CN106119422A - The PCR RFLP method of difference clade2.3.4 and clade2.3.4.4H5 AIV - Google Patents
The PCR RFLP method of difference clade2.3.4 and clade2.3.4.4H5 AIV Download PDFInfo
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- CN106119422A CN106119422A CN201610770360.9A CN201610770360A CN106119422A CN 106119422 A CN106119422 A CN 106119422A CN 201610770360 A CN201610770360 A CN 201610770360A CN 106119422 A CN106119422 A CN 106119422A
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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 distinguishing clade 2.3.4 and clade 2.3.4.4 H5AIV.Comprise the following steps: 1) from detection sample, extract virus genome RNA；2) with primer P1 and P2, clade 2.3.4 and clade 2.3.4.4 virus are carried out RT PCR amplification, obtain corresponding HA genetic fragment；3) take RT pcr amplification product and carry out rflp analysis after Bal I enzyme action.The method of the present invention only needs forward primer P1 and downstream primer P2, and only need convenient restriction restriction endonuclease Bal I to be differentiated after carrying out RFLP enzyme action, without the condition optimizing process that complexity is loaded down with trivial details, the present invention is quick accurate on clade 2.3.4 and clade2.3.4.4H5 AIV rapid differential diagnosis, and the research that can fill up domestic and international association area is blank.
Description
Technical field
The present invention relates to veterinary's infectious disease quick diagnosis technical field, particularly to one difference clade2.3.4 and
The PCR-RFLP method of clade2.3.4.4H5 AIV.
Background technology
Bird flu (Avian influneza, AI) causes by influenza A, main infringement poultry and wild bird
A kind of acute, high degree in contact sexually transmitted disease.Highly pathogenic H5 subtype avian influenza virus (H5 subtype avian influenza
Virus, H5 AIV) infection that causes is classified as, by OIE (OIE), the zoonosis that must report at present,
It is listed in a class animal epidemic in China.
Record the earliest about influenza can trace back to the once report from Italy in 1878, the most successively
There is breaking out with popular of influenza.1996, at the first strain H5N1 hypotype HPAIV [A/Goose/ that isolated in China arrives
Guangdong/1/96 (H5N1), GS/GD/96], and then Hong Kong occurred H5N1 subtype avian influenza virus directly to feel in 1997
Dye people's event, this be first fowl source influenza virus without intermediate host the evidence of direct infection people.Due to from ancestral source virus
GS/GD/96 evolves multiple pedigree and name disunity, combines for this WHO, FAO and OIE and has formulated unified point of H5N1 HPAIV
H5N1 HPAIV is divided into 0～9 to amount to 10 different clade by class criterion, and wherein clade2.3.4 is early than 2003～2006
Between be popular in the Strain on the ground such as China, Vietnam, Thailand, Laos and Malaysia, clade2.3.4 branch in 2006～2009
It is advantage epidemic strain in China much areas.In recent years, clade2.3.4 branch occurs in that new branch clade2.3.4.4's
H5N6 hypotype, H5N8 subtype virus, and be widely current in multiple provinces and cities of China.Vaccine (Re-for clade2.3.4H5 AIV
5 strains) and the vaccine (Re-8 strain) of clade2.3.4.4H5 AIV succeed in developing, to clade2.3.4 and clade2.3.4.4H5
AIV carries out Differential Diagnosis and contributes to instructing the scientifical use of the corresponding vaccine of H5 AIV.
But, clade2.3.4 and clade2.3.4.4 gene order homology therebetween is 94.4% (with classics
As a example by Reference strains compares), it is difficult to be differentiated by conventional molecular biology method, restricted enzyme fragment length is many
State property (Restriction fragment length polymorphism, RFLP) is to draw with gene loci difference in recent years
Play a kind of mark of molecular biology technology that restriction enzyme site is different, be widely used in gene of eucaryote cell group population, plant
Between Qun in the typing research work of (gene difference is the least), application RFLP technology can be to single base occurs in DNA
Suddenly change, and sudden change causes the loss of an original restriction enzyme site or one new restriction enzyme site of formation to be analyzed.
At present, it is only necessary to 1 group of primer also combines clade2.3.4 and clade2.3.4.4H5 AIV hemagglutinin gene
Nucleotide feature, utilizes Bal I enzyme to carry out RFLP restriction analysis and reflects clade2.3.4 and clade2.3.4.4H5 AIV
Do not diagnose.Detection method is fast accurate, and the research that can fill up domestic and international association area is blank.
Summary of the invention
The technical problem to be solved is to provide a kind of difference clade2.3.4 and clade2.3.4.4H5 AIV
PCR-RFLP method, the method can effectively distinguish clade2.3.4 and clade2.3.4.4H5 AIV infect, for aquatic bird industry be good for
Health cultivation offer technology ensures.
A kind of PCR-RFLP method distinguishing clade2.3.4 and clade2.3.4.4H5 AIV, comprises the following steps:
1) from detection sample, extract H5 subtype avian influenza virus clade2.3.4 branch respectively and clade2.3.4.4 divides
Prop up virus genome RNA；
2) clade2.3.4 and clade2.3.4.4H5 AIV nucleic acid RNA is carried out RT-PCR expansion with primer P1 and P2 simultaneously
Increasing, obtain corresponding HA genetic fragment, amplified production detects through agarose gel electrophoresis, and size is 538bp and 529bp, naked eyes
Cannot be carried out difference；
3) take RT-PCR amplified production, after Bal I enzyme action, carry out rflp analysis.
Preferably, in technique scheme, described step 2) the sequence of amplimer be:
Forward primer P1:5 '-TTTCCGTTGGGACATCAA-3 ',
Downstream primer P2:5 '-ACTCCATCTATTGCCTTTTG-3 '.
Preferably, in technique scheme, described step 3) in, Bal I restriction enzyme site is positioned at clade2.3.4.4 branch
The 221-226 position of amplified production of H5 subtype avian influenza virus hemagglutinin gene HA, 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
The amplified fragments of the H5 subtype avian influenza virus hemagglutinin gene HA of clade2.3.4 branch do not have Bal I restriction enzyme site, warp
Detect clip size through agarose gel electrophoresis after enzyme action constant, be still 538bp.
Technique scheme of the present invention, has the advantages that
Authentication method of the present invention is simple, quick and accuracy rate height: the present invention is dead to the flu-like symptom of 78 parts of clinical acquisitions
Duck pathological material of disease detects, and carries out RT-PCR detection, carry out after agarose gel electrophoresis carries out glue recovery after extracting nucleic acid RNA
Bal I enzyme action, wherein 4 parts of positives of clade2.3.4H5 AIV branch, positive rate is 5.13% (4/78)；clade2.3.4.4H5
7 parts of positives of AIV branch, positive rate is 8.97% (7/78), clade2.3.4 branch and clade2.3.4.4 branch H5 detected
AIV coinfection 2 parts, positive rate is 2.56% (2/78).
The present invention is in operation, it is only necessary to forward primer P1 and downstream primer P2, and only needs convenient restriction restriction endonuclease Bal I
Differentiated after carrying out RFLP enzyme action, it is not necessary to complicated loaded down with trivial details condition optimizing process, the present invention in clade2.3.4 branch and
The H5 AIV rapid differential diagnosis detection of clade2.3.4.4 branch is upper quick accurate, can fill up domestic and international association area research empty
In vain.Currently, for vaccine (Re-5 strain) and the vaccine (Re-8 strain) of clade2.3.4.4H5 AIV of clade2.3.4H5 AIV
Succeed in developing, clade2.3.4 and clade2.3.4.4H5 AIV is carried out Differential Diagnosis and contributes to instructing the corresponding epidemic disease of H5 AIV
The scientifical use of Seedling.
Accompanying drawing explanation
Fig. 1 is the upper of the PCR-RFLP method of difference clade2.3.4 and the clade2.3.4.4H5 AIV according to the present invention
Nucleotide difference figure is there is in downstream primer amplification region to the amplification region of two-strain hemagglutinin gene.
Fig. 2 is the electricity of the PCR-RFLP method of difference clade2.3.4 and the 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
The cleavage map of RT-PCR product, 6-negative control.
Fig. 3 is the sun of the PCR-RFLP method of difference clade2.3.4 and the clade2.3.4.4H5 AIV according to the present invention
The RT-PCR product cloning sequencer map of property sample.
Detailed description of the invention
Below the specific embodiment of the present invention is described in detail, in order to be further appreciated by the present invention.
In following example, the experimental technique of all uses if no special instructions, is conventional method.
Material used in following example, reagent etc., if no special instructions, all can be by being either commercially available.
Embodiment 1
1, the nucleic acid RNA of virus is extracted；
(1) viral source:
H5 subtype avian influenza virus clade2.3.4 branch (CX1 strain) and clade2.3.4.4 branch (DK10 strain) are by good fortune
Build Shanxi Academy of Agricultural Sciences's animal and veterinary institute to preserve.
(2) viral RNA is extracted:
Clade2.3.4 branch (CX1 strain) and clade2.3.4.4 branch (DK10 strain) H5 is extracted respectively by conventional method
The geneome RNA of AIV, in case RT-PCR amplification uses.
2, RT-PCR amplification
(1) design of amplimer and selection:
According to clade2.3.4H5 AIV and clade2.3.4.4H5 AIV HA gene expression characteristics design forward primer P1 and
Downstream primer P2, wherein, the sequence of primer is as follows:
Forward primer P1:5 '-TTTCCGTTGGGACATCAA-3 ',
Downstream primer P2:5 '-ACTCCATCTATTGCCTTTTG-3 '.
As shown in Figure 1: the upstream and downstream primer amplified region of present invention design is to clade2.3.4 and clade2.3.4.4H5
There is nucleotide difference (H5 subtype avian influenza virus clade2.3.4 hemagglutinin gene in 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 enzyme action recognition site).
(2) RT-PCR amplification:
Carrying out RT-PCR amplification with specific primer P1 and P2 of above-mentioned design, amplification reaction system is as follows:
Use Trans Script II One-Step RT-PCR Super Mix (purchased from Beijing full formula gold biotechnology
Company limited) the RT-PCR amplification reaction system (40 μ L, be shown in Table 1) recommended.Wherein, the viral RNA template 2 μ L of extraction, upstream
Primer P1 (10 μMs) 1 μ L, downstream primer P2 (10 μMs) 1 μ L, 2 × One-Step Reaction Mix 20 μ L, Trans
Script II One-Step Enzyme Mix 1 μ L, supplements sterilizing deionized water 15 μ L to final volume 40 μ L.
Table 1 RT-PCR reaction system
RT-PCR amplification reaction condition is: 50 DEG C of reverse transcription 30min laggard performing PCR amplifications.Condition is: 94 DEG C of denaturations
4min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, totally 35 circulations, and 72 DEG C extend 10min, and 4 DEG C of preservations are standby
With.
(3) detected through gel electrophoresis:
The agarose gel electrophoresis detection that amplified production is conventional, gel imaging, result is as shown in Figure 2.
As shown in Figure 2: under these conditions, clade2.3.4H5 AIV (swimming lane 1) and clade2.3.4.4H5 AIV (swimming
Road 2) sample amplify the fragment that size is 538bp and 529bp respectively.
3, rflp analysis
By RT-PCR product after glue reclaims, RFLP is utilized to carry out Bal I restriction analysis.Enzyme action system is as follows:
(30 μ L, are shown in Table the endonuclease reaction system using Bal I (purchased from precious biological engineering (Dalian) company limited) to recommend
2), wherein RT-PCR glue reclaims product 20 μ L, 10 × Bal I 3 μ L, Bal I enzyme 1 μ L, and 0.1%BSA0.3 μ L, supplementary sterilizing is gone
Ionized water 5.7 μ L to final volume 30 μ L.
Reaction condition: 37 DEG C of water-bath 60min.
Table 2 endonuclease reaction system
Reaction terminate after, carry out conventional agarose gel electrophoretic analysis, to detection sample be analyzed clade2.3.4 and
Clade2.3.4.4H5 AIV type, result is as shown in Figure 2.
As shown in Figure 1: Bal I restriction enzyme site is positioned at the amplified production of clade2.3.4.4 virus hemagglutinin gene HA
221-226 position (TGGCCA)；The amplified fragments of clade2.3.4H5 AIV hemagglutinin gene HA does not has Bal I restriction enzyme site,
Sequence is TGGTAA herein.
As shown in Figure 2: the amplified fragments of clade2.3.4H5 AIV hemagglutinin gene HA does not has Bal I restriction enzyme site,
Detect clip size through agarose gel electrophoresis after digested constant, be 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 practice
The flu-like symptom death duck pathological material of disease of 78 parts of clinical acquisitions is detected by the present invention, carries out RT-after extracting nucleic acid RNA
PCR detects, and carries out Bal I enzyme action, wherein 4 parts of clade2.3.4H5 AIV branch after agarose gel electrophoresis carries out glue recovery
The positive, positive rate is 5.13% (4/78)；7 parts of positives of clade2.3.4.4H5 AIV branch, positive rate is 8.97% (7/78),
Clade2.3.4 branch and clade2.3.4.4 branch H5 AIV coinfection 2 parts being detected, 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,
Positive 1 sample (the clade2.3.4.4 positive 1) of H5 subtype avian influenza virus clade2.3.4.4 and being detected as
Clade2.3.4 branch and clade2.3.4.4 branch 1 sample of H5 AIV coinfection (are designated as positive 1 He of clade2.3.4 mixing
Clade2.3.4.4 mixing positive 1) RT-PCR product carry out cloning and sequencing, result meets expection.The clade2.3.4 positive 1 exists
Nucleotides sequence is classified as TGGTAA herein；Herein, nucleotides sequence is classified as TGGCCA to the clade2.3.4.4 positive 1；It is detected as
Clade2.3.4 branch and clade2.3.4.4 branch 1 sample of H5 AIV coinfection are designated as clade2.3.4 mixing positive 1
Herein, nucleotide sequence is TGGTAA and is designated as clade2.3.4.4 mixing positive 1 nucleotide sequence is herein
TGGCCA, all meets gene expression characteristics during design of primers, and result is as shown in Figure 3.
It is an advantage of the current invention that it only needs forward primer P1 and downstream primer P2, and only need restricted enzyme Bal I
Differentiate after carrying out RFLP enzyme action, it is not necessary to complicated loaded down with trivial details 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
In detection the most accurately, domestic and international association area research can be filled up blank.Currently, for the vaccine of clade2.3.4H5 AIV
The vaccine (Re-8 strain) of (Re-5 strain) and clade2.3.4.4H5 AIV is succeeded in developing, to clade2.3.4 and
Clade2.3.4.4H5 AIV carries out Differential Diagnosis and contributes to instructing the scientifical use of the corresponding vaccine of H5 AIV.
Although the present invention is open as above with embodiment, so it is not intended to limit the present invention, any people in the art
Member, without departing from the spirit and scope of the present invention, all can make various different selection and amendment, therefore the protection model of the present invention
Enclose and limited by claims and equivalents thereof.
Claims (3)
1. the PCR-RFLP method distinguishing clade 2.3.4 and clade 2.3.4.4H5AIV, it is characterised in that include with
Lower step:
1) from detection sample, clade 2.3.4 and clade 2.3.4.4H5AIV geneome RNA are extracted；
2) clade 2.3.4 and clade 2.3.4.4H5AIV geneome RNA are carried out RT-PCR expansion with primer P1 and P2 simultaneously
Increasing, obtain corresponding HA genetic fragment, amplified production detects through agarose gel electrophoresis, and size is 538bp and 529bp, naked eyes
Cannot be carried out difference；
3) take RT-PCR amplified production, after Bal I enzyme action, carry out rflp analysis.
The PCR-RFLP method of difference clade 2.3.4 and clade 2.3.4.4H5AIV the most according to claim 1, its
Be characterised by, described step 2) the sequence of amplimer be:
Forward primer P1:5 '-TTTCCGTTGGGACATCAA-3 ',
Downstream primer P2:5 '-ACTCCATCTATTGCCTTTTG-3 '.
The PCR-RFLP method of difference clade 2.3.4 and clade 2.3.4.4H5AIV the most according to claim 1, its
It is characterised by, described step 3) in, Bal I restriction enzyme site is positioned at the H5 subtype avian influenza virus blood of clade 2.3.4.4 branch
The 221-226 position of the amplified production of solidifying plain gene HA, the H5 subtype avian influenza virus hemagglutinin gene of clade2.3.4.4 branch
The amplified production of HA can be cut into 2 sections, and size is respectively 223bp and 306bp；And the H5 hypotype fowl stream of clade 2.3.4 branch
The amplified fragments of Influenza Virus hemagglutinin gene HA do not has Bal I restriction enzyme site, digested after detect through agarose gel electrophoresis
Clip size is constant, is still 538bp.
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| CN109628643A (en) * | 2019-01-11 | 2019-04-16 | 中国动物卫生与流行病学中心 | A kind of rapid detection method of H5 subtype avian influenza virus |
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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 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109628643A (en) * | 2019-01-11 | 2019-04-16 | 中国动物卫生与流行病学中心 | A kind of rapid detection method of H5 subtype avian influenza virus |
| CN109628643B (en) * | 2019-01-11 | 2023-04-25 | 中国动物卫生与流行病学中心 | Rapid detection method for H5 subtype avian influenza virus |
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