CN106048017B - A kind of specific sequence of China's brickpox attenuated vaccine strain and application - Google Patents
A kind of specific sequence of China's brickpox attenuated vaccine strain and application Download PDFInfo
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- CN106048017B CN106048017B CN201610407489.3A CN201610407489A CN106048017B CN 106048017 B CN106048017 B CN 106048017B CN 201610407489 A CN201610407489 A CN 201610407489A CN 106048017 B CN106048017 B CN 106048017B
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Abstract
The gene order of DNA polymerase i V of swine erysipelas vaccine strain G4T10 and GC42 and being compared for brickpox street strain are found that its is distinct by specific sequence and application the invention discloses a kind of Chinese brickpox attenuated vaccine strain, applicant.The specific sequence of Chinese brickpox attenuated vaccine strain is shown in SEQ ID NO.1.Design is for the specific primer of brickpox attenuated vaccine strain, and combine brickpox species-specific primer can Rapid identification China brickpox attenuated vaccine strain and street strain, it is suitble to the mass detection of various samples, new technological means is provided for quick detection brickpox clinical separation strain and antidiastole street strain and vaccine strain.
Description
Technical field
The present invention relates to livestock contagious disease antidiastole reagent preparing technical fields, and in particular to a kind of China's brickpox is weak
The specific sequence of toxic vaccine strain and application.
Background technology
Erysipelothrix ruhsiopathiae is commonly called as bacillus rhusiopathiae suis, is Gram-positive dialister bacterium, is widespread in nature, mesh
It is preceding to have the report being separated in mammal, birds, reptiles, amphibian animal and fish species.Erysipelothrix ruhsiopathiae can make
Erysipelas occurs into a variety of domestic animals, wherein the harm particularly with pig and turkey is the most serious, while human infection can also be caused
It falls ill (erysipeloid).Brickpox is mainly shown as septicemia, endocarditis and polyarthirtis of pig etc., is brought to pig breeding industry
Huge economic losses.China cultivates G4T10 plants and GC42 plants of brickpox attenuated vaccine in the seventies in last century in succession, with 80
The popularization of age attenuated vaccine, brickpox attenuated vaccine are made that significant contribution for the control of brickpox epidemic situation.But animal epidemic disease
The purification of disease not only needs attenuated vaccine with greater need for the diagnostic method of difference street strain and vaccine strain as technical support.
Development of Molecular Biology is rapid in recent years, and round pcr has become the important method of identification pathogenic microorganism.At present
The PCR methods that vaccine strain and street strain for pathogen distinguish obtain in the epidemic diseases such as brucellosis, tuberculosis
Using.Occur the PCR method of several identification erysipelothrix ruhsiopathiaes at present, but still lacked quickly distinguish brickpox at present
Vaccine strain and the PCR methods of street strain.
In consideration of it, the DNA polymerase i V genes of swine erysipelas vaccine strain G4T10 and GC42 have been carried out clone, surveyed by applicant
Sequence and analysis find that the vaccine strain gene order has differences with street strain.We devise the spy of swine erysipelas vaccine strain accordingly
Specific primer, with reference to the species-specific primer of bacillus rhusiopathiae suis, we set up a kind of quick detection and distinguish brickpox simultaneously
The double PCR method and corresponding kit of vaccine strain and street strain.
Invention content
The purpose of the present invention is to provide a kind of specific sequence of Chinese brickpox attenuated vaccine strain, sequence SEQ
Shown in ID NO.1.Sequence presence specific in Chinese brickpox attenuated vaccine strain G4T10 and GC42, available for distinguishing
Chinese brickpox attenuated vaccine strain and street strain.
It is another object of the present invention to provide the specific sequence designs based on Chinese brickpox attenuated vaccine strain
Primer.Preferably, primer sequence is：
Primer vaccine-F sequences：5’-AGTGATGAAACTGATTCGTGCTC-3’
Primer vaccine-R sequences：5’-GCCTAAATTAAACCACTTGCACACA-3’.
Final object of the present invention is to provide the specific sequence of Chinese brickpox attenuated vaccine strain or based on it
Application of the primer of design in brickpox street strain and Chinese brickpox attenuated vaccine strain is distinguished.
To achieve these goals, the present invention adopts the following technical scheme that：
Applicant is by the gene order of the DNA polymerase i V of swine erysipelas vaccine strain G4T10 and GC42 and brickpox street strain
Be compared, it is found that its is distinct.The specific sequence of Chinese brickpox attenuated vaccine strain is shown in SEQ ID NO.1.
The protection content of the present invention further includes the primer based on sequence design shown in SEQ ID NO.1, it is preferred that design pig
Erysipelas vaccine strain specific primer is as follows：
Primer vaccine-F sequences：5’-AGTGATGAAACTGATTCGTGCTC-3’
Primer vaccine-R sequences：5’-GCCTAAATTAAACCACTTGCACACA-3’.
The specific sequence of Chinese brickpox attenuated vaccine strain is distinguishing brickpox street strain based on the primer that it is designed
With the application in Chinese brickpox attenuated vaccine strain, detect in strain to be tested and whether contain including the use of current conventional means
SEQ ID
Sequence shown in NO.1 or based on sequence design specific primer shown in SEQ ID NO.1 for detecting bacterium to be measured
Whether strain contains sequence shown in SEQ ID NO.1.
Compared with prior art, the present invention has the following advantages：
The present invention is according to brickpox attenuated vaccine DNA polymerase i V series jumps region, and design is for the weak malicious epidemic disease of brickpox
The specific primer of seedling strain, and establish quick detection brickpox clinical separation strain and simultaneously with reference to brickpox species-specific primer
Brickpox attenuated vaccine strain and the dual-PCR method of street strain are distinguished, it can be complete in 2h.Swine erysipelas vaccine strain can expand simultaneously
Increase and 453bp and 937bp segments, brickpox street strain is only capable of amplifying 937bp segments.And Escherichia coli, haemophilus parasuis
Deng not amplifying any target fragment, it was confirmed that dual PCR detection method of the present invention has preferable specificity.Present invention tool
There is the sensibility of height, the lowest detection limit is the DNA of bacteria of 50fg, based on above-mentioned double PCR rapid detection method development
Kit is suitble to the mass detection of various samples, for quick detection brickpox clinical separation strain and antidiastole street strain and
Vaccine strain provides new technological means.
Description of the drawings
Fig. 1 is the electrophoretogram of three kinds of sample products after PCR amplification.
Wherein swimming lane M, 1,2,3,4 are respectively molecular marked compound, G4T10 plants of brickpox attenuated vaccine strain, the weak poison of brickpox
GC42 plants of vaccine strain, brickpox poison epidemic strain SE38 and negative control by force.
Fig. 2 is the specific assay result electrophoretogram of the present invention.
Wherein swimming lane M, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 are respectively molecular marked compound, brickpox
Malicious epidemic strain SE38, brickpox are classical strong malicious by force for G4T10 plants of attenuated vaccine strain, GC42 plants of brickpox attenuated vaccine strain, brickpox
C43-5 plants of strain, JL03 plants of Actinobacillus pleuropneumoniae, E0630 plants of pasteurella multocida, PCN033 plants of Escherichia coli, secondary pig
SH0165 plants of haemophilus, Salmonella choleraesuis C500 strain, BS168 plants of bacillus subtilis, enterococcus faecalis ATCC29212
Strain, MG1363 plants of Lactococcus lactis, ST171 plants of Malian drainage, R61 plants of Streptococcus suis, negative control.
Fig. 3 is the sensitivity test result electrophoretogram of primer of the present invention.
Wherein swimming lane 1,2,3,4,5,6,7,8 represents that G4T10 plants of DNA of brickpox attenuated vaccine strain contain in measuring samples respectively
It measures as 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg, 5fg.M, molecular marked compound.
Fig. 4 this method is for the pcr amplification product electrophoresis detection result of the clinical separation strain of brickpox.
Swimming lane 1-34 be respectively G4T10 plants of brickpox attenuated vaccine strain, GC42 plants, popular velogen strain SE38, classical intensity
Strain C43-5 plants, clinical separation strain 041-860；M, molecular marked compound.
The testing result of the method for distinguishing brickpox street strain and vaccine strain based on SNP that Fig. 5 Japanese scholars are established.
ERH_0001, ERH_0543, ERH_0636, ERH_1398, ERH_1449, the tested base in the PCR methods based on SNP
Cause；Swimming lane 1-34 be respectively G4T10 plants of brickpox attenuated vaccine strain, GC42 plants, brickpox prevalence velogen strain SE38, brickpox warp
C43-5 plants of allusion quotation intensity strain, clinical separation strain 041-860；M, molecular marked compound.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are each fallen in protection scope of the present invention.Technical solution of the present invention, such as not
It illustrates, is the conventional scheme of this field；The reagent or material if not otherwise specified, derive from commercial channel.
Embodiment 1：
The specific sequence of Chinese brickpox attenuated vaccine strain is distinguishing brickpox street strain based on the primer that it is designed
With the application in Chinese brickpox attenuated vaccine strain：
1 materials and methods
1.1 bacterial strain
G4T10 plants of brickpox attenuated vaccine strain (hereinafter referred to as G4T10 plants), GC42 plants of brickpox attenuated vaccine strain are (following
GC42 plants of abbreviation), (hereinafter referred to as SE38 plants, be shown in document " Hubei some areas erysipelothrix ruhsiopathiae to local strong malicious epidemic strain SE38
Separation strains drug resistance analysis ") it is the bacterial strain for being located away from Hubei, strong virus force epidemic strain is identified as, more than bacterial strain is micro- by agricultural
Biology National Key Laboratory provides.
1.2 reagent
10 × PCR buffer (i.e. 10 times dilution PCR buffer solutions), dNTPs Mixture, Taq archaeal dna polymerases are purchased from
In TAKARA companies, primer vaccine-F sequences：5’-AGTGATGAAACTGATTCGTGCTC-3’
Primer vaccine-R sequences：5’-GCCTAAATTAAACCACTTGCACACA-3’
Primer ER1, ER2 sequence is shown in document " Broth cultivation-PCR combination assay for
Rapid diagnosis of swine erysipelas " are specially：
Primer ER1：5’-CGA TTATATTCTTAGCACGCAACG-3’
Primer ER2：5’-TGCTTGTGTTGTGATT TCTTGACG-3’.
It is completed by Shanghai Sheng Gong limited companies, TSB culture solutions are purchased from U.S. company BD.
1.3 measuring samples extract DNA
G4T10 plants, GC42 plants, SE38 plants of each 5mL of TSB culture solutions, TSB culture solutions as negative control, respectively according to
The DNA that TIANGEN bacterial genomes DNA extraction operations step (extracts reagent is purchased from TIANGEN companies) extracts is put in -20
DEG C preserve.
1.4 PCR systems are prepared
1 μ L DNA profilings are added in PCR premixed liquids, prepare 25 μ L reaction systems, and be uniformly mixed.PCR premixed liquids：
Contain 10 times of diluted PCR buffer solutions 2.5ul, 2.5mMdNTP mixture 2.5ul, the Taq archaeal dna polymerases 1 of a concentration of 1U/ μ L
μ L, concentration are that primer Vaccine-F, Vaccine-R, ER1 and ER2 each 1 μ L, ddH2O of 20pmol/ μ L supply 25 μ L.
1.5 reaction conditions are set
PCR reaction systems, which are placed in PCR instrument, carries out cyclic amplification reaction.Amplification condition is：95 DEG C of pre-degeneration 5min；Then
95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s carry out 35 cycles altogether；Last 72 DEG C of extensions 2min.
1.6 result judgement
PCR reaction products respectively take 5 μ L to carry out electroresis appraisal on 1% Ago-Gel.If PCR product has simultaneously
The band of 453bp and each one of the band of 937bp, then it is G4T10 plants or GC42 plants to show measuring samples；If PCR product is only
There is the band of a 937bp, then it is SE38 plants to show measuring samples, and no band appearance is feminine gender.Testing result is shown in Fig. 1.Pig
Erysipelas vaccine strain G4T10 and GC42 amplify two band of band and 937bp of 453bp, and local popular velogen strain SE38
It is only capable of amplifying mono- bands of 937bp.
Embodiment 2：
Specific assay
Using G4T10 plants, GC42 plants, SE38 as positive control, classical C43-5 plants of brickpox velogen strain, pleuropneumonia are put
JL03 plants of line bar bacterium, E0630 plants of pasteurella multocida, PCN033 plants of Escherichia coli, SH0165 plants of haemophilus parasuis, pig are suddenly
Random salmonella C500 plants, BS168 plants of bacillus subtilis, ATCC29212 plants of enterococcus faecalis, MG1363 plants of Lactococcus lactis,
ST171 plants of Malian drainage, the genome progress specific detection of R61 plants of Streptococcus suis, use the method for embodiment 1
With primer, testing result is shown in Fig. 2.It can be seen that from the result of Fig. 2 and fail to expand any specific item in addition to brickpox bacterial strain
Band shows that the double PCR primer has very high specificity.
Embodiment 3：
Sensitivity test
The DNA of G4T10 strains extracted in embodiment 1 makes 10 times of gradient dilution of sterilizing distilled water, G4T10 plants
DNA content is respectively 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg, 5fg.Each dilution respectively takes 1 μ L as mould
Plate carries out PCR amplification detection by 1 method of embodiment, observes positive band, to there is the template used amount of positive expected band most
High dilution calculates its sensibility, the results showed that minimum detectable activity is shown in Fig. 3 for 50fg.
Embodiment 4：
Clinical practice and the comparison with other methods
DNA is extracted to the brickpox clinical separation strain in table 1 respectively according to the method in example 1, carries out PCR amplification, production
Object is detected by electrophoresis detection (Fig. 4), while using the PCR methods based on SNP that Japanese scholars are established.The result shows that separation
It is street strain from the brickpox clinical separation strain in brickpox acute sepsis type case, that is to say, that the country is acute at present loses
Courageous and upright brickpox is caused by street strain, unrelated with vaccine strain.The testing result is with the foundation of Japanese scholars before based on SNP's
PCR methods are (see document " Development of an SNP-based PCR assay for rapid differentiation
of a Japanese live vaccine strain from field isolates of Erysipelothrix
Rhusiopathiae ") testing result (Fig. 5) is completely the same and more time saving and energy saving, and convenient, stability is also more preferable.
Table 1
SEQUENCE LISTING
<110>Hua Zhong Agriculture University
<120>A kind of specific sequence of China's brickpox attenuated vaccine strain and application
<130>A kind of specific sequence of China's brickpox attenuated vaccine strain and application
<160> 5
<170> PatentIn version 3.1
<210> 1
<211> 902
<212> DNA
<213>Artificial sequence
<400> 1
atggcacaag taatatttca tatagatatt aacgcgtttt atgcaagtgc tcatctcatt 60
acagattcaa gtttatatgg aaaacccgta gttgtttgta gtaaccaaag aggatctgtt 120
gttacaactg catcctatga ggcgcgctca tttggcgtta attccgcaat gccactcgca 180
catgccaaac gtctttgccc taacttggaa gtcattgaag ttgattttga gttgtatcaa 240
gagttatcgg ttaaatttat gaatattatt cgttcttatt ctgctgttat gcaaccagcg 300
agtattgatg aatgttatgt tgatatgaca gaggttataa aaaaatatga gaagccactc 360
gatcttgccg ttgaaattca gaaaaatatc tgggaagcat tgcgacttcc catttctatt 420
ggtgtagcgc caaataaatt tctagctaag atggcgagtg acatgcagaa acctcgtggt 480
attacagtac ttagaattcg tgaagtatca caaaaattat ggccgctttc gattgaatca 540
atgtacggaa ttggtaaaaa aacggtgcct aaattaaacc acttgcacac atcgatgctt 600
ataaaacggc aacccgttcc ttcacttttg atgaatatac aagagatcaa aatttagttt 660
tcgagaggct tatgagttta tatgatgaat ttgaagggga aggcggcgta tctttcattt 720
cagtaacaat gacaaactta ttgcctaaag atgaaataat tgaacagtta aatatctttg 780
atgatttgga tgaaattacg gtcaatgaca tcatccagcg acttaataag gaacttaacc 840
aagatctttt taagacgact cgatctgttc ttaaggagaa aagtcatgaa aaacaaagct 900
aa 902
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
agtgatgaaa ctgattcgtg ctc 23
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
gcctaaatta aaccacttgc acaca 25
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence
<400> 4
cgattatatt cttagcacgc aacg 24
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
tgcttgtgtt gtgatttctt gacg 24
Claims (5)
1. a kind of specific sequence of China's brickpox attenuated vaccine strain is distinguishing Chinese brickpox attenuated vaccine strain and street strain
In application, the specific sequence is shown in SEQ ID NO.1.
2. distinguishing Chinese brickpox attenuated vaccine strain and street strain for the primer that specific sequence described in claim 1 designs
In application, the specific sequence is shown in SEQ ID NO.1.
3. primer according to claim 2, primer are：
vaccine-F：5’-AGTGATGAAACTGATTCGTGCTC-3’
vaccine-R：5’-GCCTAAATTAAACCACTTGCACACA-3’.
4. a kind of kit for distinguishing state's brickpox attenuated vaccine strain and brickpox street strain, including：
vaccine-F：5 '-AGTGATGAAACTGATTCGTGCTC-3 ',
vaccine-R：5 '-GCCTAAATTAAACCACTTGCACACA-3 ',
Primer ER1：5 '-CGATTATATTCTTAGCACGCAACG-3 ',
Primer ER2：5’-TGCTTGTGTTGTGATT TCTTGACG-3’.
5. application according to claim 1 or 2, the Chinese brickpox attenuated vaccine strain is G4T10 or GC42.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397023A (en) * | 2013-08-20 | 2013-11-20 | 广东温氏食品集团股份有限公司 | Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof |
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2016
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397023A (en) * | 2013-08-20 | 2013-11-20 | 广东温氏食品集团股份有限公司 | Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas;YOSHIHIRO SHIMOJI et al;《 JOURNAL OF CLINICAL MICROBIOLOGY》;19980131;86–89 * |
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