Triple PCR (Polymerase Chain Reaction) detection method of muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4

A technology of Muscovy duck parvovirus and goose parvovirus, applied in the field of PCR detection, can solve the problems of difficult differential diagnosis and distinction of the three viruses, and achieve the effect of reducing the time and cost of electrophoresis detection and high specificity

Inactive Publication Date: 2018-12-21
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE ANHUI ACAD OF AGRI SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The genomes of GPV and MDPV have a high degree of homology, and their antigenicity is highly similar. FAdV can be divided into 5 species and 12 serotypes, among which serotype 4 is the main one. , causing difficulties in the differential diagnosis of the three viruses

Method used

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  • Triple PCR (Polymerase Chain Reaction) detection method of muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4
  • Triple PCR (Polymerase Chain Reaction) detection method of muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4
  • Triple PCR (Polymerase Chain Reaction) detection method of muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A triple PCR detection method for Muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4, comprising the steps of:

[0031] S1. Design 3 pairs of specific amplification primers through the Primer Premier 5.0 software; select specific primers in the conserved region of the viral genome according to the primer design principles, avoid the formation of dimers or hairpin structures between the primers, and avoid the formation of Mismatches, and the annealing temperature of the three pairs of primers should be consistent to maximize the efficiency of PCR.

[0032] Muscovy duck parvovirus forward primer sequence: 5'-AACTGAGAGATGACATGAAT-3', Muscovy duck parvovirus reverse primer sequence: 5'-ACTTGCTTCTCTCTCCACTAT-3', the annealing temperature is 48°C, and the length of the amplified fragment is 628bp;

[0033] Goose parvovirus forward primer sequence: 5′-GTCCGGGTAGACCAGAAA-3′, goose parvovirus reverse primer sequence: 5′-CTCAGGAGTCACAGGAAT-3′, the annealing tem...

Embodiment 2

[0042] Triple PCR Specificity Experiment

[0043] Prepare 7 samples, the first one is a mixed template of Muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4, and the second to seventh samples are respectively Muscovy duck parvovirus, goose parvovirus, avian adenovirus type 4, duck yellow Virus, duck type I hepatitis virus and avian reovirus template, above-mentioned 8 samples are carried out triple PCR amplification respectively, PCR amplification system and amplification condition are the same as embodiment 1, and the mass concentration is 2.0% agarose gel PCR products were detected by electrophoresis.

[0044] The result is as figure 2 As shown, using this method to detect Muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4, all can amplify bands of 628bp, 432bp, and 979bp that match the size of the experimental design, while duck yellow virus, duck Hepatitis I virus and avian reovirus have no specific amplification bands at the same p...

Embodiment 3

[0046] Triple PCR sensitivity test

[0047] Prepare a mixed nucleic acid template of Muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4 with a total concentration of 20ng / μL, dilute it to 200fg / μL with a 10-fold progressive gradient, and perform triple PCR on the above-mentioned nucleic acid templates with different concentrations Amplification, the PCR amplification system and amplification conditions are the same as in Example 1, and the PCR products are detected by agarose gel electrophoresis with a mass concentration of 2.0%.

[0048] The result is as image 3 As shown, the triple PCR can detect at least 2pg / μL of Muscovy duck parvovirus and avian adenovirus nucleic acid templates, and 20pg / μL of goose parvovirus templates simultaneously.

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Abstract

The invention belongs to the field of PCR (Polymerase Chain Reaction) detection, and particularly relates to a triple PCR detection method of muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4. The triple PCR detection method comprises the following steps: S1, respectively designing three pairs of specific amplification primers according to gene conserved sequences of the muscovy duck parvovirus, the goose parvovirus and the avian adenovirus type 4; S2, extracting DNAs of the muscovy duck parvovirus, the goose parvovirus and the avian adenovirus type 4; S3, mixing all the DNAs obtained in S2 and then carrying out PCR amplification; S4, detecting PCR products in S3 through electrophoresis. The triple PCR detection method established can simultaneously detect the muscovy duck parvovirus, the goose parvovirus and the avian adenovirus type 4; the minimum detection amounts of three viral nucleic acid templates are 2pg/mu L, 20pg/mu L and 2pg/mu L respectively.

Description

technical field [0001] The invention belongs to the field of PCR detection, in particular to a triple PCR detection method for Muscovy duck parvovirus, goose parvovirus and avian adenovirus type 4. Background technique [0002] Muscovy duck parvovlrus (MDPV) and goose parvovirus (GPV) are members of the family Parvoviridae, the subfamily Parvoviridae, and the genus Reliant Virus. The parvovirus disease of young muscovy ducks that MDPV can cause is also known as the 3-week disease of muscovy ducks. The disease is highly contagious and mainly causes lesions in the digestive tract and intestinal tract of young muscovy ducks. Sick ducks often show symptoms such as panting, weight loss, food refusal, soft feet and loose stools, and the morbidity and mortality are high. Gosling plague is a severe infectious disease mainly caused by GPV. Goose parvovirus is an acute or subacute septic infectious disease that spreads rapidly. The disease is mainly characterized by intestinal les...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 戴银张丹俊赵瑞宏胡晓苗周学利沈学怀侯宏艳潘孝成李默
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE ANHUI ACAD OF AGRI SCI
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