DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method

Abstract

The invention discloses a DHAV (duck hepatitis A virus) typing detection method based on a fluorescent quantitative PCR (polymerase chain reaction) melting curve method. The DHAV typing detection method comprises steps as follows: virus RNA (ribonucleic acid) extraction and cDNA (complementary deoxyribonucleic acid) synthesis; preparation of positive standard forms; establishment of the DHAV typing detection method based on the fluorescent quantitative PCR melting curve method; method validation adopting a specificity test, a sensitivity test and a reproducibility test. According to the DHAV typing detection method, all that is required is to add a pair of primers, A and C gene types are detected by single tube, the method is simple and feasible, and time consumption is low; the whole amplification and detection process adopts closed tube operation, so that pollution of PCR amplification products is avoided, and false positive results are reduced; amplified fragments are short, higher annealing temperature is adopted, and amplification specificity is guaranteed. The fast DHAV typing detection method is established, great convenience is provided for correct diagnosis and control of the DHAV, and economic benefits of agricultural production are achieved.

Description

technical field [0001] The invention relates to a DHAV (duck hepatitis A virus) typing detection method based on a fluorescent quantitative PCR melting curve method. Background technique [0002] Duck hepatitis A virus (DHAV) is a major pathogen that causes viral hepatitis in ducklings. It is mainly characterized by acute necrotizing hepatitis in ducklings aged 1 to 21 days. It has rapid onset, high mortality and high infectivity. It has been distributed worldwide. According to the distance of genetic evolution, DHAV can be divided into three genotypes: DHAV-A, DHAV-B, and DHAV-C. Among them, the dominant strains in my country are mainly DHAV-A and DHAV-C. Because the DHAV-specific antibody is difficult to transmit to ducklings through laying ducks, it is currently mainly relying on the injection of attenuated vaccines or specific yolk antibodies to ducklings to prevent and treat the disease. However, a large number of experiments have confirmed that different genotypes of...

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Problems solved by technology

[0003] At present, the detection of gene fragments of DHAV using reverse transcription polymerase chain reaction (RT-PCR) is the main rapid detection method, but this method cannot quantify the virus

It is also reported that two pairs of primers are used simultaneously to detect and distinguish DHAV-A and DHAV-C, but the sensitivity of this method and the simplicity of operation are lacking, and nucleic acid electrophoresis is required to complete, which increases the time-consuming detection

However, there is no report about using one RRT-PCR to differentially diagnose different genotypes of DHAV

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