Method for detecting nucleic acid based on hybridization trapping in single tube

A technology of nucleic acid trapping, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, sugar derivatives, etc., and can solve problems such as low sensitivity and nucleic acid contamination

Inactive Publication Date: 2004-08-18
国家质量监督检验检疫总局动植物检疫实验所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, non-specific amplification is prone to occur in PCR operations, etc.
Nucleic acid extraction by trapping can remove non-target nucleic acids, but most of the probes used for trapping are non-covalently immobilized on the hybridization membrane, and hybridization is performed directly after trapping, so it is difficult to use the trapped nucleic acid as a template for PCR amplification
Moreover, this hybridization is easy to cause nucleic acid contamination
Sensitivity is much lower than the sensitivity of detection after amplification detection using trapped nucleic acid as a template

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Hybridization detection of trapped nucleic acids

[0017] 1. Preparation of Trap PCR Tubes

[0018] (1) Add 100ul of newly prepared coating solution to each PCR well, and incubate at 50°C for 5h (4-24h).

[0019] (2) Discard the coating solution, wash with lotion 3 times, soak for 5 minutes, and then wash 3 times.

[0020] (3) Wash 3 times with sterile double distilled water, soak for 5 minutes, and then wash 3 times.

[0021] (4) Dry at room temperature and store at 4°C or lower.

[0022] Coating solution: 100 nM 5'-aminated primer, 10 mM EDC, 10 mM methylimidazole (1-Melm) (pH7.0). EDC: (Ethyl-3-(3-dimethylaminopropyl)-carbodiimide carbodiimide)

[0023] Wash solution: 100 mM Tris-HCl (pH7.5), 150 mM NaCl, 0.1% Tween-20.

[0024] 2. Hybrid trapping of nucleic acids

[0025] (1) Take an appropriate amount of sample and add 5 times the volume of DNA extraction solution, grind, bathe in 95°C water for 5 minutes, add an equal volume of phenol / chloroform / heterogeneou...

Embodiment 2

[0035] Hybrid trap PCR-ELISA detection

[0036] 1. Preparation of trapping PCR tubes (step same as above)

[0037] 2. Hybrid trapping of nucleic acids

[0038] (1) Take an appropriate amount of sample and add 5 times the volume of DNA extraction solution, grind, bathe in 95°C water for 5 minutes, add an equal volume of phenol / chloroform / heterogeneous alcohol for extraction once.

[0039] (2) Extract 100ul of the supernatant, add to the coated trap PCR tube, incubate at 95°C for 2min, place on ice for 1min, and incubate at 45°C for 10-60min.

[0040] (3) Washing with the washing solution for 3 times to complete the trapping of nucleic acids.

[0041] DNA extraction solution: 0.8M NaCL, 100mM Tris-HCL (PH8.0), 20mM EDTA, 1% SDS

[0042] 3.PCR amplification

[0043] Amplification conditions vary with different sequences. In order to balance the liquid-phase products and solid-phase products, the concentration ratio of liquid-phase primers and solid-phase primers is 8:1, and t...

Embodiment 3

[0059] Hybrid trap real-time fluorescence detection

[0060] 1. The preparation of trapping PCR tubes (the steps are the same as above).

[0061] 2. Hybrid trapping of nucleic acids (steps as above).

[0062] 3. Real-time fluorescent PCR

[0063] Perform real-time fluorescent PCR in a PCR tube with nucleic acid trapped, reaction system: 10×PCR buffer 2.5μl, 25mmol / LMgCl2 5μl, 10mM dATP, dUTP, dGTP, dCTP each 0.5μl, 20μmol / L primer 0.5μl each, 20μlmol / L Probe 1μl, 1U / μl UNGase 0.15μl, 5U / μl Taq DNA polymerase 0.5μl, template DNA 1μl, add sterilized double distilled water to make the total volume 25μl. Put the PCR tube with the PCR reaction solution into the ABIPRISM7700 96-well reaction plate, open Sequence Detection 1.71, and set the PCR reaction conditions. The first cycle is 50°C, 2min, 95°C, 10min; the last 40 cycles are 94°C , 15S; 68°C, 1min. Click Run to carry out the PCR reaction. After the reaction ends in 1 hour and 56 minutes, save the file, open the analysis sof...

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Abstract

Trap chain covalent united to PCR tube wall is used in directional trap of destination segment in nucleic acid coarse body fluid, and the destination segment is hybrid combined onto the inner wall of PCR tube. After the impurity is washed out, the trapped nucleic acid may be hybrid detected directly with marker probe or the trapped nucleic acid may be used as template for PCR in the tube and the solid phase PCR product is hybrid detected or for real-time fluorescent PCR in the tube.

Description

1. Technical field: [0001] This article relates to a nucleic acid extraction and detection method 2. Technical background: [0002] Nucleic acid extraction is the basis for many nucleic acid manipulations. At present, there are many nucleic acid extraction methods, such as SDS method, CTAB method, spin column method, etc. Each of these methods has advantages and disadvantages. These nucleic acid crude extracts often contain a large amount of protein, polysaccharides, tannins and pigments, etc. These impurities are sometimes difficult to remove from the nucleic acid. Most proteins can be denatured by treatment with chloroform or phenol and removed by precipitation. RNA in DNA extraction can be removed by RNase, but polysaccharides, tannins and other substances are difficult to remove. When the concentration of these impurities is high, it often makes the nucleic acid extract colloidal, and more importantly, even at very low concentrations, they can interfere with subsequen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12P19/34C12Q1/25C12Q1/68
Inventor 朱水芳赵文军黄文胜陈红运
Owner 国家质量监督检验检疫总局动植物检疫实验所
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