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Sample conditioning fluid based on denaturation precipitation as well as application thereof and nucleic acid releasing method

A technology for sample treatment solution and nucleic acid release, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms. , the effect of reducing pollution sources

Active Publication Date: 2015-03-25
亚能生物技术(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The invention solves the problems of low sample preparation and recovery efficiency, complicated operation and high cost in nucleic acid amplification samples, and provides a method that does not require centrifugation and proteinase K digestion, is suitable for long fragments and multiplex PCR, and has high amplification efficiency and stability. Sample treatment solution and nucleic acid release method based on denaturing precipitation

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  • Sample conditioning fluid based on denaturation precipitation as well as application thereof and nucleic acid releasing method
  • Sample conditioning fluid based on denaturation precipitation as well as application thereof and nucleic acid releasing method

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Embodiment 1

[0034] Embodiment 1 Technical principle of the present invention

[0035] Polyethylene glycol can induce the aggregation of macromolecules in the aqueous solution. Under the action of polyethylene glycol, it can increase the hydrophilicity of the solution and increase the OH-ion concentration of the solution. The strong alkaline environment provided is helpful for rapid cell lysis It also destroys the structure of the nucleoprotein, and when it is added to the PCR reaction solution, its pH value will drop to a normal level without inhibiting amplification.

[0036] High salt solution is beneficial to the precipitation of nucleoprotein in the cell. The solid-phase chelating resin can integrate multivalent metal ions, especially divalent ions. It has higher metal ion selectivity and stronger binding than ordinary ion exchangers. It can combine many other exogenous substances (such as ferrous ions) that may affect one-step analysis, and remove the solid particle resin and impurities b...

Embodiment 2

[0043] The sample treatment solution of the present invention is composed of (the mass percentage of the sample treatment solution) 1-15% PEG400, 10-40mmol / L potassium hydroxide (KOH), 0.1-2% surfactant (Teween-20, TritonX-100 ), 1-20% solid phase resin chelex-100, 3-20% PCR enhancer (glycerin, DMSO) and 100-400mmol / L KCL.

[0044] Configuration method: use sterile water, add 1-15% PEG400 and 10-40mmol / L potassium hydroxide (KOH), add 0.1-2% surfactant (Teween-20, TritonX-100) and 100-400mmol / L KCL, then add 3-20% PCR synergist (glycerin, DMSO), and finally add 1-20% solid phase resin chelex-100 to mix well to get the sample treatment solution.

[0045] The configured sample treatment solution and the blood sample are added and mixed in a certain ratio. The blood sample is preferably added in a ratio of 20 μL to the 20-500 μL of the lysate, and vortexed to mix. More preferably, 20 μL of the blood sample is added to 50 -150μL of lysis buffer, vortex and mix well, add 10% of the PCR...

Embodiment 3

[0048] A preferred sample treatment solution of the present invention consists of the following components (accounting for the mass percentage of the sample treatment solution): 3% PEG400, 0.5-1.2% surfactant, 5-15% solid phase resin chelex-100, 5- 8% PCR enhancer, 150-300mmol / LKCL and 20mmol / LKOH. The sample processing solution solvent is sterile water.

[0049] Use of the above-mentioned sample treatment liquid of the present invention:

[0050] 1. Take out the sample treatment solution (the reagent is a suspension, shake well before use to suspend the particles), add 300μL of the sample treatment solution to a 1.5mL centrifuge tube, and pipette should be used from time to time during the process of aspirating the treatment solution The liquid device is blown to make the particles evenly suspended.

[0051] 2. Add 20.0ul of anticoagulant blood to the sample processing solution, vortex for 25sec, and centrifuge briefly to avoid residual droplets on the inner wall of the tube.

[00...

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Abstract

The invention belongs to the biotechnology field, and in particular relates to sample conditioning fluid based on denaturation precipitation as well as application thereof and a nucleic acid releasing method. The sample conditioning fluid comprises the components in percentage by mass: 1-15 percent of PEG400, 0.1-2 percent of surfactant, 1-20 percent of solid phase resin chelex-100, 3-20 percent of polymerase chain reaction (PCR) enhancer, 10-40 mmol / L of potassium hydroxide and 100-400 mmol / L of potassium chloride (KCL), wherein the solvent of the conditioning fluid is sterile water. The sample conditioning fluid is used for removing substances inhibiting PCR amplification based on denaturation precipitation, and the treated product can be subjected to direct amplification so as to guarantee the amplification stability; because of non-selectivity of the treated product based on the principle on the amplification, the fluid is suitable for taq enzymes, chemically-decorated and antibody-decorated hot start enzymes and the like and is particularly suitable for performing amplification on long sections and gene sections with complicated structure.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a sample processing solution based on denaturation precipitation, its application, and a nucleic acid release method. Background technique [0002] With the deepening of clinical genetic diagnosis, genetic diagnosis has been widely used in prenatal diagnosis, newborn screening, and detection of genetic and metabolic diseases. [0003] In clinical, pathological and other fields, PCR is widely used in molecular testing for the purpose of genetic diagnosis. Generally speaking, the PCR method uses one strand of the dissociated strand of the double-stranded DNA as a template, binds primers to a specific region of the DNA strand, and performs repeated DNA synthesis reactions by DNA polymerase to amplify the target DNA fragments exponentially. Nucleic acid amplification method. In addition, in addition to PCR amplification methods, there are also nucleic acid amplification methods suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q2523/308C12Q2523/113
Inventor 李长远刘晶晶任维
Owner 亚能生物技术(深圳)有限公司
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