CN103602759A - PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method for differentiating duck circovirus from goose circovirus - Google Patents

PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method for differentiating duck circovirus from goose circovirus Download PDF

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CN103602759A
CN103602759A CN201310591203.8A CN201310591203A CN103602759A CN 103602759 A CN103602759 A CN 103602759A CN 201310591203 A CN201310591203 A CN 201310591203A CN 103602759 A CN103602759 A CN 103602759A
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goose
pcv
pcr
circovirus
duck circovirus
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CN103602759B (en
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万春和
黄瑜
陈红梅
施少华
傅光华
程龙飞
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Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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Abstract

The invention discloses a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method for differentiating duck circovirus from goose circovirus. The PCR-RFLP method is used for differentiating by utilizing Rep protein gene sequence enzyme cutting site difference and comprises the following steps of: extracting DNA (Deoxyribonucleic Acid), carrying out PCR amplification to obtain a Rep protein gene segment, and carrying out RFLP analysis after carrying out XhoI digestion. The PCR-RFLP method disclosed by the invention is simple and higher in efficiency and accuracy.

Description

A kind of PCR-RFLP method of distinguishing duck circovirus and goose PCV-II
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of polymerization integrated enzyme reaction (PCR)-restriction fragment length polymorphism (RFLP) method of utilizing aquatic bird PCV-II (duck circovirus and goose PCV-II) Rep protein gene sequence restriction enzyme site difference difference duck circovirus and goose PCV-II.
Background technology
Duck infects PCV-II (duck circovirus, DuCV) and by Hattermann, equals report in 2003 the earliest.The sample detection that China Taiwan's scholars Chen etc. gather 2002～2003 Nian China Taiwans shows, PCV-II recall rate is 38.2 %.First Fu Guanghua etc. have duck circovirus to infect at China mainland area report, Jiang etc. have from the report of duck circovirus being detected in duck body, its positive rate is 33.29%, and with duck 1 C-type virus C hepatitis (DHV-I), sick (E. coli) coinfection of infectious serositis of duck (RA) and E. coli isolated from ducks.At present, nearly all kind duck all sees has DuCV to infect positive detection report.
Goose PCV-II (GoCV) was observed by German scholar Soike etc. early than 1999 from ill goose pathological tissue.The dysplasia of sick goose main manifestations, weight loss, feather are in disorder etc., histopathologic examination finds the lymphopenia of the fabricius bursa, spleen and thymus gland, wherein fabricius bursa pathology is the most obvious, even occurs whole capsule structural damage, and can be observed basophilous occlusion body.Calendar year 2001 Todd etc., according to the conserved sequence design degenerated primer of the Rep albumen of BFDV and PCV, have measured the whole genome sequence of GoCV.9 pairs of primers of TaiWan, China scholar Chen in 2002 etc. application are amplification gene group different fragments respectively, after order-checking splicing, have obtained the whole genome sequence of 12 strain GoCV, and find to exist between GoCV genome sequence certain difference.Genome structure and Epidemiology that remaining rising sun equality detects goose PCV-II ，Bing Dui Zhejiang Province goose PCV-II as far back as China Zhejiang are analyzed.Ten thousand spring detected GoCV infection first with waiting from bright moral goose, and carried out full gene sequencing.There is coded albumen (Rep albumen) nucleotide homology of ORF-V1 of report DuCV and GoCV to reach more than 80.0% can to design a kind of primer and detect DuCV and GoCV infection.
At present, studies have reported that multiple duck viral transmissible disease is across kind of a report for propagation infected goose, as duck hepatitis virus, muscovy duck reovirus and flavivirus; Also there is goose viral infectious to infect across kind of a report for propagation infected duck, as reports such as goose parvovirus and goose hemorrhagic polyomavirus infection kind duck and half kind of ducks.
At present, have coded albumen (Rep albumen) nucleotide homology of ORF-V1 of report DuCV and GoCV to reach more than 80.0% can to design a kind of primer and detect DuCV and GoCV infection, but to may occur across kind of propagation still without corresponding deposit detection method.This research is set up difference duck circovirus and goose PCV-II PCR-RFLP method relevant report for aquatic bird PCV-II (duck circovirus and goose PCV-II) Rep protein gene difference characteristic, and it is blank that foundation of the present invention can be filled up domestic and international association area.
Summary of the invention
The object of this invention is to provide a kind of PCR-RFLP method of utilizing aquatic bird PCV-II (duck circovirus and goose PCV-II) Rep protein gene sequence restriction enzyme site difference difference duck circovirus and goose PCV-II.The method can effectively distinguish duck circovirus and goose PCV-II infects (or coinfection), for aquatic bird healthy aquaculture provides technical guarantee.
The present invention is according to Rep protein gene feature in duck circovirus and goose PCV-II genome, design one group of primer and can to duck circovirus and goose PCV-II, carry out positive amplification simultaneously, according to having specific Xho I restriction enzyme site in goose PCV-II amplification PCR product, and in duck circovirus amplification PCR product, there is no Xho I restriction enzyme site to setting up a kind of PCR-RFLP method of duck circovirus and goose PCV-II being carried out to rapid detection.
The present invention is by the following technical solutions:
A PCR-RFLP method of distinguishing duck circovirus and goose PCV-II, is characterized in that comprising the following steps:
(1) extract duck circovirus and goose PCV-II genomic dna;
(2) with primer P1 and P2, duck circovirus and goose PCV-II are carried out to pcr amplification simultaneously, obtain corresponding Rep albumen (replication protein, replication protein) gene fragment;
(3) get PCR product and carry out rflp analysis after Xho I enzyme is cut.
Wherein, PCR primer need meet following requirement:
(1) this PCR product need select the conservative region in duck circovirus and goose PCV-II Rep protein gene to design, so that PCR reaction of energy can all can positively be increased to duck circovirus genomic dna and goose PCV-II genomic dna;
(2) when this PCR product need select conservative region in duck circovirus and goose PCV-II Rep protein gene to design, must stride across in goose PCV-II Rep protein gene gene the Xho I restriction enzyme site of 309, to glue is reclaimed to product, can carry out restriction fragment length polymorphism analysis.
According to above requirement, the amplimer P1 of described step (2) and the sequence of P2 are:
Upstream primer P1:5 '-CATGATGGGCAGTGGCTTCCT-3 ',
Downstream primer P2:5 '-ACCTCCGTCTTCCAATCA-3 '.
Wherein, described step (2) pcr amplification product is 623bp through glue recovery size.
Wherein, described step (3) Xho I restriction enzyme site is positioned at 309 of goose PCV-II genome Rep protein gene sequence, and it is 2 sections that goose PCV-II can be cut by agarose gel electrophoresis detection fragment 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, duck circovirus is cut by agarose gel electrophoresis detection clip size constant through Xho I enzyme.
Beneficial effect of the present invention: authentication method is simple, efficiency and accuracy rate are higher.Use present method to carry out PCR-RFLP detection to 20 parts of this clinical censorships (wherein duck and goose are organized each 10 parts of pathological material of diseases) doubtful aquatic bird PCV-II (duck circovirus and goose PCV-II), wherein positive 4 parts, 3 strains of goose PCV-II of duck circovirus, do not detect duck circovirus and goose PCV-II coinfection.According to the PCR-RFLP method of setting up, can, to determining the aquatic bird PCV-II type infecting in 5 hours, can also carry out Correct Analysis to duck circovirus and goose PCV-II type simultaneously.
Accompanying drawing explanation
fig. 1 isduck circovirus and goose PCV-II are carried out the electrophorogram of the PCR-RFLP method of rapid detection.M is DL2000 DNA Marker, and 1 is that duck circovirus PCR glue reclaims product, and 2 is that goose PCV-II CR glue reclaims product, and 3 reclaim product for cut duck circovirus PCR glue through Xho I enzyme, and 4 reclaim product for cut goose PCV-II PCR glue through Xho I enzyme.
Embodiment
The present invention will be further described for embodiment below.
Embodiment 1
1, strain: duck circovirus (GenBank accession number: GQ423744) with goose PCV-II (GenBank accession number: GU320569) by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute isolation identification and preservation.
2, design of primers and synthetic
According to duck circovirus and goose PCV-II NS gene expression characteristics design primer P1 and P2, wherein primer P1 and P2 sequence are: upstream primer P1:5 '-CATGATGGGCAGTGGCTTCCT-3 ', downstream primer P2:5 '-ACCTCCGTCTTCCAATCA-3 '.
3, pcr amplification
With ordinary method, extract duck circovirus and goose PCV-II genomic dna.With designed Auele Specific Primer P1 and P2, carry out pcr amplification, the about 623bp of amplified fragments size.Amplification system is 50 μ L, and wherein 2 * GoTaq Master Green Mix, 25 μ L, upstream and downstream primer (20 μ M/mL) each 1 μ L, DNA profiling 1 μ L, supplement sterilizing deionized water to final volume 50 μ L.Reaction conditions is 94 ℃ of denaturation 5min, carries out subsequently 94 ℃ of 50s, 53 ℃ of 35s, 72 ℃ of 45s, carries out after 35 circulations, and 72 ℃ are extended 10min.
4, rflp analysis
After PCR reaction finishes, duck circovirus and goose PCV-II PCR product are carried out to Xho I enzyme respectively after glue reclaims test kit purifying and cut.The enzyme system of cutting is 20 μ L, 10 * H Buffer, 2 μ L wherein, and glue reclaims product 10 μ L, Xho I enzyme 2 μ L, supplements sterilizing deionized water to final volume 20 μ L.After mixing, through 37 ℃ of water-baths 1 hour, add 2 μ L10 * Loading Buffer to stop, carry out agarose gel electrophoresis analysis, to detecting sample, analyze duck circovirus and goose PCV-II type.Xho I restriction enzyme site is positioned at 309 of goose PCV-II genome Rep protein gene sequence, and it is 2 sections that goose PCV-II can be cut by agarose gel electrophoresis detection fragment by Xho I enzyme, and size is 364bpbp and 259bp; And in duck circovirus genome Rep protein gene, there is no Xho I restriction enzyme site, duck circovirus is cut by agarose gel electrophoresis detection clip size constant through Xho I enzyme.
5, clinical application
Use present method to carry out PCR-RFLP detection to 20 parts of this clinical censorships (wherein duck and goose are organized each 10 parts of pathological material of diseases) doubtful aquatic bird PCV-II (duck circovirus and goose PCV-II), wherein positive 4 parts, 3 strains of goose PCV-II of duck circovirus, do not detect duck circovirus and goose PCV-II coinfection.According to the PCR-RFLP method of setting up, can, to determining the aquatic bird PCV-II type infecting in 5 hours, can also carry out Correct Analysis to duck circovirus and goose PCV-II type simultaneously.
SEQUENCE LISTING
<110> Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120> PCR-RFLP method of distinguishing duck circovirus and goose PCV-II
<160> 2
<170> BiSSAP 1.2
<210> 1
<211> 21
<212> DNA
<213> Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<220>
<221> source
<222> 1..21
<223>/organism=" Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute "
/mol_type="unassigned DNA"
<400> 1
catgatgggc agtggcttcc t 21
<210> 2
<211> 18
<212> DNA
<213> Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<220>
<221> source
<222> 1..18
<223>/organism=" Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute "
/mol_type="unassigned DNA"
<400> 2
acctccgtct tccaatca 18

Claims (2)

1. a PCR-RFLP method of distinguishing duck circovirus and goose PCV-II, is characterized in that comprising the following steps:
(1) extract duck circovirus and goose PCV-II genomic dna;
(2) with upstream primer P1 and downstream primer P2, duck circovirus and goose PCV-II are carried out to pcr amplification 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, what can not enzyme cut is duck circovirus.
2. according to the PCR-RFLP method of the difference duck circovirus described in claims 1 and goose PCV-II, it is characterized in that: described step (2) pcr amplification product reclaims size for 623bp through glue; In described step (3), rflp analysis 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 can be cut by agarose gel electrophoresis detection fragment 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, duck circovirus is cut by agarose gel electrophoresis detection clip size constant through Xho I enzyme.
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CN105132589A (en) * 2015-10-12 2015-12-09 福建省农业科学院畜牧兽医研究所 PCR-RFLP primer for distinguishing DHV-1 and new serotype and method
CN105177186A (en) * 2015-10-12 2015-12-23 福建省农业科学院畜牧兽医研究所 PCR-RFLP primer and method for rapidly identifying genotypes of duck circoviruses
CN106367532A (en) * 2016-08-30 2017-02-01 福建省农业科学院畜牧兽医研究所 PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for distinguishing clade 2.3.4 and clade 2.3.4.4 H5 AIVs (H5 subtype avian influenza viruses) based on sequence polymorphism
CN106929607A (en) * 2017-04-25 2017-07-07 华南农业大学 A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit
CN113234855A (en) * 2021-04-22 2021-08-10 河北农业大学 Primer and probe combination for RAA-LFD detection of duck circovirus and application thereof

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105132589A (en) * 2015-10-12 2015-12-09 福建省农业科学院畜牧兽医研究所 PCR-RFLP primer for distinguishing DHV-1 and new serotype and method
CN105177186A (en) * 2015-10-12 2015-12-23 福建省农业科学院畜牧兽医研究所 PCR-RFLP primer and method for rapidly identifying genotypes of duck circoviruses
CN105132589B (en) * 2015-10-12 2018-08-14 福建省农业科学院畜牧兽医研究所 A kind of the PCR-RFLP primers and method of difference 1 type of duck hepatitis virus and new serotype
CN106367532A (en) * 2016-08-30 2017-02-01 福建省农业科学院畜牧兽医研究所 PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for distinguishing clade 2.3.4 and clade 2.3.4.4 H5 AIVs (H5 subtype avian influenza viruses) based on sequence polymorphism
CN106929607A (en) * 2017-04-25 2017-07-07 华南农业大学 A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit
CN113234855A (en) * 2021-04-22 2021-08-10 河北农业大学 Primer and probe combination for RAA-LFD detection of duck circovirus and application thereof

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