Method for preparing visual gene identification and detection reagent based on G4DNAzyme coloration

A technology for identification and detection of genes, applied in the field of genetic engineering, can solve the problems of unsuitability for mass detection, poor specificity and sensitivity, difficulty in identification and detection of swine fever, etc., and achieves the effects of low detection cost, intuitive results, and fast detection speed.

Active Publication Date: 2011-05-04
巨星农牧有限公司 +1
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  • Abstract
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Problems solved by technology

[0007] The common identification and detection methods of swine fever mainly include: virus isolation and biological tests, immunofluorescence technology, nucleic acid sequencing, fluorescent quantitative PCR, etc., but these methods generally have obvious

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  • Method for preparing visual gene identification and detection reagent based on G4DNAzyme coloration
  • Method for preparing visual gene identification and detection reagent based on G4DNAzyme coloration
  • Method for preparing visual gene identification and detection reagent based on G4DNAzyme coloration

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Embodiment Construction

[0043] Based on G 4 A method for preparing a visual gene identification detection reagent for DNase color development, characterized in that it comprises the following steps:

[0044] A. Extraction of target genes

[0045] Select the cells infected with classical swine fever virus, freeze and thaw three times at -70°C, each time for 10-30 minutes, take 200 μl and add 1ml RNA lysate (Trizol) to mix, let stand at room temperature for 10-15 minutes, add 0.2 ml chloroform, let stand at room temperature for 3-5 minutes, centrifuge at 12000g / min at 4°C for 10-15 minutes, transfer the supernatant to another centrifuge tube, add 0.5ml isopropanol, precipitate at room temperature for 10-20 minutes, and then Centrifuge at 12000g / min at 4°C for 10 minutes, pour off the supernatant, add 1ml of 75% ethanol and centrifuge at 7500g / min at 4°C for 5 minutes, discard the supernatant, dry the RNA pellet at room temperature, add DEPC-treated water 20μl to get target gene RNA;

[0046] The 75%...

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Abstract

The invention relates to a visual classical swine fever virus (CSFV) gene identification and detection reagent based on G4DNAzyme coloration and a preparation method thereof. The reagent is asymmetric (polymerase chain reaction) PCR reaction liquid. The 100mul reaction liquid comprises: 2mul of positive primerW3(+) (0.4-4mumol/L) and 2mul of negative primer W3(-) (20mumol/L), 10mul of PCR solution (10*buffer), 8mul of MgCl2 solution (2.5mmol/L), 2mul of dNTPs solution (10mmol/L), 2mul of TaDNA polymerase (2U) and balance of sterile double distilled water. A sequence of the positive primer W3(+) in the asymmetric PCR reaction liquid is: 5'-gcgcgggtaacccgggat-3', and the sequence of the gemeative primer W3(-) is 5'-GTCCAACTGTGGACGTCAGGAA-3'. The method has the beneficial effects of high detection speed, accuracy, stability, good repeatability, simple and easy-to-operate detection steps, capability of directly observing the coloration reaction with naked eyes, intuitionistic results and low cost.

Description

technical field [0001] The present invention relates to a kind of preparation method of gene identification detection reagent, specifically, relates to the preparation method based on G 4 A method for preparing a visual gene identification detection reagent developed by DNase color development. The reagent can not only quickly identify and detect the classical swine fever virus, and distinguish the virulent classical swine fever virus from the vaccine virus, but also is suitable for the identification and detection of nucleic acids of other pathogenic microorganisms, and belongs to the technical field of genetic engineering. Background technique [0002] Nucleic acid is the key substance that determines the nature of heredity. Every organism has its own specific nucleic acid sequence, and various biological traits can eventually find their roots in specific nucleic acid sequences. Therefore, nucleic acid analysis technology has a wide range of applications and plays an impo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/78
Inventor 王毅鲁小璐
Owner 巨星农牧有限公司
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