CN105002173A - Kit for identifying Brucella S2 vaccine strain and wild strain - Google Patents

Kit for identifying Brucella S2 vaccine strain and wild strain Download PDF

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Publication number
CN105002173A
CN105002173A CN201510484411.7A CN201510484411A CN105002173A CN 105002173 A CN105002173 A CN 105002173A CN 201510484411 A CN201510484411 A CN 201510484411A CN 105002173 A CN105002173 A CN 105002173A
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brucella
vaccine strain
sample
strain
pcr
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何洪彬
赵贵民
王洪梅
贾春涛
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DAIRY CATTLE RESEARCH CENTER OF SHANDONG ACADEMY OF AGRICULTURAL SCIENCES
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DAIRY CATTLE RESEARCH CENTER OF SHANDONG ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

The invention relates to a kit for identifying a Brucella S2 vaccine strain and a wild strain. The kit comprises a specific primer pair SEQ ID No.1 and SEQ ID No.2 for identifying the Brucella vaccine strain S2. Sequencing analysis is further performed on a PCR amplification product after the kit is used for distributing Brucella and other conventional bacterial strains through a PCR amplification-electrophoresis detection area, and the Brucella S2 vaccine strain in a clinic sample can be rapidly and effectively identified and diagnosed according to the sequencing result.

Description

Differentiate the test kit of brucella S2 vaccine strain and street strain
Technical field
The invention belongs to biological technical field, be specifically related to a kind of test kit differentiating brucella S2 vaccine strain and street strain.
Background technology
Brucellosis (Brucellosis) suffers from transmissible disease altogether by the bacterial a kind of people and animals (beast) be distributed widely in all over the world of Brucella, and it not only affects sound development but also the harm humans public health health of livestock industry.Defend the national Epidemic Situation of Notifiable Infectious Diseases data of planning commission's announcement according to country, it is 57222 that Chinese people in 2014 infects brucellosis number, and sickness rate increased 31.48% than 2013.The contagium that people infects brucellosis is mainly derived from the animal of infection and contaminated livestock product, and therefore, controlling and eliminate animal brucellosis, is the basic guarantee preventing human infection's cloth Lu Shi disease.
Current vaccine immunity is still one of major measure controlling animal brucellosis.The brucella vaccine strain that present mainly uses is ox kind S19 strain and RB51 strain and sheep kind Rev.1 strain, and these vaccine strain China not yet introduce at present and use.Vaccine strain mainly B. abortus A19 vaccine strain and the pig kind brucella S2 vaccine strain that China uses, also once used sheep kind M5-90 vaccine strain, in the past because virulence problem does not temporarily re-use now.The use of S2 vaccine strain provides important leverage for the prevention and control of China's fauna brucellosis, this vaccine virus force rate S19 and Rev.1 is weak, all can produce good immunity to pig, ox, sheep, what it was outstanding be also advantageous in that can not cause miscarriage by oral way immunity pregnant female.But also there is the gordian technique bottleneck problem cannot differentiating vaccine strain and wild virus infection strain.Although competitive ELISA (cELISA) and fluorescence polarization experiment (FPT) are with the advantage of its high-throughput and operating aspect, the serology infected at brucella attenuated vaccine and wild strain differentiates certain application, but concerning the serum sample of Unknown Background, single detects still can not definitely differentiate vaccine immunity or wild virus infection.From etiology aspect differential diagnosis brucella street strain nucleic acid, accurately can judge whether animal body carries disease germs, the quarantine for brucellosis animals showing positive is eliminated and is provided support.Therefore, utilize molecular biology method, according to S2 vaccine strain and other brucella strain genome difference base sequences, by pcr amplification and PCR primer sequencing, effectively can realize the molecular identificalion of S2 vaccine strain and street strain, be applied to clinical discriminating, technical guarantee can be provided for the discriminating of China S2 vaccine strain.
Summary of the invention
Contriver is by genome sequence comparison, find compared with other Brucella bacterial genomes, pig kind brucella S2 vaccine strain is in genome 246967 site, namely 369 site of SEQ ID No.3 lack 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA), according to this deletion sequence, design of amplification primers of the present invention, by whether there is overlapping peak in sequence alignment and peak figure just itself and other brucella street strain or vaccine strain can be differentiated, be more than propose technical basis of the present invention.
The object of this invention is to provide a kind of test kit and the using method thereof of differentiating brucella S2 vaccine strain and street strain, this test kit can differentiate brucella S2 vaccine strain in clinical sample specifically.
For realizing the object of the invention, adopt following technical scheme:
Differentiate the test kit of brucella S2 vaccine strain, this PCR kit is made up of PCR reaction solution, archaeal dna polymerase, positive quality control standard substance and negative quality control standard product;
Described PCR reaction solution comprises to be identified and the primer pair of the S2 vaccine strain specific nucleotide sequences that checks order, and wherein differentiate that the upstream primer sequence of S2 vaccine strain is as shown in SEQ ID No.1, downstream primer sequence is as shown in SEQ IDNo.2.As follows respectively:
SEQ ID No.1:5'-CCTGCTGAGCGATGACCACCA-3'
SEQ ID No.2:5'-CCGCCAGAACATGCGATTTGA-3'
Described positive quality control standard substance are pig kind brucella S2 vaccine strain positive quality control standard substance;
The pEASY-T3 recombinant plasmid that described pig kind brucella S2 vaccine strain positive quality control standard substance are made up of the nucleotide fragments containing 539 bases forms.
The described sequence containing the nucleotide fragments of 539 bases is as SEQIDNo.3:
SEQIDNo.3:
5'-CCTGCTGAGCGATGACCACCACCGAGCCGCGGTCGAACACGGCAAGCTGATTGGCCTGTTTGGAACGGTCGGCAAGCTCGCGCATGAAGGGCGTGGCGAAGGAGGCAAGCCTGCGCACCGGCGCATGGAGTTGCGCAAGCCCGAACAGCTTCAGCGTCAGTGAATAACGGTCGCCATCGAGTTTGGTGACATAGCCGCGCTTCACCAGCCGGTCCAGCATCCGGTAAAATTCGTTGGGGCTGCGGTCGAGATGCTTGGCGATCTCGGCCTGTGTCAGCCCGCCATCCACACTCGCCAGCAATTCCAATATGTCCAGCCCCTTGTCCAGCGCGGGCGCGCGATAGCGATCGTCAGTTTCCAAGGTCGGCTACGAACAGCGTAAAGGGTGAAAGGAATAACCACCCGCAAATCGCCTTGACCATGTTTGAATAATATGTTTGCATATAAATGGAAGCCCCACAATCGGGGAACGTGGACGGGGGAGCGTCCGCTAATAAGGAGGAGACGAATGCAGGATTTCAAATCGCATGTTCTGGCGG-3'
Described PCR reaction solution also comprises containing dNTPs, Mg 2+, distilled water PCR damping fluid.
The described nucleotide fragments containing 539 bases clones to obtain from pig kind brucella S2 vaccine strain.
Described negative quality control standard product are aseptic double-distilled water (ddH 2o).
Invention further provides the using method of the PCR kit differentiating brucella S2 vaccine strain, condition and the step of the method are as follows:
(1) genomic dna of sample to be tested is extracted;
Described sample to be detected is blood, milk sample, tissue samples, aerosol sample etc.
(2) with the sample genomic dna extracted for template, sample DNA, positive quality control standard substance and negative quality control standard product are joined respectively in the PCR reaction tubes of the 50 μ L systems containing PCR reaction solution and archaeal dna polymerase, increase according to the PCR reaction conditions after optimizing, concrete reaction conditions is: 94 DEG C of 3min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations, 72 DEG C of 10min;
Described PCR reaction solution is by identifying the primer pair of S2 vaccine strain and containing dNTPs, Mg 2+, distilled water PCR damping fluid composition;
Described primer pair is respectively SEQ ID No.1 and SEQ ID No.2, respectively called after S2-F and S2-R, and sequence is as follows:
Forward primer S2-F:5'-CCTGCTGAGCGATGACCACCA-3'
Reverse primer S2-R:5'-CCGCCAGAACATGCGATTTGA-3'
Described PCR reaction system is 50 μ L, specifically comprises following component:
(3) get 50 μ L PCR primer, detect with 1.5% agarose gel electrophoresis, observe and differentiate that pig kind brucella S2 vaccine strain primer pair amplifies goes out 539bp and size is corresponding with it band; Those skilled in the art should know, when agarose gel electrophoresis detects, show the band corresponding with object stripe size, mean pcr amplification product size close, because of sample source or target kind difference, PCR primer size has less difference, but display band is corresponding, and the such as band differed within the scope of 30bp/20bp all shows size correspondence.
(4) if above-mentioned PCR detected result and object band are not inconsistent, then pattern of descriptive parts is that brucella is negative, if PCR test strip size is correct, then cuts glue to positive band and reclaims purifying, and then carry out sequencing.
(5) differentiate that the result of brucella S2 vaccine strain judges: use the 539bp of S2-F primer pair purifying and size is corresponding with it product to check order, if sequencing result peak figure is simple spike, mate completely with after SEQIDNo.3 comparison, then this sample is S2 vaccine strain; If sequencing result peak figure is simple spike, have more 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA) with after SEQIDNo.3 comparison in the 369th site, then this sample is other brucella street strain or the vaccine strains except S2 vaccine strain; If sequencing result and SEQIDNo.3 comparison, occur cover peak behind peak graphic sequence the 369th site, then this sample is S2 vaccine strain and other brucella strains mixing.
It should be understood by those skilled in the art that above-mentioned discrimination method, its direct object is strain identification kind, and non-acquisition medical diagnosis on disease result.
The present invention achieves following effect:
(1) for the discriminating of China S2 vaccine strain provides new technique means;
(2) primer pair of the present invention has higher specificity, by amplification-electrophoresis detection, effectively can distinguish brucella and other Conventional bacteria bacterial strains;
(3) other brucella street strains and S2 vaccine strain are made a distinction, have high accuracy rate by discrimination method of the present invention effectively.
Accompanying drawing explanation
Fig. 1 S2 vaccine strain deletion segment place upstream and downstream sequence pcr amplification, +: template is the pcr amplification product 539bp – of S2 vaccine strain positive quality control standard substance: template is negative quality control standard product, 1-5: template is respectively the pcr amplification product 539bp of pig kind brucella vaccine strain S2 strain, B. abortus vaccine strain A19 strain, brucella melitensis vaccine strain M5-90 strain, Br.ovis 63/290 strain, dog brucella RM6/66 pnca gene group DNA; 6-10: template is respectively Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus, cow mycobacteria genomic dna.
Fig. 2 S2 vaccine strain deletion segment place sequencing peak figure, A: order-checking peak figure is simple spike, and mate completely with after SEQIDNo.3 comparison, detection template is S2 vaccine strain.B: order-checking peak figure is simple spike, and have more 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA) at 369 places with after SEQ ID No.3 comparison, detection template is other brucella street strain or vaccine strains except S2 vaccine strain.C: sequencing result and SEQ ID No.3 comparison, peak figure in sequence site 369 after there is cover peak, detection template is S2 vaccine strain and the mixing of other brucella strains.
Embodiment
Following embodiment is used for further illustrating the present invention, but is not used for limiting the scope of the invention.
Following examples all conveniently test conditions and method are carried out, or according to the test conditions that manufacturer advises.
1. test materials
Pig kind brucella vaccine strain S2 strain (B.suis S2), B. abortus vaccine strain A19 strain (B.abortus A19), brucella melitensis vaccine strain M5-90 strain (B.melitensis M5-90), Br.ovis 63/290 strain (B.ovis 63/290), dog brucella RM6/66 strain (B.canisRM6/66), Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus (CMCC25623), cow mycobacteria (M.bovis ATCC 19210), be diagnosed as the blood of the brucella positive clinically, milk sample, the DNA samples such as aborted fetus tissue and aerosol are preserved by Cow Research Center, Shandong Academy of Agricultural Sciences's disease research room.
Archaeal dna polymerase (TaKaRa LA Taq) is purchased from precious biotechnology (Dalian) company limited, agarose is purchased from Beijing Quanshijin Biotechnology Co., Ltd, Fast DNA extraction test kit, DNA of bacteria extract test kit, sepharose recovery test kit etc. all purchased from TIANGEN Biotech (Beijing) Co., Ltd., and other biochemical reagents are import packing or domestic analytical pure.PCR primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and determined dna sequence is completed by Shandong Academy of Agricultural Sciences center of checking order.
2. laboratory apparatus
Grads PCR instrument (TaKaRa TP-600), electrophoresis apparatus (Beijing Liuyi Instrument Factory DYY-6C type), Alpha human-like gel imaging system red (ProteinSimple company of the U.S.), DNA sequencer (ABI3730XL DNA Analyzer).
Embodiment one, differentiate the using method of the PCR kit of brucella S2 vaccine strain and street strain
(1) genomic dna of sample to be tested is extracted: use Fast DNA extraction test kit to carry out the extraction of genomic dna to blood to be detected, milk sample, tissue samples, aerosol equal samples.
(2) with the sample genomic dna extracted for template, S2-F and S2-R primer pair is used to carry out pcr amplification, simultaneously using positive quality control standard substance and negative quality control standard product as positive and negative control, successively each component of 50 μ L reaction systems is added in PCR reaction tubes, be respectively 10 × PCR Buffer:5.0 μ L, dNTP Mixture (2.5mM): 8.0 μ L, the forward primer of 10 μMs: 2.0 μ L, the reverse primer of 10 μMs: 2.0 μ L, sample to be tested DNA:5.0 μ L, archaeal dna polymerase: 0.5 μ L, finally add 27.5 μ L distilled water (ddH 2o) 50 μ L are supplied.The expanding effect of PCR directly has influence on specificity and the susceptibility of detected result, therefore needs to be optimized the correlation parameter in PCR reaction conditions, comprises the concentration of primer, Mg 2+concentration, annealing temperature, annealing time, cycle index and extension time etc.The optimization of PCR reaction conditions adopts prior art, and the PCR reaction conditions after final optimization pass is: primer concentration is 0.4 μm of oL/L, Mg 2+concentration is 0.2mmoL/L, PCR reaction conditions: 94 DEG C of 3min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations, 72 DEG C of 10min.Pcr amplification is carried out with the reaction conditions that above-mentioned optimization is good.
(3) after reaction terminates, get 50 μ L PCR primer, carry out electrophoresis (120V25min) with 1.5% sepharose and detect, observe and differentiate that pig kind brucella S2 vaccine strain amplifies the band of 539bp.
(4) if above-mentioned PCR detected result and object band are not inconsistent, then pattern of descriptive parts is that brucella is negative, if PCR test strip size is correct, then cuts glue to positive band and reclaims purifying, then carry out sequencing by DNA sequencer.
(5) differentiate that the result of brucella S2 vaccine strain judges: use the 539bp product of S2-F primer pair purifying to check order, if sequencing result peak figure is simple spike, mate completely with after SEQ ID No.3 comparison, then this sample is S2 vaccine strain; If sequencing result peak figure is simple spike, have more 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA) with after SEQIDNo.3 comparison in the 369th site, then this sample is other brucella street strain or the vaccine strains except S2 vaccine strain; If sequencing result and SEQID No.3 comparison, occur cover peak behind peak graphic sequence the 369th site, then this sample is S2 vaccine strain and other brucella strains mixing.
The assembling of embodiment two, PCR kit
(1) PCR reaction solution is prepared:
1. 1 is designed to the primer identifying pig kind brucella S2 vaccine strain: be SEQ ID No.1 and SEQ IDNo.2, called after S2-F and S2-R, sequence is as follows:
S2-F:5'-CCTGCTGAGCGATGACCACCA-3'
S2-R:5'-CCGCCAGAACATGCGATTTGA-3'
2. the preparation of PCR reaction solution: PCR reaction solution is by the primer of pig kind brucella S2 vaccine strain with containing dNTPs, Mg 2+, distilled water (ddH 2o) PCR damping fluid composition, the cumulative volume of PCR reaction solution is 44.5 μ L, and the amount of every bar primer is 2 μ L (primer concentration is 10 μm of oL/L), containing dNTPs, Mg 2+, distilled water (ddH 2the total amount of PCR buffer reagent O) is 40.5 μ L, wherein distilled water (ddH 2o) be 27.5 μ L.
(2) preparation of positive quality control standard substance:
1. the extraction of template DNA: respectively test kit is extracted to the bacterium liquid application DNA of bacteria of pig kind brucella vaccine strain S2 and extract DNA.
2. pcr amplification: with the DNA extracted for template, according to PCR reaction system: DNA profiling 5 μ L, archaeal dna polymerase 0.5 μ L, PCR reaction solution 44.5 μ L, PCR reaction conditions is: 94 DEG C of 3min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations, 72 DEG C of 10min, object that PCR instrument increases fragment, the agarose gel electrophoresis (120V 25min) with 1.5% identifies PCR object product.
3. the structure of recombinant plasmid and the foundation of positive quality control standard substance: PCR primer is carried out gel-purified recovery, then connects pEASY-T3 carrier, transforms, and after extracting plasmid, double digestion is identified, is checked order by positive recombinant plasmid, and carries out sequence alignment.The recombinant plasmid that sequence is correct is the positive quality control standard substance of acquisition.
Said positive quality control standard substance are pig kind brucella S2 vaccine strain positive quality control standard substance.
Said pig kind brucella S2 vaccine strain positive quality control standard substance are formed by containing the recombinant plasmid after the nucleotide fragments of 539 bases is connected to pEASY-T3 carrier.
(3) negative quality control standard product: negative quality control standard product are aseptic double-distilled water, volume is 5 μ L.
Embodiment three, brucella S2 vaccine strain identification reagent box specific test
Adopt PCR kit of the present invention respectively to pig kind brucella S2, B. abortus A19, brucella melitensis M5-90, Br.ovis 63/290 strain, dog brucella RM6/66 strain, Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus, cow mycobacteria genomic dna increase, and establish the positive and negative control, the specificity of checking PCR reaction, PCR reaction conditions is: 94 DEG C of 5min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations, 72 DEG C of 10min.Purifying and the order-checking of PCR primer is carried out again after reaction terminates.
Pig kind brucella S2 vaccine strain differentiates specific outcome as shown in Figure 1, in agarose gel electrophoresis result :+be positive quality control standard substance, 1-5: be respectively pig kind brucella S2, B. abortus A19, brucella melitensis M5-90, Br.ovis 63/290 strain, dog brucella RM6/66 strain all amplifies band , Er –, 6-10 of 539bp: be respectively negative quality control standard product, Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus and cow mycobacteria all without band.After sequencing, sequence and peak figure comparison result are as shown in Figure 2, S2 vaccine strain is only had to there is disappearance (Fig. 2 of 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA) in 369 site, A), other brucella strains and vaccine strain result are shown in Fig. 2-B.
The discriminating of embodiment four, brucella positive clinical sample S2 vaccine strain
Respectively to the brucella positive sample (through pcr amplification sequence verification) that this laboratory is preserved, comprise 2 parts of blood samples, the cowshed aerosol sample of 2 parts of milk samples, 2 parts of aborted fetus tissue samples and 2 parts of different dairy cow farms carries out the discriminating of vaccine strain, result is as shown in the table.

Claims (9)

1. for differentiating a primer pair of pig kind brucella vaccine strain S2, it is characterized in that, two primer sequences are respectively SEQ ID No.1 and SEQ ID No.2.
2. primer pair according to claim 1 for the preparation of differentiate and/detect the application of brucellar test kit, chip.
3. primer pair according to claim 1 for the preparation of differentiate and/detect the test kit of pig kind brucella vaccine strain S2, the application of chip.
4. comprise the test kit of primer pair according to claim 1.
5. test kit according to claim 4, is characterized in that, also comprises: PCR reaction solution, archaeal dna polymerase, positive quality control standard substance and negative quality control standard product.
6. for differentiating a brucellar method, it is characterized in that, comprising the steps:
(1) genomic dna of sample to be tested is extracted;
(2) with the sample genomic dna extracted for template, carry out pcr amplification, the primer adopted is primer pair according to claim 1;
(3) get PCR primer, detect with agarose gel electrophoresis, observe whether occur the 539bp that pig kind brucella vaccine strain S2 amplifies and the band that size is corresponding with it;
(4) if above-mentioned PCR detected result and object band are not inconsistent, then pattern of descriptive parts is that brucella is negative, and object band conforms to, be then brucella.
7. differentiate brucella S2 vaccine strain and other brucellar methods, it is characterized in that,
(1) genomic dna of sample to be tested is extracted;
(2) with the sample genomic dna extracted for template, carry out pcr amplification, the primer adopted is primer pair according to claim 1;
(3) get PCR primer, detect with agarose gel electrophoresis, observe whether occur the 539bp that pig kind brucella vaccine strain S2 amplifies and the band that size is corresponding with it;
(4) if above-mentioned PCR detected result and object band are not inconsistent, then pattern of descriptive parts is that brucella is negative, if PCR test strip size is correct, then cuts glue to positive band and reclaims purifying, and then carry out sequencing;
(5) result judges: check order to using the 539bp of SEQ ID NO:1 and SEQ ID NO:2 primer pair purifying and size is corresponding with it product, if sequencing result peak figure is simple spike, mate completely with after SEQ ID No.3 comparison, then this sample is S2 vaccine strain; If sequencing result peak figure is simple spike, have more 25 nucleotide sequences with after SEQID No.3 comparison in the 369th site, then this sample is other brucella street strain or the vaccine strains except S2 vaccine strain; If sequencing result and SEQ ID No.3 comparison, occur cover peak behind peak graphic sequence the 369th site, then this sample is S2 vaccine strain and other brucella strains mixing.
8. method described in claim 6 or 7, is characterized in that, described sample to be tested is blood, milk sample, tissue samples, aerosol sample.
9. method described in claim 6 or 7, is characterized in that, pcr amplification condition is: 94 DEG C of 3min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations, 72 DEG C of 10min.
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CN105693831B (en) * 2016-02-06 2019-01-11 内蒙古农业大学 A kind of detection reagent and its application of brucella S2 vaccine immunity antibody
CN108048586A (en) * 2017-12-22 2018-05-18 中国动物卫生与流行病学中心 A kind of detection method of ox kind Brucella sp attenuated vaccine strain S19
CN108034739A (en) * 2018-01-29 2018-05-15 中国农业科学院兰州兽医研究所 Differentiate primer, kit and its application of brucella gene-deleted vaccine and natural infection strain based on RPA methods
CN112899385A (en) * 2021-03-19 2021-06-04 广东省农业科学院动物卫生研究所 Primer group and probe for identifying Brucella S2 vaccine strain and wild strain and application of primer group and probe

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