Method for preparing visual gene detection reagent based on G4DNAzyme coloration

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...

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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 disease diagnosis and import and export inspection and quarantine. However, there are obvious deficiencies in general, such as long period, cumbersome operation, poor specificity and sensitivity, non-standard, not suitable for large-scale detection, which brings great difficulties to the detection of swine fever

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