CN103602759B - A kind of PCR-RFLP method distinguishing duck circovirus and goose PCV-II - Google Patents

Abstract

The invention discloses a PCR-RFLP method for distinguishing duck circovirus and goose circovirus. The method utilizes the difference in the enzyme cutting site of the Rep protein gene sequence to distinguish, including DNA extraction and PCR amplification to obtain Rep Protein gene fragments were digested with XhoI and analyzed by RFLP. The identification method of the invention is simple, and the efficiency and accuracy are high.

Description

A PCR-RFLP method for distinguishing duck circovirus and goose circovirus

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to a polymerization method for distinguishing duck circovirus and goose circovirus by utilizing the difference in enzyme cleavage sites of the Rep protein gene sequence of waterfowl circovirus (duck circovirus and goose circovirus) Enzyme-linked reaction (PCR)-restriction fragment length polymorphism (RFLP) method.

Background technique

Duck infection with circovirus (duck circovirus, DuCV) was first reported by Hattermann et al. in 2003. Chen et al., a Taiwan scholar in my country, tested samples collected in Taiwan from 2002 to 2003 and showed that the detection rate of circovirus was 38.2%. Fu Guanghua and others first reported duck circovirus infection in mainland China. Jiang et al. reported that duck circovirus was detected from ducks. The positive rate was 33.29%, accompanied by duck type 1 viral hepatitis (DHV- I), duck infectious serositis (RA) and duck colibacillosis (E. coli) co-infection. At present, almost all breeds of ducks have positive detection reports of DuCV infection.

Goose circovirus (GoCV) was first observed in 1999 by German scholar Soike et al. from diseased goose pathological tissue. The sick goose mainly showed dysplasia, weight loss, messy feathers, etc. Histopathological examination found that the lymphocytes in the bursa, spleen and thymus decreased, and the bursal lesion was the most obvious, and even the entire sac structure was destroyed, and it could be observed to basophilic inclusion bodies. In 2001, Todd et al. designed degenerate primers based on the conserved sequences of the Rep proteins of BFDV and PCV, and determined the whole genome sequence of GoCV. In 2002, Chen et al., a scholar from Taiwan, China, used 9 pairs of primers to amplify different fragments of the genome respectively. After sequencing and splicing, they obtained the whole genome sequences of 12 GoCV strains, and found that there were certain differences among the GoCV genome sequences. Yu Xuping first detected goose circovirus in Zhejiang, my country, and analyzed the genome structure and epidemiology of goose circovirus in Zhejiang Province. Wan Chunhe et al. detected GoCV infection from Lande geese for the first time, and performed whole gene sequencing. It has been reported that the nucleotide homology of the protein (Rep protein) encoded by ORF-V1 of DuCV and GoCV can reach more than 80.0%, and a primer can be designed to detect DuCV and GoCV infection.

At present, there have been reports of cross-species transmission of various duck viral infectious diseases, such as duck hepatitis virus, muscovy duck reovirus and flavivirus; there are also reports of cross-species transmission of goose viral infectious diseases and infection of ducks , Such as goose parvovirus and goose hemorrhagic polyomavirus infection of muscovy ducks and half muscovy ducks and other reports.

At present, it has been reported that the nucleotide homology of the protein (Rep protein) encoded by ORF-V1 of DuCV and GoCV can reach more than 80.0%. A primer can be designed to detect DuCV and GoCV infection, but the possible cross-species transmission There is still no corresponding reserve detection method. This study aims at the difference characteristics of Rep protein gene of waterfowl circovirus (duck circovirus and goose circovirus) and establishes related reports on the PCR-RFLP method for distinguishing duck circovirus and goose circovirus. Field blank.

Contents of the invention

The object of the present invention is to provide a kind of PCR-RFLP method that utilizes water fowl circovirus (duck circovirus and goose circovirus) Rep protein gene sequence enzyme cleavage site difference to distinguish duck circovirus and goose circovirus. The method can effectively distinguish duck circovirus and goose circovirus infection (or co-infection), and provide technical guarantee for healthy breeding of waterfowl.

According to the characteristics of the Rep protein gene in the genomes of duck circovirus and goose circovirus, the present invention designs a set of primers that can simultaneously carry out positive amplification of duck circovirus and goose circovirus, and according to the amplification PCR product of goose circovirus There is a specific XhoI restriction site, but there is no XhoI restriction site in the amplified PCR product of duck circovirus to establish a PCR-RFLP method for rapid detection of duck circovirus and goose circovirus.

The present invention adopts following technical scheme:

A PCR-RFLP method for distinguishing duck circovirus and goose circovirus is characterized in that it comprises the following steps:

(1) Extract duck circovirus and goose circovirus genome DNA;

(2) Simultaneously amplify duck circovirus and goose circovirus by PCR using primers P1 and P2 to obtain corresponding Rep protein (replication protein, replication protein) gene fragments;

(3) The PCR product was digested by Xho I and then analyzed by RFLP.

Among them, PCR primers need to meet the following requirements:

(1) The PCR product needs to be designed by selecting the conserved region in the Rep protein gene of duck circovirus and goose circovirus, so that one PCR reaction can be positive for both duck circovirus genomic DNA and goose circovirus genomic DNA amplify;

(2) The PCR product needs to select the conserved region in the Rep protein gene of duck circovirus and goose circovirus for design. Gel recovered products are capable of restriction fragment length polymorphism analysis.

According to the above requirements, the sequences of the amplification primers P1 and P2 in the step (2) are:

Upstream primer P1: 5'-CATGATGGGCAGTGGCTTCCT-3',

Downstream primer P2: 5'-ACCTCCGTCTTCCAATCA-3'.

Wherein, the size of the PCR amplification product in step (2) recovered by gel is 623bp.

Wherein, the XhoI restriction site in the step (3) is located at position 309 of the Rep protein gene sequence of the goose circovirus genome, and the goose circovirus can be digested by XhoI and then detected by agarose gel electrophoresis as two fragments, The sizes are 364bp and 259bp; however, there is no XhoI restriction site in the Rep protein gene of duck circovirus genome, and the size of the fragments detected by agarose gel electrophoresis after duck circovirus digestion with XhoI remains unchanged.

The beneficial effect of the invention is that the identification method is simple, and the efficiency and accuracy are high. Using this method, PCR-RFLP detection was carried out on 20 specimens (including 10 duck and goose tissue disease materials) suspected waterfowl circovirus (duck circovirus and goose circovirus) submitted for inspection in this clinic, and duck circovirus was positive for 4 There were 3 strains of goose circovirus and duck circovirus, and co-infection of duck circovirus and goose circovirus was not detected. According to the established PCR-RFLP method, the type of waterfowl circovirus that is infected can be determined within 5 hours, and the type of duck circovirus and goose circovirus can be correctly analyzed at the same time.

Description of drawings

Fig. 1 is the electrophoresis diagram of the PCR-RFLP method for rapid detection of duck circovirus and goose circovirus. M is DL2000 DNA Marker, 1 is the product of duck circovirus PCR gel recovery, 2 is the product of goose circovirus CR gel recovery, 3 is the product of duck circovirus PCR gel recovery after XhoⅠ enzyme digestion, 4 is the product of goose circovirus digestion by XhoⅠ enzyme Circovirus PCR gel recovery products.

Detailed ways

The following examples further describe the present invention.

Example 1

1. Virus strains: Duck circovirus (GenBank accession number: GQ423744) and goose circovirus (GenBank accession number: GU320569) were isolated, identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

2. Primer design and synthesis

Primers P1 and P2 were designed according to the NS gene characteristics of duck circovirus and goose circovirus, wherein the sequences of primers P1 and P2 were: upstream primer P1: 5'- CATGATGGGCAGTGGCTTCCT -3', downstream primer P2: 5'- ACCTCCGTCTTCCAATCA -3' .

3. PCR amplification

 The genomic DNA of duck circovirus and goose circovirus was extracted by conventional methods. Using the designed specific primers P1 and P2 for PCR amplification, the size of the amplified fragment is about 623bp. The amplification system was 50 μL, including 25 μL of 2×GoTaq Master Green Mix, 1 μL of upstream and downstream primers (20 μM/mL), 1 μL of DNA template, and supplemented with sterilized deionized water to a final volume of 50 μL. The reaction conditions were pre-denaturation at 94°C for 5 min, followed by 50 s at 94°C, 35 s at 53°C, and 45 s at 72°C for 35 cycles, followed by extension at 72°C for 10 min.

4. RFLP analysis

After the PCR reaction, the duck circovirus and goose circovirus PCR products were purified by gel recovery kits and then digested with XhoI. The enzyme digestion system is 20 μL, including 2 μL of 10×H Buffer, 10 μL of the product recovered from the gel, 2 μL of XhoⅠ enzyme, and supplemented with sterilized deionized water to a final volume of 20 μL. After mixing, put in a water bath at 37°C for 1 hour, add 2 μL of 10×Loading Buffer to stop, perform agarose gel electrophoresis analysis, and analyze the duck circovirus and goose circovirus types of the test samples. The Xho I restriction site is located at position 309 of the Rep protein gene sequence of the goose circovirus genome. The goose circovirus can be digested by Xho I and then detected by agarose gel electrophoresis. There is no XhoⅠ restriction site in the Rep protein gene of the circovirus genome, and the size of the fragments detected by agarose gel electrophoresis after duck circovirus digestion with XhoⅠ remains unchanged.

5. Clinical application

Using this method, PCR-RFLP detection was carried out on 20 specimens (including 10 duck and goose tissue disease materials) suspected waterfowl circovirus (duck circovirus and goose circovirus) submitted for inspection in this clinic, and duck circovirus was positive for 4 There were 3 strains of goose circovirus and duck circovirus, and co-infection of duck circovirus and goose circovirus was not detected. According to the established PCR-RFLP method, the type of waterfowl circovirus that is infected can be determined within 5 hours, and the type of duck circovirus and goose circovirus can be correctly analyzed at the same time.

SEQUENCE LISTING

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<110> Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences

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Claims (1)

1. distinguish a PCR-RFLP method for duck circovirus and goose PCV-II, it is characterized in that comprising the following steps:

(1) duck circovirus and goose PCV-II genomic dna is extracted;

(2) pcr amplification is carried out to duck circovirus and goose PCV-II with upstream primer P1 and downstream primer P2 simultaneously, obtain corresponding Rep protein gene fragment; The sequence of upstream primer P1 and downstream primer P2 is respectively:

Upstream primer P1: 5 '-CATGATGGGCAGTGGCTTCCT-3 ',

Downstream primer P2: 5 '-ACCTCCGTCTTCCAATCA-3 ';

(3) get pcr amplification product and carry out rflp analysis after Xho I enzyme cut, can be digested be goose PCV-II, can not enzyme cut then for duck circovirus;

It is 623bp that described step (2) pcr amplification product reclaims size through glue; In described step (3), RFLP analyzes and is specially: Xho I restriction enzyme site is positioned at 309 of goose PCV-II Rep protein sequence, and it is 2 sections that goose PCV-II detects fragment through agarose gel electrophoresis after can being cut by Xho I enzyme, and size is 364bp and 259bp; And in duck circovirus genome Rep protein gene, there is no Xho I restriction enzyme site, it is constant that duck circovirus detects clip size through agarose gel electrophoresis after Xho I enzyme is cut.