M; peptide bovine serum 0-10g / mL; chloride Tetramethylammonium 0-100mM; Trehalose 0-50mM; dNTP mixture 1.0-3.0mM; MgSO 4 4-10mM; Bst DNA polymerase 0.1-0.5U / μL.

Embodiment 3

[0040] Example 3 - Kit for LAMP loop-mediated isothermal amplification

[0041] This embodiment is another preferred form of kit, which includes the following components: 10× constant temperature buffer, dNTP mix (25mM), MgSO 4 (100mM), Bst DNA polymerase (8U / μL), neutral red dye, nanoparticles (0.1mg / mL), DTT (2M), BSA (0.25g / mL); wherein, the material of nanoparticles is gold nano Particles, the surface of which is positively charged or uncharged, and whose diameter is 1-200nm. The kits described above may also optionally comprise an amplification primer composition.

[0042] In the ring-mediated isothermal amplification reaction system prepared by the kit, the final concentration of each component is as follows: gold nanoparticles 0-2mg / mL; dithiothreitol 0-2M; peptide bovine serum 0-2g / mL; dNTP mix 1.0-3.0mM; MgSO 4 4-10mM; Bst DNA polymerase 0.1-0.5U / μL.

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Abstract

The invention relates to an agent for loop-mediated isothermal amplification. The agent comprises at least one of nanoparticles, dithiothreitol, peptide bovine serum, tetramethylammonium chloride andtrehalose. The invention also relates to a kit containing the abovementioned agent and the application thereof. The kit further comprises a 10x constant-temperature buffer, a dNTP mixture, MgSO4, a Bst DNA polymerase, an indicator and water; a loop-mediated isothermal amplification reaction system prepared through the kit comprises the following components by final concentration: 0-10mg / mL of nanoparticles, 0-10M of dithiothreitol, 0-10g / mL of peptide bovine serum, 0-100mM of tetramethylammonium chloride, 0-50mM of trehalose, 1.0-3.0mM of dNTP mixture, 4-10mM of MgSO4, and 0.1-0.5U / uL of DNA polymerase. According to the agent, the kit and the application thereof, the detection quality is ensured while the problem of high cost of an LAMP agent is high is solved; the kit has the same detection effect as an imported agent; the sensitivity, the specificity and the stability meet the clinical test demand; the detection cost is greatly decreased; and the application prospect is good.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and in particular relates to a reagent for loop-mediated isothermal amplification, a kit containing the reagent and an application of the kit. Background technique [0002] Loop-mediated isothermal amplification (LAMP) is a new technique for in vitro nucleic acid gene amplification that is different from conventional PCR. LAMP mainly uses two or three pairs of specific primers and Bst DNA polymerase with strand displacement activity to continuously and rapidly amplify under constant temperature conditions. Compared with traditional PCR technology, this technology has higher sensitivity, specificity and amplification efficiency, strong anti-inhibitor ability, and low requirement for the quality of nucleic acid template, so it can greatly simplify the steps of nucleic acid extraction. This technology can be used in Amplified 10 within 1 hour 9 ~10 10 Double the target sequence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119
Inventor 关明曹国君
Owner 关明
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