Preservation method of loop-mediated isothermal amplification reaction reagent mixture

A loop-mediated isothermal and amplification reaction technology is applied in biochemical equipment and methods, and the determination/inspection of microorganisms.

Inactive Publication Date: 2009-12-02
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the LAMP kits sold on the market usually pack the reagents required for the LAMP reaction separately, and then store and transport them at low temperatur...

Method used

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  • Preservation method of loop-mediated isothermal amplification reaction reagent mixture
  • Preservation method of loop-mediated isothermal amplification reaction reagent mixture
  • Preservation method of loop-mediated isothermal amplification reaction reagent mixture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1. Vacuum drying treatment after adding different concentrations and different types of dry protection agents to the LAMP reaction reagent mixture

[0013] Mix 3744 μL of the mixture of various LAMP reaction reagents except the nucleic acid of the test sample (including 1.6 μM each of LAMP primers FIP and BIP, 0.2 μM each of primers F3 and B3, 1.4 mM each of dATP, dTTP, dGTP and dCTP, MgCl 2 6mM, Betaine 1M, Tris-HCl 20mM, KCl 10mM, MgSO 4 2mM, (NH 4 ) 2 SO 4 10mM, TritonX-1000.1%, BstDNA polymerase 1248U, AMV reverse transcriptase 19.5U) in 26 1.5mL centrifuge tubes according to 144μL per tube, as shown in the first column of Table 1 (26 rows in total) Concentration and type Add dry protectant to each 1.5mL centrifuge tube, mix well, then divide the mixture in each 1.5mL centrifuge tube into 6 0.2mL centrifuge tubes according to the amount of 24μL, and then divide the packed The 0.2ml centrifuge tube with the mixture is placed in a low-temperature vac...

Embodiment 2

[0014] Example 2. Rapid air-drying after adding different concentrations and different types of drying protectants to the LAMP reaction reagent mixture

[0015] Mix 3744 μL of the mixture of various LAMP reaction reagents except the nucleic acid of the test sample (including 1.6 μM each of LAMP primers FIP and BIP, 0.2 μM each of primers F3 and B3, 1.4 mM each of dATP, dTTP, dGTP and dCTP, MgCl 2 6mM, Betaine 1M, Tris-HCl 20mM, KCl 10mM, MgSO4 2mM, (NH 4 ) 2 SO 4 10mM, BSA 100μg / mL, Bst DNA polymerase 1248U, AMV reverse transcriptase 19.5U) in 26 1.5mL centrifuge tubes according to 144μL per tube, as shown in column 1 of Table 2 (26 rows in total) The concentration and type of each 1.5mL centrifuge tube was added to dry protective agent, mixed, and then the mixture in each 1.5mL centrifuge tube was divided into six 0.2mL centrifuge tubes according to the amount of 24μL, and then The 0.2ml centrifuge tube containing the mixture is placed in a normal temperature air dryer ...

Embodiment 3

[0016] Example 3. Vacuum drying treatment after adding two or more drying protectants to the LAMP reaction reagent mixture

[0017] Mix 1440 μL of the mixture of various LAMP reaction reagents except for the nucleic acid of the test sample (including 1.6 μM each of LAMP primers FIP and BIP, 0.2 μM each of primers F3 and B3, 1.4 mM each of dATP, dTTP, dGTP and dCTP, 1 M betaine, Tris-HCl 20mM, KCl 10mM, MgSO 4 8mM, (NH 4 ) 2 SO 4 10mM, TritonX-1000.1%, BstDNA polymerase 480U, AMV reverse transcriptase 7.5U) in 10 centrifuge tubes of 1.5mL according to 144μL per tube, as shown in the first column of Table 3 (10 rows in total) Concentration and type Add dry protectant to each 1.5mL centrifuge tube, mix well, then divide the mixture in each 1.5mL centrifuge tube into 6 0.2mL centrifuge tubes according to the amount of 24μL, and then divide the packed Place the 0.2ml centrifuge tube of the mixture in a low-temperature vacuum freeze dryer (HetoDrywinne, Denmark), control the t...

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PUM

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Abstract

The invention discloses a preservation method of a loop-mediated isothermal amplification reaction reagent mixture. The preservation method realizes long-term preservation of the loop-mediated isothermal amplification reaction reagent mixture at normal temperature or room temperature by adopting the following steps: adding a specific drying protective agent in the loop-mediated isothermal amplification reaction reagent mixture; and then carrying out vacuum drying or quick air drying of the mixture at a temperature lower than 80 DEG C. The preservation method has the advantages of low cost, simple operation and stable persistent activity of dried loop-mediated isothermal amplification reaction reagent mixture at the normal temperature, and the like. Therefore, the preservation method can effectively promote the application of loop-mediated isothermal amplification technology in the fields of medical treatment, inspection and quarantine, and the like, as well as the popularization of loop-mediated isothermal amplification kits.

Description

technical field [0001] The present invention relates to a preservation method of a biochemical reagent composition, specifically a method for preservation of a mixture of various reagents in a ring-mediated isothermal amplification reaction system in molecular biology except the nucleic acid (DNA or RNA) of the sample to be tested—the ring Method for storing reagent mixtures for mediated isothermal amplification reactions. Background technique [0002] Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology is a new isothermal amplification technology invented by Notomi et al. in 2000. This technology designs 4 specific primers for 6 segments of the target gene and uses a DNA polymerase with strand displacement activity to perform isothermal amplification of nucleic acid at about 65°C. Due to the advantages of high specificity, simple operation, and no need for complicated instruments and equipment, LAMP technology has been widely us...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张庆利黄倢史成银杨冰高强梁艳
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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