CN113481199A

CN113481199A - Method for identifying two genotypes of waterfowl circovirus

Info

Publication number: CN113481199A
Application number: CN202110953455.5A
Authority: CN
Inventors: 于静; 刘�东; 刘红祥; 许春雨; 任衍倍; 王玉超; 李彬
Original assignee: Qingdao Yebio Bioengineering Co Ltd
Current assignee: Qingdao Yebio Bioengineering Co Ltd
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2021-10-08

Abstract

The invention provides a method for identifying two genotypes of a waterfowl circovirus, wherein a primer group used comprises an upstream primer and two downstream primers, wherein the sequence of the upstream primer is SEQ ID NO. 1, the sequence of the downstream primer is SEQ ID NO. 2, and the sequence of the downstream primer is SEQ ID NO. 3. The primer combination of the invention can specifically amplify the Cap gene segment of the waterfowl circovirus in the sample, has high detection sensitivity, can distinguish two genotypes according to the size of the amplified gene segment, has quick and accurate result, and greatly reduces the time cost and the reagent cost.

Description

Method for identifying two genotypes of waterfowl circovirus

Technical Field

The invention belongs to the technical field of molecular detection, and particularly relates to a method for identifying two genotypes of a waterfowl circovirus.

Background

Duck circovirus (DuCV) is a single-stranded circular DNA virus with the diameter of 15-16 nm, no envelope, icosahedral symmetry and a genome of about 1.9kb, belongs to the circovirus family and belongs to the genus of circovirus. DuCV was first reported in 2003 by Hattermann et al, Germany, and was subsequently discovered or reported in Hungary, the United states, Korea, Taiwan, and mainland China, among others. DuCV infected ducks show feather disorder, growth retardation, weight loss and other symptoms. Based on the genomic analysis of DuCV and the sequence analysis of Cap gene, DuCV is currently divided into two genotypes: DuCV-1 and DuCV-2. PCR, fluorescent quantitative PCR, nucleic acid probe hybridization and ELISA methods for detecting DuCV have been established and epidemiological investigation has been completed.

At present, circovirus is ubiquitous in duck groups, and mixed infection of DuCV-1 and DuCV-2 is ubiquitous, but problems exist in the identification of DuCV-1 and DuCV-2.

Disclosure of Invention

The invention provides a method for identifying two genotypes of a waterfowl circovirus, thereby making up the defects of the prior art.

The invention firstly provides a primer group, which comprises 1 upstream primer and 2 downstream primers, wherein the information of the upstream primer is as follows:

DuCV-F：5′-GCACGCTCGACAATTGCAAG-3′(SEQ ID NO:1)，

the information of the downstream primer is as follows:

DuCV-R1：5′-TGTTAATTCTGCGGTACAGA-3′(SEQ ID NO:2)，

DuCV-R2：5′-AGATAATGCGACCGGCGACG-3′(SEQ ID NO:3)。

the primer group provided by the invention is used for identifying two genotypes DuCV-1 and DuCV-2 of the waterfowl circovirus; or for preparing a preparation for detecting two genotypes of DuCV-1 and DuCV-2;

the invention also provides a method for detecting two genotypes of the waterfowl circovirus, namely DuCV-1 and DuCV-2, which utilizes the primer group to carry out PCR amplification detection;

the PCR reaction system comprises: an Additive for high Specificity at a final concentration of 1X, a MightyAmp Buffer (Mg) at a final concentration of 1X²⁺dNTP), MightyAmp DNA Polymerase at a concentration of 1.25U/. mu.L, and a primer mix concentration of 20. mu.M.

The conditions of the PCR reaction were: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 68 ℃ for 2min, 32 cycles, and extension at 72 ℃ for 10 min.

The primer combination of the invention can specifically amplify the Cap gene segment of the waterfowl circovirus in the sample, has high detection sensitivity, can distinguish two genotypes according to the size of the amplified gene segment, has quick and accurate result, and greatly reduces the time cost and the reagent cost.

Drawings

FIG. 1: multiple primer specificity verification electrophoretogram, wherein M is DL2000 DNA Marker, 1, DuCV-1 genotype positive sample; 2, DuCV-1 genotype positive samples; 3, GPV samples; 4, EDS sample; 5, ILTV samples; 6, ALV samples; 7, MDV samples; 8, FPV samples; 9, MDPV samples; 10, negative control; 10, negative control.

FIG. 2: the sensitivity of DuCV-1 genotype sample amplified by multiple primers is verified by electrophoresis, wherein M is DL2000 DNA Marker, 1, the concentration of DuCV-1 nucleic acid is 200ng/uL, 2, and the concentration of DuCV-1 nucleic acid is 40 ng/uL; 3, the concentration of DuCV-1 nucleic acid is 8 ng/uL; 4, DuCV-1 nucleic acid concentration of 1.6 ng/uL; 5, DuCV-1 nucleic acid concentration of 0.32 ng/uL; 6, DuCV-1 nucleic acid concentration of 0.064 ng/uL; 7, DuCV-1 nucleic acid concentration of 0.012 ng/uL; DuCV-1 nucleic acid concentration of 0.002 ng/uL; 9, DuCV-1 nucleic acid concentration is negligible; 10, DuCV-1 nucleic acid concentration is negligible.

FIG. 3: the sensitivity of DuCV-2 genotype sample amplified by multiple primers is verified by electrophoresis, wherein M is DL2000 DNA Marker, 1, the concentration of DuCV-2 nucleic acid is 200ng/uL, 2, and the concentration of DuCV-2 nucleic acid is 40 ng/uL; 3, the concentration of DuCV-2 nucleic acid is 8 ng/uL; 4, the concentration of DuCV-2 nucleic acid is 1.6 ng/uL; 5, DuCV-2 nucleic acid concentration is 0.32 ng/uL; 6, the concentration of DuCV-2 nucleic acid is 0.064 ng/uL; 7, DuCV-2 nucleic acid concentration of 0.012 ng/uL; the concentration of DuCV-2 nucleic acid is 0.002 ng/uL; 9, DuCV-2 nucleic acid concentration is negligible; 10, DuCV-2 nucleic acid concentration is negligible.

FIG. 4: DuCV-1 and DuCV-2 genotype homology analysis plots.

FIG. 5: and (3) verifying the genotype of a suspected waterfowl circovirus positive sample in a laboratory.

Detailed Description

In the method for distinguishing the circovirus type 1 from the circovirus type 2, the type 1 and the type 2 can be effectively distinguished only by a single target strip. In the embodiment, the positive sample is waterfowl circovirus positive morbid material in a laboratory, the positive morbid material sample is taken as a template, and the negative control is SPF duck embryo allantoic fluid; PCR amplification is carried out by using the primer group, and PCR products of 700bp and 1080bp are respectively obtained by DuCV-1 and DuCV-2 genotypes. Sequence analysis shows that the amplified DNA fragments are all corresponding circovirus genotype sequences.

The method of using the primer combination of the present invention will be described in detail with reference to specific examples.

Example 1: primer design screening and detection method

1. Primer design

DuCV genome is about 1.9kb single-chain circular DNA molecule, DuCV genome has been identified in the gene has 3 genes, respectively located in the positive chain of the coding replication protein ORF1 (also called Rep gene), located in the complementary chain of the coding capsid protein ORF2 (also called Cap gene), and located in the complementary chain of the apoptosis activity protein ORF 3. DuCV is classified into DuCV-1 and DuCV-2 according to the sequence analysis of the genomic chicken Cap gene of DuCV. The nucleotide homology of the two genotypes is 82% to 84% (FIG. 4). Primers are designed according to two genotype reference strains DuCV-1 (accession numbers AY228555, EF451157, EU022375 and GU131340) and DuCV-2 (accession numbers AY394721, DQ166836, DQ166837 and EF370476), a universal upstream primer is designed in an N-terminal conserved region, and specific primers for amplifying two genotypes are designed in a C-terminal base difference region. The primer sequences are as follows:

F：5’-GCACGCTCGACAATTGCAAG-3’，

R1：5’TGTTAATTCTGCGGTACAGA--3’，

R2：5’-AGATAATGCGACCGGCGACG-3’，

DuCV-1 was expected to show a 700bp band and DuCV-2 a 1080bp band.

2. Primer specificity and sensitivity validation

Firstly, verification of primer specificity

The specificity of the multiplex primer is verified by carrying out PCR detection on seven virus genomes of GPV, EDS, ILTV, ALV, MDV, FPV and MDPV by using the multiplex primer. The PCR product was electrophoresed through 1.0% agarose gel, stained with golden view, and observed under ultraviolet light, and the results are shown in FIG. 1, and the amplification results of seven viruses are all negative.

② verification of primer sensitivity

Taking 10uL of each of the DuCV-1 positive disease material and the DuCV-2 positive disease material, respectively diluting by 5 times, leading the template concentration to be 200 ng/uL-0.002 ng/uL, and verifying the sensitivity of the primer. As a result, as shown in FIGS. 2 and 3, the minimum nucleic acid concentration of each of DuCV-1 and DuCV-2 was 1.6 ng/uL.

3. PCR reaction (25uL system)

0.5uL of the supernatant was added with additional for high Specificity 2.5uL, 12.5uL of MightyAmp Buffer (Mg2+, dNTP), 0.5uL of MightyAmp DNA polymerase0.5uL, and 1.5uL of 20uM primer mix. The conditions of the PCR reaction were: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 68 ℃ for 2min, 32 cycles, and extension at 72 ℃ for 10 min.

After electrophoresis of the PCR products on a 1.0% agarose gel, golden view stained and observed under UV light, the results are shown in FIG. 1, and DNA samples of different serotypes show bright bands at the expected targets.

4. PCR product sequencing validation

Cutting the gel to recover a PCR product, connecting the recovered PCR product to a T vector, sending the T vector to a sequencing company for sequencing, and analyzing a BLAST sequence to show that a 700bp target gene band is DuCV-1, and the sequence of the DuCV-1 target gene band is shown as SEQ ID NO. 4; the 1080bp target gene band is DuCV-2, and the sequence is shown in SEQ ID NO. 5.

Example 2 genotyping of waterfowl circovirus infection disease Agents

The suspected positive disease material of water bird circovirus in the laboratory is 6 parts, 0.5uL of supernatant of the disease material is taken for each part, added for high Specificity 2.5uL, 12.5uL of MightyAmp Buffer (Mg2+, dNTP) is added, 0.5uL of MightyAmp DNA polymerase0.5uL is added, and 2.5uL of 20uM primer mixture is added. The conditions of the PCR reaction were: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 68 ℃ for 2min, 32 cycles, and extension at 72 ℃ for 10 min. After the PCR product is subjected to 1.0% agarose gel electrophoresis, golden view staining is carried out, and the result is shown in figure 5, wherein the DNA samples of 5 samples have bright bands at 780bp, all belong to the

circovirus type

1, and 1 sample is negative; the result shows that the primer group and the method provided by the invention can effectively detect two genotypes of the waterfowl circovirus.

Sequence listing

<110> Qingdao Yibang bioengineering Co., Ltd

<120> a method for identifying two genotypes of waterfowl circovirus

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<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcacgctcga caattgcaag 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgttaattct gcggtacaga 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agataatgcg accggcgacg 20

<210> 4

<211> 714

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gcacgctcga caattgcaag tttgctatcg tcggcgagga gaagggcgcg aatggtacgc 60

ctcaccttca gggattcctg aaccttcgaa gtaacgcgcg agctgccgcc cttgaggagt 120

cgctgggagg aagagcctgg ctctctcgtg cccggggatc tgatgaagat aatgaagaat 180

attgcgccaa agagtcgaca taccttcgag ttggtgagcc agtctccaag ggtcgatcct 240

ctgatctggc cgaagcgaca tccgctgtga tggctggcgt cccgctgact gaggtggccc 300

ggaagttccc cacgacttat gtaatctttg ggcgtggcct ggaacgcctc cggcacctga 360

tcgtcgagac gcaacgtgat tggaagaccg aagttatcgt tctgattggt cctcctggca 420

ccggaaagag ccgttatgca tttgaatttc ccgccgaaaa caagtattac aaaccacgcg 480

ggaagtggtg ggacggttac tcgggaaatg acgtagtcgt catggacgac ttttatggtt 540

ggttgccgta tgatgatttg ctgagaatta ccgaccgtta cccgttacgg gttgaattca 600

aaggcggtat gactcagttt gtggctaaga ccctgatcat aacaagcaac cgcgagcctc 660

gtgattggta caagagcgag tttgaccttt cagctctgta ccgcagaatt aaca 714

<210> 5

<211> 1100

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<400> 5

gcacgctcga caattgcaag ttcgcaatcg tcggagagga aaagggcgcg aacggtacac 60

ctcaccttca gggattcctg aatctccgaa gcaatgcgag agctgccgcc cttgaagagt 120

cgctgggagg aagagcctgg ctctctcgtg cccggggatc tgacgaagat aacgaagaat 180

attgctccaa agagtcgaca tacctgagag ttggtgaacc aggcggtaag ggtcgatcct 240

cccagctcgc cgatgcgaca tccgctgtgc tcgctggtct cccgctgact gacgttgccc 300

ggaagtatcc gacgacttat gttatctttg ggcgtggcct cgaacgtctt cgtcacctga 360

tcgttgaaac gcaacgtgat tggaagacag aagtcatcgt cttgattggt ccgccaggga 420

ccggaaagag ccgttatgcg tttgaatttc ccgccgaaaa caagtattac aaaccacgcg 480

ggaagtggtg ggacggttac tcgggaaatg acgtagtcgt catggacgac ttttatggtt 540

gggtacctta cgatgatctg cttcggatca ctgaccgtta tccgttacgt gttgagttta 600

aaggtggcat gacccagttt gttgctaaga cgttgatcac aactagcaac aaagagcccc 660

gtgattggta tagaagtgag ttcgatctgt ccgccttata tcggcgcatt aataagtatc 720

ttgtctataa tgttgacaag tatgaacctg cccccgcttg ttccctcccc tttccaataa 780

actactgaga caaagttgtc gttttatgtc tttattaagg tgaacactcg aaagggatat 840

acacttgggc agctggttat ggtgaacact tgaaagggat atacacttgg gcagctggtt 900

atggtgaaca ctcgaaaggg atatacactt gggcagctgg ttatggtgaa cactcgaaag 960

ggatatacac ttgggcagct gggttaaggt taggacagga ccacgtttat gcctagaacc 1020

cggtgaactg accaaacttg atgtaaaacg taaattgagc ttctatatca tatttcccct 1080

cgtcgccggt cgcattatct 1100

Claims

1. A primer group is characterized by comprising 1 upstream primer and 2 downstream primers, wherein the sequence of the upstream primer is SEQ ID NO. 1, the sequence of the downstream primer is SEQ ID NO. 2, and the sequence of the downstream primer is SEQ ID NO. 3.

2. The use of the primer set of claim 1 in the preparation of a product for detecting two genotypes of waterfowl circovirus.

3. The use of claim 2, wherein the article of manufacture is a PCR assay kit.

4. Use of the primer set of claim 1 for identifying two genotypes of waterfowl circovirus, DuCV-1 and DuCV-2.

5. A method for detecting two genotypes of waterfowl circovirus, DuCV-1 and DuCV-2, wherein the method comprises the step of carrying out PCR amplification detection on a sample by using the primer set according to claim 1.

6. The method of claim 5, wherein the PCR reaction system comprises: the final concentration was 1X Additive for high Specificity, the final concentration was 1X MightyAmp Buffer, the concentration was 1.25U/. mu.L MightyAmp DNA Polymerase, and the concentration of the primer mixture was 20. mu.M.

7. The method of claim 6, wherein the PCR reaction conditions in said method are as follows: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 68 ℃ for 2min, 32 cycles, and extension at 72 ℃ for 10 min.