CN103397023A - Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof - Google Patents
Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and an application thereof, belonging to the fields of molecular biology and production of animal medicines. The nucleotide sequence of the Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer is shown in SEQIDNO:1-2. The Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer in the invention can be used to prepare a detection reagent of Bacillus erysipelatos-suis, can distinguish swine erysipelas attenuated vaccine strain G4T10 strain and other wild prevalent strains, and is contributed to monitoring the epidemic prevention condition of vaccines and diagnosing swine erysipelas condition in production.
Description
Technical field
The invention belongs to molecular biology and the production field of animal medicine, be specifically related to be mainly used in differentiating pcr amplification primer and the application thereof of the G4T10 strain of pig erysipelas attenuated vaccine strain and wild malicious epidemic strain (other pig erysipelas strains that comprise the non-pig erysipelas attenuated vaccine strain G4T10 strains such as strong malicious epidemic strain GD1201).
Background technology
Pig erysipelas (Swine erysipelas, SE) be by bacillus rhusiopathiae suis (
Erysipelothrix rhusiopathiaeER) a kind of acute heat sexually transmitted disease that causes, its principal character are high heat, acute sepsis, skin rash piece (subacute), chronic libman-Sack endocarditis and cutaneous necrosis and multiple non suppurative arthritis (Wood R.L., Erysipelas, in:Leman, et al. (Eds.), Diseases of swine, Iowa State University Press, Ames, Iowa, 1992, pp., 475 – 486).From the bacillus rhusiopathiae suis that is separated in the pig body of these symptoms, be mostly serum 1 type (1a hypotype and 1b hypotype) and 2 types (Takashi et al., Protection Effect of NaOH-Extracted
Erysipelothrix rhusiopathiaeJ.Vet.Med.Sci.60 (1): 9-14.1998).This disease once was called as one of China's " three large transmissible diseases ", in recent years, along with fade out gradually people's the visual field of the intensivization development pig erysipelas of raising pigs, but should disease not be cleaned fully, there is fragmentary appearance in district always throughout the country, to pig farmer caused serious financial loss (Gong Pingyang, Wang Lian thinks, sun will perfume (or spice). the characteristics of incidence of In Guangdong Province Pig Farm erysipelas and comprehensive measures for the prevention and control ［ J ］. poultry industry is total, 2010(11): 11-12.).
Bacillus rhusiopathiae suis surface membrane protein (Surface protective antigen A, SpaA) full length gene is about 1881bp, the albumen of the big or small 64KD of coding.SpaA albumen is its main immunogenic protein, and in course of infection, work (Takahashi et al., DNA relatedness amongst
ErysipelothrixrhusiopathiaeStrains representing all twenty-three serovars and Erisiperothrix tonsillarum. Int.J.Syst.Bacteriol.42,469-473.1992, Sou-ichi Makino et al., Surface antigen, SpaA, of Erysipelothrix rhusiopathiae binds to Gram-positive bacterial cell surfaces, FEMS Microbiology Letters 186 (2000) 313-317).
This disease of prevention mainly adopts the bacillus rhusiopathiae suis attenuated vaccine to carry out immune prevention and control at present, the vaccine that China adopts is mainly pig erysipelas attenuated vaccine strain G4T10 strain, this vaccine strain is to be formed at 20 century 70 joint research and developments by Jiangsu Province Agriculture Science Institute and Nanjing biologics factory, and to be virulent strain uploaded for 10 generations and obtain through 370 generations of cavy with containing 0.01% ~ 0.04% trypaflavine blood agar culture-medium pig erysipelas low virulent strain G4T10.
At present, owing to producing upper pig erysipelas attenuated vaccine strain G4T10 strain commonly used, carry out the immune prevention and control to pig erysipelas, for the epidemic prevention situation of monitoring vaccine and the diagnosis of the pig erysipelas state of an illness, in the urgent need to a kind of differential diagnostic method that vaccine low virulent strain and wild malicious epidemic strain are infected.
Summary of the invention
The object of the invention is to for the problems referred to above of the prior art, SpaA gene order by pig erysipelas attenuated vaccine G4T10 strain (the Genbank accession number: KF150604) and virulent strain Fujishawa, GD1201 strain (the Genbank accession number: KF177344) etc. the contrast of strain sequence finds that the vaccine low virulent strain, at 1369-1488 topagnosis 119bp, sets up the pcr amplification method of the G4T10 strain of vaccine low virulent strain and street strain's differential diagnosis for this characteristic Design primer of sequence.
The PCR primer of bacillus rhusiopathiae suis provided by the invention, its nucleotide sequence is as shown in SEQ ID NO:1 ~ 2.
The present invention also provides the application of PCR primer in the detection reagent of preparation bacillus rhusiopathiae suis of above-mentioned bacillus rhusiopathiae suis.
Preferably, the application of the PCR primer of above-mentioned bacillus rhusiopathiae suis in the detection reagent of preparation bacillus rhusiopathiae suis, described bacillus rhusiopathiae suis is the wild malicious epidemic strain of the G4T10 strain of pig erysipelas attenuated vaccine strain and/or pig erysipelas.
The present invention also provides the detection kit of a kind of bacillus rhusiopathiae suis, comprises following component: the primer sequence shown in SEQ ID NO:1 ~ 2, archaeal dna polymerase, PCR damping fluid, dNTP mixture and aseptic double-distilled water.
Preferably, the detection kit of above-mentioned bacillus rhusiopathiae suis, concrete component is: the PCR damping fluid 1.5-3 μ L of 10 times of dilutions, dNTPs mixture 1.5-3 μ L, concentration is the Taq archaeal dna polymerase 1 μ L of 2.5M/ μ L, concentration is each 2 μ L of primer shown in the SEQ ID NO:1 of 10pmol/ μ L ~ 2, ddH
2O supplies 25 μ L.
Beneficial effect of the present invention: primer sequence of the present invention, the bacillus rhusiopathiae suis disease is carried out to differential diagnosis, susceptibility is high, specificity is good, and this primer can be distinguished the G4T10 strain of pig erysipelas attenuated vaccine strain and the wild malicious epidemic strain of pig erysipelas infects, for the epidemic prevention situation of monitoring vaccine and the diagnosis of the pig erysipelas state of an illness in producing provide foundation.
The accompanying drawing explanation
Fig. 1 is the electrophorograms of two kinds of samples through the pcr amplification after product, and wherein swimming lane 1,2,3 is respectively pig erysipelas attenuated vaccine strain G4T10 strain, strong malicious epidemic strain GD1201 and negative control.
Fig. 2 is the specificity assay electrophorogram of primer of the present invention, wherein swimming lane 1,2,3,4,5,6,7,8 is respectively pig erysipelas attenuated vaccine strain G4T10 and strong malicious epidemic strain GD1201, Actinobacillus pleuropneumoniae, haemophilus parasuis, swine streptococcus, bowel oedema disease intestinal bacteria, pig pasteurellosis bacillus and negative control.
Fig. 3 is the sensitivity test electrophorogram as a result of primer of the present invention, and wherein swimming lane 1,2,3,4,5,6,7,8 represents that respectively in sample to be checked, pig erysipelas attenuated vaccine strain G4T10 strain DNA content is 55.8 μ g, 55.8 μ g, 0558 μ g, 0.0558 μ g, 5.58ng, 0.558ng, 0.0558ng, 5.58pg.
Fig. 4 is the sensitivity test electrophorogram as a result of primer of the present invention, and wherein swimming lane 1,2,3,4,5,6,7,8 represents that respectively the DNA content of strong malicious epidemic strain GD1201 in sample to be checked is 23.8 μ g, 23.8 μ g, 0.238 μ g, 0.0238 μ g, 2.38ng, 0.238ng, 0.0238ng, 2.38pg.
The pcr amplification product electrophoresis detection result of the different serotypes type strain of Fig. 5 pig erysipelas and clinical separation strain, wherein swimming lane 1,2,3,4,5,6 is respectively pig erysipelas attenuated vaccine strain G4T10 strain, strong malicious epidemic strain GD1201, CVCC123(serotype 1a), CVCC127(serotype 1b), CVCC140(serotype 2), clinical separation strain HB and clinical separation strain GX.
Embodiment
Below in conjunction with embodiment, the present invention is further described, but is not used for limiting practical range of the present invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to the skilled person, agents useful for same be commercial goods (10 times of dilution PCR damping fluids of 10 * PCR buffer<namely >, dNTPs Mixture, Taq archaeal dna polymerase be all purchased from TAKARA company, primer is synthetic to be completed by Shanghai Sheng Gong limited-liability company).Pig erysipelas attenuated vaccine strain G4T10 strain (hereinafter to be referred as the G4T10 strain) is purchased from Zhongmu Industry Co.,Ltd, strong malicious epidemic strain GD1201(is hereinafter to be referred as the GD1201 strain) for being located away from the bacterial strain in Guangdong, through being accredited as the strong virus force epidemic strain, the BHI nutrient solution is purchased from U.S. company BD.
(1) the sample extraction DNA:1.5mL to be checked BHI nutrient solution, the 1.5mL that contain the G4T10 strain contains the BHI nutrient solution of GD1201 strain, the BHI nutrient solution is as negative control, and the DNA that extracts according to TIANGEN bacterial genomes DNA extraction operation steps (extracting reagent purchased from TIANGEN company) respectively is put in-20 ℃ of preservations.
(2) 1 μ L DNA profiling is joined in the PCR premixed liquid, preparation 25 μ L reaction systems, and mix.
The PCR premixed liquid: PCR damping fluid 1.5 ~ 3 μ L of 10 times of dilutions, dNTP mixture 1.5 ~ 3 μ L, concentration is the Taq archaeal dna polymerase 1 μ L of 2.5M/ μ L, concentration is each 2 μ L of primer shown in the SEQ ID NO:1 of 10pmol/ μ L ~ 2, ddH
2O supplies 25 μ L.
(3) the PCR reaction system of step (2) is placed on the PCR instrument and carries out cyclic amplification reaction.Amplification condition is: 95 ℃ of denaturation 10min; Then 95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 45s carry out 30 circulations altogether; Last 72 ℃ are extended 7min.The PCR reaction product is respectively got 5 μ L at the 1%(mass ratio) sepharose on carry out the electrophoresis evaluation.
(4) result is judged: if the PCR product only has the band of 379 bp, show that sample to be checked is the G4T10 strain; If the PCR product only has the band of a 498bp, show that sample to be checked is the GD1201 strain, without band occur negative.Detected result is shown in Fig. 1 and table 1.
Table 1 sample detection result to be checked
| Sample to be checked | Electrophoretic band | The extension increasing sequence order-checking is identified |
| G4T10 strain nutrient solution | 379bp | The G4T10 strain |
| GD1201 strain nutrient solution | 498bp | The GD1201 strain |
| The BHI nutrient solution | Without amplified band | Nothing |
Using the BHI nutrient solution that contains the G4T10 strain and contain the BHI nutrient solution of GD1201 strain as positive control, the BHI nutrient solution of Actinobacillus pleuropneumoniae, haemophilus parasuis, swine streptococcus, bowel oedema disease intestinal bacteria and pig pasteurellosis bacillus carries out specific detection, use method and the primer of embodiment 1, detected result is shown in Fig. 2.From the result of Fig. 2, can find out, except positive control, all fail to expand any band, positive pathological material of disease amplifies respectively 379bp band and 498bp band through PCR, shows that the primer of SEQ ID NO:1 ~ 2 has very high specificity.
The DNA that extracts the two kinds of bacterial strains (G4T10 strain and GD1201 strain) that obtain in embodiment 1 makes respectively the gradient dilution of 10 times of sterilizing distilled water, the DNA content of G4T10 strain is respectively 55.8 μ g, 55.8 μ g, 0558 μ g, 0.0558 μ g, 5.58ng, 0.558ng, 0.0558ng, 5.58pg, and after the DNA that extracts in GD1201 strain culture sample dilution, content is respectively 23.8 μ g, 23.8 μ g, 0.238 μ g, 0.0238 μ g, 2.38ng, 0.238ng, 0.0238ng, 2.38pg.Each extent of dilution is respectively got 2 μ L as template, by embodiment 1 method, carry out the pcr amplification detection, observe positive band, with the high dilution that the template used amount of positive expection band occurs, calculate its susceptibility, result shows that minimum detectable activity is respectively 0.56ng and 2.4 ng, sees Fig. 3, Fig. 4.
The check of embodiment 4 different serotypes reference cultures and clinical separation strain
to clinical separation epidemic strain HB strain (being located away from pig farm, Hubei morbidity swinery), clinical separation epidemic strain GX(is located away from pig farm, Guangxi morbidity swinery) and purchased from the bacillus rhusiopathiae suis reference culture CVCC123(serotype 1a of DSMZ of China Veterinary Drugs Supervisory Inst.), CVCC127(serotype 1b), CVCC140(serotype 2), according to the method in example 1, extract respectively DNA, use primer shown in SEQ ID NO:1 ~ 2, carry out pcr amplification, product passes through electrophoresis detection, result shows that primer sequence of the present invention both can distinguish G4T10 vaccine strain and clinical popular street strain, also can distinguish the capable serotype bacillus rhusiopathiae suis of G4T10 vaccine strain and common flow strain, has broad applicability.
SEQUENCE LISTING
<110 > Guangdong Wen Shi food Group Plc
<120 > PCR primer and the application thereof of bacillus rhusiopathiae suis
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213 > artificial sequence
<400> 1
aagggtggat taagaaagat aataagtg 28
<210> 2
<211> 28
<212> DNA
<213 > artificial sequence
<400> 2
caatagattt tgaacctgta accatcat 28
Claims (5)
1. the PCR primer of a bacillus rhusiopathiae suis, is characterized in that, nucleotide sequence is as shown in SEQ ID NO:1 ~ 2.
2. the application of the PCR primer of bacillus rhusiopathiae suis claimed in claim 1 in the detection reagent of preparation bacillus rhusiopathiae suis.
3. the application of the PCR primer of bacillus rhusiopathiae suis according to claim 2 in the detection reagent of preparation bacillus rhusiopathiae suis is characterized in that described bacillus rhusiopathiae suis is the G4T10 strain of pig erysipelas attenuated vaccine strain and/or the wild epidemic isolates of pig erysipelas.
4. the detection kit of a bacillus rhusiopathiae suis, is characterized in that, comprises following component: the primer sequence shown in SEQ ID NO:1 ~ 2, archaeal dna polymerase, PCR damping fluid, dNTP mixture and aseptic double-distilled water.
5. the detection kit of bacillus rhusiopathiae suis according to claim 4, it is characterized in that, concrete component is: PCR damping fluid 1.5 ~ 3 μ L of 10 times of dilutions, dNTP mixture 1.5 ~ 3 μ L, concentration is the Taq archaeal dna polymerase 1 μ L of 2.5M/ μ L, concentration is each 2 μ L of primer shown in the SEQ ID NO:1 of 10pmol/ μ L ~ 2, ddH
2O supplies 25 μ L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048017A (en) * | 2016-06-08 | 2016-10-26 | 华中农业大学 | Specific sequence of Chinese swine erysipelas attenuated vaccine strains and application of specific sequence |
| CN107937573A (en) * | 2017-11-03 | 2018-04-20 | 华中农业大学 | Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method |
| CN112626245A (en) * | 2020-12-31 | 2021-04-09 | 中国科学院北京基因组研究所(国家生物信息中心) | Primer combination, kit and detection method for detecting related strains of erysipelothrix |
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| CN101031647A (en) * | 2004-02-27 | 2007-09-05 | 财团法人化学及血清疗法研究所 | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101031647A (en) * | 2004-02-27 | 2007-09-05 | 财团法人化学及血清疗法研究所 | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli |
Non-Patent Citations (3)
| Title |
|---|
| NAGAI S等: "Differentiation of Erysipelothrix rhusiopathiae strains by nucleotide sequence analysis of a hypervariable region in the spaA gene:discrimination of a live vaccine strain from field isolates", 《J VET DIAGN INVEST》 * |
| 晏鹏飞: "猪丹毒丝菌SpaA蛋白的功能区分析及其抗原表位嵌合质粒的构建", 《中国优秀硕士学位论文-农业科技辑》 * |
| 林琳等: "猪丹毒杆菌ＳｐａＡ基因的克隆与生物信息学分析", 《中国兽医学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048017A (en) * | 2016-06-08 | 2016-10-26 | 华中农业大学 | Specific sequence of Chinese swine erysipelas attenuated vaccine strains and application of specific sequence |
| CN106048017B (en) * | 2016-06-08 | 2018-06-29 | 华中农业大学 | A kind of specific sequence of China's brickpox attenuated vaccine strain and application |
| CN107937573A (en) * | 2017-11-03 | 2018-04-20 | 华中农业大学 | Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method |
| CN112626245A (en) * | 2020-12-31 | 2021-04-09 | 中国科学院北京基因组研究所(国家生物信息中心) | Primer combination, kit and detection method for detecting related strains of erysipelothrix |
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Address after: 527400 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Patentee after: Winson food group Limited by Share Ltd Address before: 527439 Yunfu, Xinxing County, Guangdong Patentee before: Guangdong Wens Foodstuff Group Co., Ltd. |