PCR-HRM primer and method for quickly distinguishing canine parvoviruses of different genotypes

A canine parvovirus, genotype technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of complex operation, limited application, high cost, and achieve good repeatability, The effect of good degeneracy and low cost

Active Publication Date: 2014-08-27
广东宠健生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] None of the above methods can overcome the shortcomings of complex operation, time-consuming, high cost, etc., and their application in clinical practice is limited

Method used

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  • PCR-HRM primer and method for quickly distinguishing canine parvoviruses of different genotypes
  • PCR-HRM primer and method for quickly distinguishing canine parvoviruses of different genotypes
  • PCR-HRM primer and method for quickly distinguishing canine parvoviruses of different genotypes

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Experimental program
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Embodiment 1

[0061] (1) PCR-HRM primers

[0062] After screening a large number of designed primers, it was found that the base sequences of the primers, SEQ ID NO: 1 and SEQ ID NO: 2, have the best effect on the PCR-HRM method for distinguishing different genotypes of canine parvovirus, and the base sequences are as follows Show.

[0063] P1 : 5'-CCAGAAGGAGATTGGATTCA-3' (SEQ ID NO: 1),

[0064] P2: 5'-TTAATGCAGTTAAAGGACCAT-3' (SEQ ID NO: 2).

[0065] Among them, the primer P1 is on the 4014-4033 position of the canine parvovirus VP2 gene, and the primer P2 is on the 4138-4158 position of the canine parvovirus VP2 gene (the accession number of this gene in Genbank is M38245).

[0066] (2) Construction of standard plasmid samples and PCR-HRM analysis

[0067] 1) Extraction of canine parvovirus DNA:

[0068] Canine parvovirus DNA in disease samples was extracted with MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0. Disease samples can be whole blood, feces, rectal swabs and other samp...

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Abstract

The invention discloses a PCR-HRM primer and method for quickly distinguishing canine parvoviruses of different genotypes. The method comprises the following steps: extracting virus DNA (deoxyribonucleic acid) from a sample; by taking the virus DNA as a template, using a designed specific primer pair and fluorescent saturated dye to perform amplification reaction, thus obtaining an amplification product; and finally, performing HRM analysis on the amplification product, thus determining the genotype of a canine parvovirus. According to the invention, the method is simple to operate, only the fluorescent saturated dye needs to be added before PCR reaction, and the method is high in detection speed and high in flux; the whole operation process only costs 3 hours, and cell culture of viruses is not needed, thus greatly shortening the time required for typing; the expense is low, specific probes are not needed, and the cost of the saturated dye for each sample is 1.6RMB; and the accuracy is high, the specificity and repetitiveness are favorable, and analysis can be accurately and quickly performed at high flux, thereby ensuring that the invention is beneficial to popularization and application in clinical practice.

Description

technical field [0001] The invention relates to a virus genotyping method, in particular to a PCR-HRM primer and method for quickly distinguishing different genotypes of canine parvoviruses. Background technique [0002] Canine parvovirus (CPV) is a single-stranded small DNA virus, which is the main cause of acute hemorrhagic gastroenteritis in dogs and acute myocarditis in puppies. In 1977, Eugster and Nairn first isolated canine parvovirus from the feces of dogs suffering from hemorrhagic enteritis and named it CPV-2. Since the first isolation of CPV-2 by Eugster et al., the antigenicity of CPV has changed over time, and new CPV genotypes and antigenic types have emerged continuously, and have spread widely around the world. Currently known CPV genotypes include CPV-2a, CPV-2b, and CPV-2c, and the original CPV-2 genotype has been replaced by the emerging antigenic variants. The differences in the nucleic acid sequences of the three genotypes of CPV-2a, CPV-2b and CPV-2c ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 郭鹏举嘎利兵嘎张建峰刘志成朱余军陈琴苓
Owner 广东宠健生物科技有限公司
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