CN106119422B - Distinguish the PCR-RFLP method of clade2.3.4 and clade2.3.4.4H5 AIV - Google Patents
Distinguish the PCR-RFLP method of clade2.3.4 and clade2.3.4.4H5 AIV Download PDFInfo
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- CN106119422B CN106119422B CN201610770360.9A CN201610770360A CN106119422B CN 106119422 B CN106119422 B CN 106119422B CN 201610770360 A CN201610770360 A CN 201610770360A CN 106119422 B CN106119422 B CN 106119422B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The present invention discloses a kind of PCR-RFLP method for distinguishing clade 2.3.4 and clade 2.3.4.4 H5AIV.The following steps are included: 1) extract virus genome RNA from test sample；2) RT-PCR amplification is carried out to clade 2.3.4 and clade 2.3.4.4 virus with primer P1 and P2, obtains corresponding HA genetic fragment；3) RT-PCR amplified production is taken to carry out rflp analysis after Bal I digestion.Method of the invention only needs upstream primer P1 and downstream primer P2, and identified after only needing convenient restriction restriction endonuclease Bal I to carry out RFLP digestion, without complicated cumbersome condition optimizing process, the present invention is quick accurate in clade 2.3.4 and clade2.3.4.4H5 AIV rapid differential diagnosis, can fill up the research blank of domestic and international related fields.
Description
Technical field
The present invention relates to animal doctor's infectious disease quick diagnosis technical field, in particular to a kind of difference clade2.3.4 and
The PCR-RFLP method of clade2.3.4.4H5 AIV.
Background technique
Bird flu (Avian influneza, AI) is as caused by influenza A, mainly encroaches on poultry and wild bird
A kind of acute, highly contagious disease.Highly pathogenic H5 subtype avian influenza virus (H5 subtype avian influenza
Virus, H5 AIV) caused by infection the zoonosis that must be reported has been classified as by World Organization for Animal Health (OIE) at present,
Our country is listed as a kind of animal epidemics.
Earliest record about influenza can be traced to the primary report from Italy in 1878, then all over the world successively
Occur influenza break out and it is popular.1996, in first plant of H5N1 hypotype HPAIV [A/Goose/ that isolated in China arrives
Guangdong/1/96 (H5N1), GS/GD/96], and then Hong Kong was directly felt in generation H5N1 subtype avian influenza virus in 1997
Contaminate occurrences in human life part, this be first fowl source stream Influenza Virus without intermediate host the evidence of direct infection people.Due to viral from ancestral source
GS/GD/96 evolves multiple pedigrees and name disunity, and WHO, FAO and OIE, which combine, thus has formulated unified point of H5N1 HPAIV
Class criterion amounts to 10 difference clade for H5N1 HPAIV points for 0~9, and wherein clade2.3.4 is most earlier than 2003~2006 years
Between be popular in the Strain on the ground such as China, Vietnam, Thailand, Laos and Malaysia, 2006~2009 years clade2.3.4 branches
It is advantage epidemic strain in many areas in China.In recent years, clade2.3.4 branch occurs new branch clade2.3.4.4's
H5N6 hypotype, H5N8 subtype virus, and be widely current in multiple provinces and cities, China.For the vaccine (Re- of clade2.3.4H5 AIV
5 plants) and the vaccine (Re-8 plants) of clade2.3.4.4H5 AIV succeeded in developing, to clade2.3.4 and clade2.3.4.4H5
AIV, which carries out antidiastole, to be helped to instruct the scientifical use of the corresponding vaccine of H5 AIV.
However, gene order homology clade2.3.4 and clade2.3.4.4 between the two is 94.4% (with classics
For Reference strains compare), it is difficult to be identified by conventional molecular biology method, restriction enzyme fragment length is more
State property (Restriction fragment length polymorphism, RFLP) is to be drawn in recent years with gene loci difference
A kind of different mark of molecular biology technology of restriction enzyme site is played, is widely used in gene of eucaryote cell group population, kind
It, can be to single base occurs in DNA chain using RFLP technology between group in the parting research work of (gene difference often very little)
Mutation, and mutation causes one new restriction enzyme site of loss or formation of an original restriction enzyme site to be analyzed.
At present, it is only necessary to which 1 group of primer simultaneously combines clade2.3.4 and clade2.3.4.4H5 AIV hemagglutinin gene
Nucleotides feature carries out RFLP restriction analysis using Bal I enzyme and reflects to clade2.3.4 and clade2.3.4.4H5 AIV
It does not diagnose.Detection method is fast accurate, can fill up the research blank of domestic and international related fields.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of difference clade2.3.4 and clade2.3.4.4H5 AIV
PCR-RFLP method, this method can effectively distinguish clade2.3.4 and clade2.3.4.4H5 AIV infection, strong for aquatic bird industry
Health cultivates offer technology and guarantees.
A kind of PCR-RFLP method for distinguishing clade2.3.4 and clade2.3.4.4H5 AIV, comprising the following steps:
1) H5 subtype avian influenza virus clade2.3.4 branch and clade2.3.4.4 points are extracted respectively from test sample
Branch virus genome RNA；
2) RT-PCR expansion is carried out to clade2.3.4 and clade2.3.4.4H5 AIV nucleic acid RNA simultaneously with primer P1 and P2
Increase, obtain corresponding HA genetic fragment, amplified production is detected through agarose gel electrophoresis, and size is 538bp and 529bp, naked eyes
It can not be distinguished；
3) RT-PCR amplified production is taken, carries out rflp analysis after Bal I digestion.
Preferably, in above-mentioned technical proposal, the sequence of the amplimer of the step 2) are as follows:
Upstream primer P1:5 '-TTTCCGTTGGGACATCAA-3 ',
Downstream primer P2:5 '-ACTCCATCTATTGCCTTTTG-3 '.
Preferably, in above-mentioned technical proposal, in the step 3), Bal I restriction enzyme site is located at clade2.3.4.4 branch
H5 subtype avian influenza virus hemagglutinin gene HA amplified production the position 221-226, the H5 hypotype of clade2.3.4.4 branch
The amplified production of avian influenza virus hemagglutinin gene HA can be cut into 2 sections, and size is respectively 223bp and 306bp；And
There is no Bal I restriction enzyme site in the amplified fragments of the H5 subtype avian influenza virus hemagglutinin gene HA of clade2.3.4 branch, passes through
It is constant through agarose gel electrophoresis detection clip size after digestion, it is still 538bp.
Above-mentioned technical proposal of the present invention, has the following beneficial effects:
Identification method of the present invention is simple, and quick and accuracy rate is high: the present invention is dead to the flu symptom of 78 parts of clinical acquisitions
Duck pathological material of disease is detected, and carries out RT-PCR detection after extracting nucleic acid RNA, is carried out after agarose gel electrophoresis carries out glue recycling
Bal I digestion, wherein 4 parts of positives of clade2.3.4H5 AIV branch, positive rate are 5.13% (4/78)；clade2.3.4.4H5
7 parts of positives of AIV branch, positive rate are 8.97% (7/78), detect clade2.3.4 branch and clade2.3.4.4 branch H5
2 parts of AIV coinfection, positive rate is 2.56% (2/78).
The present invention is in operation, it is only necessary to upstream primer P1 and downstream primer P2, and only need convenient restriction restriction endonuclease Bal I
Identified after carrying out RFLP digestion, without complicated cumbersome condition optimizing process, the present invention in clade2.3.4 branch and
The detection of clade2.3.4.4 branch H5 AIV rapid differential diagnosis is upper quick accurate, and it is empty can to fill up domestic and international related fields research
It is white.Currently, for the vaccine (Re-8 plants) of the vaccine of clade2.3.4H5 AIV (Re-5 plants) and clade2.3.4.4H5 AIV
It has been succeeded in developing that, carrying out antidiastole to clade2.3.4 and clade2.3.4.4H5 AIV helps to instruct the corresponding epidemic disease of H5 AIV
The scientifical use of seedling.
Detailed description of the invention
Fig. 1 is the upper of the PCR-RFLP method of difference clade2.3.4 and clade2.3.4.4H5 AIV according to the present invention
There are nucleotide difference figures for amplification region of the downstream primer amplification region to two kinds of viral hemagglutinin HA genes.
Fig. 2 is the electricity of the PCR-RFLP method of difference clade2.3.4 and clade2.3.4.4H5 AIV according to the present invention
Swimming figure.
Wherein: swimming lane M-DNA molecular weight standard 2000, the RT-PCR product of 1-clade2.3.4H5 AIV, 2-
The RT-PCR product of clade2.3.4.4H5 AIV, the cleavage map of the RT-PCR product of 3-clade2.3.4H5 AIV, 4-
The cleavage map of the RT-PCR product of clade2.3.4.4H5 AIV, 5-clade2.3.4 and clade2.3.4.4H5 AIV coinfection
RT-PCR product cleavage map, 6- negative control.
Fig. 3 is the sun of the PCR-RFLP method of difference clade2.3.4 and clade2.3.4.4H5 AIV according to the present invention
The RT-PCR product cloning sequencer map of property sample.
Specific embodiment
Specific embodiments of the present invention are described in detail below, in order to further understand the present invention.
All experimental methods used are conventional method unless otherwise specified in following embodiment.
Material used in following embodiment, reagent etc. can be obtained through commercial channels unless otherwise specified.
Embodiment 1
1, the nucleic acid RNA of virus is extracted；
(1) viral source:
H5 subtype avian influenza virus clade2.3.4 branch (CX1 plants) and clade2.3.4.4 branch (DK10 plants) are by good fortune
Build the preservation of animal and veterinary research institute, Shanxi Academy of Agricultural Sciences.
(2) viral RNA is extracted:
Extract clade2.3.4 branch (CX1 plants) and clade2.3.4.4 branch (DK10 plants) H5 respectively with conventional method
The geneome RNA of AIV, in case RT-PCR amplification uses.
2, RT-PCR is expanded
(1) design and selection of amplimer:
According to the HA gene expression characteristics of clade2.3.4H5 AIV and clade2.3.4.4H5 AIV design upstream primer P1 and
Downstream primer P2, wherein the sequence of primer is as follows:
Upstream primer P1:5 '-TTTCCGTTGGGACATCAA-3 ',
Downstream primer P2:5 '-ACTCCATCTATTGCCTTTTG-3 '.
The upstream and downstream primer amplification region that the present invention designs as shown in Figure 1: is to clade2.3.4 and clade2.3.4.4H5
There are nucleotide difference (H5 subtype avian influenza virus clade2.3.4 hemagglutinin genes for the amplification region of AIV hemagglutinin gene
HA nucleotides sequence is classified as TGGTAA, and H5 subtype avian influenza virus clade2.3.4.4 hemagglutinin gene HA nucleotides sequence is classified as
TGGCCA (Bal I digestion recognition site).
(2) RT-PCR is expanded:
RT-PCR amplification is carried out with the specific primer P1 and P2 of above-mentioned design, amplification reaction system is as follows:
Quan Shijin biotechnology in Beijing (is purchased from using II One-Step RT-PCR Super Mix of Trans Script
Co., Ltd) recommend RT-PCR amplification reaction system (1) 40 μ L, are shown in Table.Wherein, 2 μ L of viral RNA template of extraction, upstream
Primer P1 (10 μM) 1 μ L, downstream primer P2 (10 μM) 1 μ L, 2 × One-Step Reaction Mix, 20 μ L, Trans
II One-Step Enzyme Mix of Script, 1 μ L supplements 15 μ L of sterile deionized water to 40 μ L of final volume.
1 RT-PCR reaction system of table
RT-PCR amplification reaction condition are as follows: carry out PCR amplification after 50 DEG C of reverse transcription 30min.Condition are as follows: 94 DEG C of initial denaturations
4min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 circulations, 72 DEG C of extension 10min, 4 DEG C of preservations are standby
With.
(3) detected through gel electrophoresis:
The agarose gel electrophoresis of amplified production routine detects, gel imaging, as a result as shown in Figure 2.
It is as shown in Figure 2: under the above conditions, clade2.3.4H5 AIV (swimming lane 1) and clade2.3.4.4H5 AIV (swimming
Road 2) sample amplify respectively size be 538bp and 529bp segment.
3, rflp analysis
By RT-PCR product after glue recycles, Bal I restriction analysis is carried out using RFLP.Digestion system is as follows:
(30 μ L, are shown in Table the endonuclease reaction system recommended using Bal I (purchased from precious bioengineering (Dalian) Co., Ltd)
2), wherein 20 μ L of RT-PCR glue recovery product, 10 × Bal I, 3 μ L, 1 μ L, 0.1%BSA0.3 μ L of Bal I enzyme, supplement sterilizing are gone
5.7 μ L of ionized water to 30 μ L of final volume.
Reaction condition: 37 DEG C of water-bath 60min.
2 endonuclease reaction system of table
After reaction, carry out conventional agarose gel electrophoretic analysis, to test sample carry out analysis clade2.3.4 and
Clade2.3.4.4H5 AIV type, as a result as shown in Figure 2.
Bal I restriction enzyme site is located at the amplified production of clade2.3.4.4 virus hemagglutinin gene HA as shown in Figure 1:
221-226 (TGGCCA)；There is no Bal I restriction enzyme site in the amplified fragments of clade2.3.4H5 AIV hemagglutinin gene HA,
Sequence is TGGTAA herein.
It is as shown in Figure 2: there is no Bal I restriction enzyme site in the amplified fragments of clade2.3.4H5 AIV hemagglutinin gene HA,
It is constant through agarose gel electrophoresis detection clip size after being digested, it is still 538bp (swimming lane 3), and clade2.3.4.4H5
AIV can be cut into 2 sections, and size is respectively 223bp and 306bp (swimming lane 4).
4, clinical application
The present invention detects the flu symptom death duck pathological material of disease of 78 parts of clinical acquisitions, carries out RT- after extracting nucleic acid RNA
PCR detection carries out Bal I digestion after agarose gel electrophoresis carries out glue recycling, wherein 4 parts of clade2.3.4H5 AIV branch
The positive, positive rate are 5.13% (4/78)；7 parts of positives of clade2.3.4.4H5 AIV branch, positive rate are 8.97% (7/78),
Detect 2 parts of clade2.3.4 branch and clade2.3.4.4 branch H5 AIV coinfection, positive rate is 2.56% (2/78).
Randomly select be detected as positive 1 sample (the clade2.3.4 positive 1) of H5 subtype avian influenza virus clade2.3.4,
It positive 1 sample (the clade2.3.4.4 positive 1) of H5 subtype avian influenza virus clade2.3.4.4 and is detected as
Clade2.3.4 branch and clade2.3.4.4 branch 1 sample of H5 AIV coinfection (are denoted as clade2.3.4 and mix positive 1 He
The positive RT-PCR product 1) of clade2.3.4.4 mixing carries out cloning and sequencing, as a result meets expection.The clade2.3.4 positive 1 exists
Nucleotides sequence is classified as TGGTAA herein；Nucleotides sequence is classified as TGGCCA to the clade2.3.4.4 positive 1 here；It is detected as
The clade2.3.4 mixing positive 1 is denoted as in clade2.3.4 branch and clade2.3.4.4 branch 1 sample of H5 AIV coinfection
Here nucleotide sequence be TGGTAA and be denoted as clade2.3.4.4 mixing the positive 1 here nucleotide sequence be
TGGCCA meets gene expression characteristics when design of primers, as a result as shown in Figure 3.
It is an advantage of the current invention that it only needs upstream primer P1 and downstream primer P2, and only need restriction enzyme Bal I
Identified after carrying out RFLP digestion, without complicated cumbersome condition optimizing process.
The present invention is in H5 subtype avian influenza virus clade2.3.4 branch and clade2.3.4.4 branch rapid differential diagnosis
It is quick accurate in detection, domestic and international related fields research blank can be filled up.Currently, for the vaccine of clade2.3.4H5 AIV
The vaccine (Re-8 plants) of (Re-5 plants) and clade2.3.4.4H5 AIV have been succeeded in developing, to clade2.3.4 and
Clade2.3.4.4H5 AIV, which carries out antidiastole, to be helped to instruct the scientifical use of the corresponding vaccine of H5 AIV.
Although the present invention is disclosed as above with embodiment, so it is not intended to limit the present invention, any those skilled in the art
Member, without departing from the spirit and scope of the present invention, can make a variety of different selections and modification, therefore protection model of the invention
It encloses and is limited by claims and its equivalents.
Claims (1)
1. it is a kind of distinguish clade 2.3.4 and clade 2.3.4.4H5AIV PCR-RFLP method, which is characterized in that including with
Lower step: 1) clade 2.3.4 and clade 2.3.4.4H5AIV geneome RNA from test sample are extracted；2) primer P1 is used
RT-PCR amplification is carried out to clade 2.3.4 and clade 2.3.4.4H5AIV geneome RNA simultaneously with P2, obtains corresponding HA
Genetic fragment, amplified production are detected through agarose gel electrophoresis, and size is 538bp and 529bp, can not visually be distinguished；3)
RT-PCR amplified production is taken, carries out rflp analysis after Bal I digestion；
The sequence of the amplimer of the step 2) are as follows: upstream primer P1:5 '-TTTCCGTTGGGACATCAA-3 ', downstream primer
P2:5 '-ACTCCATCTATTGCCTTTTG-3 '；
In the step 3), Bal I restriction enzyme site is located at the H5 subtype avian influenza virus hemagglutinin base of clade 2.3.4.4 branch
Because of the position 221-226 of the amplified production of HA, the expansion of the H5 subtype avian influenza virus hemagglutinin gene HA of clade2.3.4.4 branch
Volume increase object can be cut into 2 sections, and size is respectively 223bp and 306bp；And the H5 subtype avian influenza virus of clade 2.3.4 branch
There is no Bal I restriction enzyme site in the amplified fragments of hemagglutinin gene HA, it is big through agarose gel electrophoresis detection segment after being digested
It is small constant, it is still 538bp.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851355A (en) * | 2012-03-20 | 2013-01-02 | 华南农业大学 | Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken |
| 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 |
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| CN102851355A (en) * | 2012-03-20 | 2013-01-02 | 华南农业大学 | Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction;Hans G Heine等;《Virology Journal》;20151231;第12卷(第18期);1-4 * |
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