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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 liquid and nucleic acid release, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of complex operation, low recovery efficiency, and high cost, and achieves reduction of pollution sources, guaranteeing stability, and avoiding errors. and the effects of pollution

Active Publication Date: 2014-03-19
亚能生物技术(深圳)有限公司
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  • 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 aqueous solution. Under the action of polyethylene glycol, it can increase the hydrophilicity of the solution and increase the concentration of OH- ions in the solution. The strong alkaline environment provided is conducive to the rapid lysis of cells. And destroy the nucleoprotein structure, and when it is added to the PCR reaction solution, its pH value will be reduced to the normal level, and will not inhibit the amplification.

[0036] High salt solution is conducive to the precipitation of nucleoproteins in cells. Solid-phase chelating resin can integrate multivalent metal ions, especially divalent ions selectively. It has higher metal ion selectivity and stronger binding than ordinary ion exchangers. It can combine many other foreign substances (such as ferrous ions) that may affect the one-step analysis, and remove solid-phase par...

Embodiment 2

[0043] The sample treatment solution of the present invention is composed of (accounting for 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 (glycerol, DMSO) and 100-400mmol / L KCL.

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

[0045] The prepared sample processing solution and the blood sample are added and mixed according to a certain ratio. The preferred adding ratio of blood sample is 20 μL of blood sample added to 20-500 μL of lysate, vortexed and mixed, and more preferably 20 μL of blood sample is added to 50 -150 μL of the lysate, vor...

Embodiment 3

[0048] A preferred sample treatment solution of the present invention has 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 booster, 150-300mmol / LKCL and 20mmol / LKOH. The sample processing liquid solvent is sterile water.

[0049] The 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 it well before use to suspend the particles), add 300 μL of the sample treatment solution to the 1.5mL centrifuge tube, and use a pipette from time to time during the process of absorbing the sample treatment solution. Pipette the liquid to make the particles evenly suspended.

[0051] 2. Add 20.0 ul of anticoagulated blood to the sample treatment solution, vortex for 25 sec, 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 in particular relates to a denaturing precipitation-based sample treatment liquid, 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 metabolic diseases. [0003] In the clinical and pathological fields, the PCR method is widely used in molecular detection for the purpose of gene diagnosis. In general, the PCR method uses one strand of the dissociated strand of the DNA double strand as a template, primer binding to a specific region of the opposite DNA strand, and repeated DNA synthesis reactions by DNA polymerase to exponentially amplify the target DNA fragment. Nucleic Acid Amplification Methods. In addition, in addition to PCR amplification methods, there are nucleic acid amplification methods such as RT-PCR,...

Claims

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Application Information

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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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