A pcr-hrm primer and method for rapidly distinguishing different genotypes of canine parvovirus

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 Currently known CPV genotypes include CPV-2a, CPV-2b, and CPV-2c, and the original CPV-2 genotype has been replaced by the emerging antigenic variants.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 ...

AI Technical Summary

Problems solved by technology

[0003]The various traditional genotyping methods have their own deficiencies. For example, the virus isolation and identification method needs to rely on 3 different monoclonal antibodies to complete the genotyping. Time-consuming and labor-intensive, it is not easy to quickly distinguish different genotypes of canine parvovirus; the hemagglutination inhibition test method has higher requirements on the gradient of canine parvovirus. Generally, the corresponding monoclonal antibody can be used to identify the gradient greater than 1:64, and the gradient is less than 1:32. Monoclonal antibodies can be used for detection after amplification on MDCK or F81 cells; while ordinary PCR methods are not specific, although fluorescent quantitative PCR (MGB probes) can accurately distinguish different genotypes of canine parvovirus, it requires two Pair probes are expensive, limiting their practical application in production, etc.

[0004] Traditional CPV typing methods include virus isolation and identification (VI), hemagglutination inhibition test (HI), common PCR method, restriction fragment length polymorphism ( RFLP) method, MGB probe method and sequencing, among which the virus isolation and identification method needs to rely on three different types of monoclonal antibodies to complete the typing, which is time-consuming and laborious and difficult to quickly identify different subtypes of CPV; the hemagglutination inhibition test method is effective for CPV virus The gradient requirements are relatively high. Generally, the corresponding monoclonal antibody can be used to identify the gradient greater than 1:64, and the gradient is less than 1:32, and the monoclonal antibody can only be detected after amplification on MDCK or F81 cells; the PCR method uses CPV CPV-2a and CPV-2b types can be distinguished by base mismatch at the end of 3′ of the primer of -2b sequence. Although this method can distinguish CPV-2a and CPV-2b types, the specificity is not high and it is easy to amplify two types at the same time.

In addition, the emerging CPV variants show that the 3ˊ terminal base mismatch method is poorly conserved and is easily affected by the mutation of the viral nucleic acid sequence. Therefore, it is difficult to accurately type CPV by ordinary PCR methods; the restriction fragment length polymorphism method can Distinguish between CPV-2b and CPV-2c, but cannot distinguish between CPV-2a and CPV-2b, because MboⅡenzyme has the same digestion results on CPV-2a and CPV-2b, this method can only be confirmed as CPV with monoclonal antibody The genotypes of CPV-2b and CPV-2c can only be identified under the precursor of -2b; although the MGB probe method can distinguish different genotypes of CPV, but synthesizing two or more probes at the same time is expensive and difficult to use in clinical testing; sequencing Although the accuracy of the method is high, but the price is high and it takes a long time, generally it takes 24 hours to complete

[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

Images