Method for preparing visual gene detection reagent based on G4DNAzyme coloration

A technology of gene detection and reagents, applied in the field of genetic engineering, can solve problems such as poor specificity and sensitivity, inapplicability to large-scale detection, difficulty in detection of swine fever, etc., achieve intuitive results, low detection cost, and facilitate popularization and use

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

[0005] Traditional swine fever detection methods mainly include: virus isolation and biological tests, electron microscope technology, immunofluorescence technology, complement fixation test, nucleic acid hybridization, polyacrylamide gel electrophoresis, etc. These methods are widely used in virus dise...

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

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Embodiment Construction

[0040] Based on G 4 The preparation method of the visual gene detection reagent of DNase color development is characterized in that: comprising the following steps:

[0041] A. Extraction of target genes

[0042] Select PK-15 cells infected with CSFV Shimen virus, blank PK-15 cells, use sterile double distilled water as control, freeze and thaw at -70°C for 3 times, each time for 10-30 minutes, take 200 μl Add 1ml of RNA Lysis Solution (Trizol) and mix well, let stand at room temperature for 10-15 minutes, add 0.2ml of chloroform, let stand at room temperature for 3-5 minutes, centrifuge at 12000g / min at 4°C for 10-15 minutes, and transfer the supernatant to In another centrifuge tube, add 0.5ml of isopropanol, precipitate at room temperature for 10-20 minutes, 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,...

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Abstract

The invention relates to a method for preparing a visual classical swine fever virus (CSFV) gene detection reagent based on G4DNAzyme coloration. The method comprises the following steps: selecting cells infected with viruses, after freeze thawing and lysing, adopting a gene extraction kit for extracting a target gene and obtaining cDNA after carrying out reverse transcription and inactivation onMoloney murine leukemia virus (MMLV) reverse transcriptase; adding the cDNA to an asymmetric polymerase chain reaction (PCR) system and obtaining an asymmetric PCR product through degeneration, annealing and extension amplification for 50-100 cycles; and adding upstream and downstream probes and the asymmetric PCR product to G4DNAzyme coloration reaction buffer, and after degeneration and annealing, adding Hemin, ATBS and H2O2 to carry out coloration reaction and observing whether macroscopic green color appears, thus judging whether CSFV infection exists. 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, intuitionisticresults and low cost.

Description

technical field [0001] The present invention relates to a kind of preparation method of gene detection reagent, specifically, relates to the preparation method based on G 4 A method for preparing a visual gene detection reagent for DNase color development. The reagent not only can quickly and qualitatively detect swine fever virus, but also is suitable for qualitative detection of nucleic acids of other pathogenic microorganisms, and belongs to the technical field of genetic engineering. Background technique [0002] Since the last century, it has been recognized that nucleic acid is a key substance that determines the nature of heredity. For most organisms, DNA is the carrier of genetic information; in some viruses, this role is played by RNA. Every organism has its own specific nucleic acid sequence, and various biological traits can eventually find their roots in specific nucleic acid sequences. Therefore, the detection of specific sequence nucleic acids has broad appl...

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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