CN105018489B

CN105018489B - Differentiate brucella street strain and vaccine strain A19 and S2 kit

Info

Publication number: CN105018489B
Application number: CN201510483012.9A
Authority: CN
Inventors: 何洪彬; 赵贵民; 王洪梅
Original assignee: Dairy Cattle Research Center Shandong Academy of Agricultural Science
Current assignee: Dairy Cattle Research Center Shandong Academy of Agricultural Science
Priority date: 2015-08-07
Filing date: 2015-08-07
Publication date: 2018-03-20
Anticipated expiration: 2035-08-07
Also published as: CN105018489A

Abstract

Differentiate brucella street strain and vaccine strain A19 and S2 kit the present invention relates to a kind of, including the specific primer for differentiating brucella vaccine strain A19 to SEQ ID No.1 and SEQ ID No.2；For differentiating brucella vaccine strain S2 specific primer to SEQ ID No.3 and SEQ ID No.4；Using the kit after electrophoresis detection is expanded by PCR and distinguishes brucella and other Conventional bacteria bacterial strains,, being capable of fast and effectively brucella A19 and S2 vaccine strain in antidiastole clinical sample by sequencing result for the further sequencing analysis of pcr amplification product.

Description

Differentiate brucella street strain and vaccine strain A19 and S2 kit

Technical field

The invention belongs to biological technical field, and in particular to one kind differentiates brucella street strain and vaccine strain A19 and S2 Kit.

Background technology

Brucellosis (Brucellosis) is distributed widely in all over the world by the bacterial one kind of Brucella People and animals (beast) suffer from infectious disease altogether, its not only influence the sound development of animal husbandry and also endanger the mankind's public health health.According to Country defends the national Epidemic Situation of Notifiable Infectious Diseases data of planning commission's announcement, and Chinese people infection brucellosis number is 57222 within 2014, The incidence of disease increased 31.48% than 2013.People infect brucellosis the infection sources be mainly derived from infection animal and by The livestock products of pollution, therefore, control and elimination animal brucellosis, are the basic guarantees for preventing human infection's cloth Lu Shi diseases.

Vaccine immunity is still one of major measure for controlling animal brucellosis at present.The cloth that current foreign countries mainly use Shandong Salmonella vaccine strain is that ox kind S19 strains and RB51 strains and sheep kind Rev.1 strains, these vaccine strain China not yet introduce and made at present With.The vaccine strain that China uses is mainly B. abortus A19 vaccine strains and pig kind brucella S2 vaccine strains, in the past also once Sheep kind M5-90 vaccine strains were used, because virulence problem does not use temporarily now.The use of A19 and S2 vaccine strains is China The prevention and control of fauna brucellosis provide important leverage, but there is also can not differentiate vaccine strain and wild virus infection strain Key technology bottleneck problem.Although competitive ELISA (cELISA) and fluorescence polarization experiment (FPT) are with its high flux and operation side The advantage in face, differentiate there is certain application in the serology of brucella attenuated vaccine and wild strain infection, but to unknown For the serum sample of background, single detection still can not definitely differentiate vaccine immunity or wild virus infection.Differentiate in terms of aetology Brucella street strain nucleic acid is diagnosed, whether can be carried disease germs with accurate judgement animal body, is the inspection of cloth brucellosis animals showing positive Epidemic disease, which is eliminated, provides support.Therefore, using molecular biology method, according to vaccine strain and other brucella strain genome differences Base sequence, by PCR amplifications and PCR primer sequencing, vaccine strain and the molecular identificalion of street strain can be effectively realized, by it Differentiate applied to clinic, technical guarantee can be provided for the discriminating of China's A19 and S2 vaccine strain.

The content of the invention

Inventor is compared by genome sequence, is found different from other Brucella bacterial genomes DNA sequence dnas It is that A19 vaccine strains are at the site of genome 22840, i.e., at SEQ ID No.5 344 sites, lacks 7 nucleotide sequences, should Site postorder is classified as AGATTTCAGA, and other brucella sequences are AGATTTCAGATTTCAGA, because being herein AGATTTC Repetitive sequence, so two kinds of sequence deletion phenomenons (AGATTTC or TTTCAGA) occur in sequence alignment.In the missing Primer, sequencing after being expanded by PCR, sequence alignment and the different cans spy that peak figure is sequenced are designed at the upstream and downstream of site The opposite sex differentiates B. abortus A19 vaccine strains.Likewise we find that pig kind brucella S2 vaccine strains are in genome 246967 At site, compared with other Brucella bacterial genomes, S2 vaccine strains are at the site, i.e., 369 of SEQ ID No.6 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA) are lacked at point, according to the deletion sequence, present invention design expands Increase primer, by whether occurring covering peak can in sequence alignment and peak figure by itself and other brucella street strains or vaccine strain Differentiated, above is proposing the technical basis of the present invention.

It is an object of the invention to provide a kind of kit for differentiating brucella street strain and vaccine strain A19 and S2 and its make With method, the kit can specifically differentiate brucella A19 and S2 vaccine strain in clinical sample.

To realize the object of the invention, adopt the following technical scheme that：

Differentiate brucella street strain and vaccine strain A19 and S2 kit, the PCR kit is by PCR reaction solutions, DNA Polymerase, positive quality control standard items and negative quality control standard product composition；

The PCR reaction solutions include identifying and being sequenced the primer of A19 vaccine strains and S2 vaccine strain specific nucleotide sequences It is right, wherein differentiate A19 vaccine strains upstream primer sequence as shown in SEQ ID No.1, downstream primer sequence such as SEQ ID No.2 It is shown, differentiate the upstream primer sequence of S2 vaccine strains as shown in SEQ ID No.3, downstream primer sequence such as SEQ ID No.4 institutes Show.It is as follows respectively：

SEQ ID No.1：5'-TTCACCGTCATCGGATAATAGGTGCCA-3'

SEQ ID No.2：5'-GGCGGGCTGCATTCTTTACTCTGC-3'

SEQ ID No.3：5'-CCTGCTGAGCGATGACCACCA-3'

SEQ ID No.4：5'-CCGCCAGAACATGCGATTTGA-3'

The positive quality control standard items include B. abortus A19 vaccine strain positive quality control standard items and pig kind cloth Lu Shi Bacterium S2 vaccine strain positive quality control standard items；

The B. abortus A19 vaccine strain positive quality control standard items are by the nucleotide fragments structure containing 562 bases Into pEASY-T3 recombinant plasmids composition, the pig kind brucella S2 vaccine strain positive quality control standard items are by containing 539 alkali The pEASY-T3 recombinant plasmids composition that the nucleotide fragments of base are formed.

The sequence of the nucleotide fragments containing 562 bases is as shown in SEQ ID No.5：

SEQ ID No.5：

5'-TTCACCGTCATCGGATAATAGGTGCCACCCTTTACCGTCGCATCATTGCAGACGATCATGCATTCG CGGCCCGATACGCGCCCGATGCCGGTGATGAAGCCAGCGGCGGGCGCTGCCCCGCCATACATGCCATGCGCTGCCGT CAGCCCCAGTTCCAGAAAGGGGGAACCCGGATCGAGCAATTGCGCCACGCGCTCACGGGGCAGAAGTTTTCCGCGCG AAACATGGCGGTCGCGCGCTTTCTCGCCGCCGCCGTCAATGGCAATGCGCGAGGCTTCCGCCACCACGTCGATTGCC GCCAGCATGGCCTTGCGGTTGGCCTCGAAGCTTGCGCTGCGCGTCGAGATTTCAGAACCGGCATCAACGGGTCTCCT GAAAGAGTTCCCGTCCGATCAGCATACGCCGGATTTCCGACGTGCCCGCGCCGATTTCATAAAGCTTGGCATCGCGC AAAAGGCGGCCCGTCGGATAATCGTTGATATAGCCGTTGCCGCCGAGAGACTGGATCGCCTGCAAGGCCATCTGCGT GGCATTTTCCGCAGAGTAAAGAATGCAGCCCGCC-3'

The sequence of the nucleotide fragments containing 539 bases such as SEQ ID No.6：

SEQ ID No.6：

5'-CCTGCTGAGCGATGACCACCACCGAGCCGCGGTCGAACACGGCAAGCTGATTGGCCTGTTTGGAAC GGTCGGCAAGCTCGCGCATGAAGGGCGTGGCGAAGGAGGCAAGCCTGCGCACCGGCGCATGGAGTTGCGCAAGCCCG AACAGCTTCAGCGTCAGTGAATAACGGTCGCCATCGAGTTTGGTGACATAGCCGCGCTTCACCAGCCGGTCCAGCAT CCGGTAAAATTCGTTGGGGCTGCGGTCGAGATGCTTGGCGATCTCGGCCTGTGTCAGCCCGCCATCCACACTCGCCA GCAATTCCAATATGTCCAGCCCCTTGTCCAGCGCGGGCGCGCGATAGCGATCGTCAGTTTCCAAGGTCGGCTACGAA CAGCGTAAAGGGTGAAAGGAATAACCACCCGCAAATCGCCTTGACCATGTTTGAATAATATGTTTGCATATAAATGG AAGCCCCACAATCGGGGAACGTGGACGGGGGAGCGTCCGCTAATAAGGAGGAGACGAATGCAGGATTTCAAATCGCA TGTTCTGGCGG-3'

The PCR reaction solutions also include containing dNTPs, Mg²⁺, distilled water PCR buffer solutions.

The nucleotide fragments containing 562 bases are to clone to obtain from B. abortus A19 vaccine strains, described Nucleotide fragments containing 539 bases are to clone to obtain from pig kind brucella S2 vaccine strains.

The negative quality control standard product are aseptic double-distilled water (ddH₂O)。

Invention further provides differentiate brucella street strain and the use of vaccine strain A19 and S2 PCR kit Method, the condition and step of this method are as follows：

(1) genomic DNA of sample to be tested is extracted；

The sample to be detected is blood, milk sample, tissue samples, aerosol sample etc..

(2) using the sample genomic dna of extraction as template, by sample DNA, positive quality control standard items and negative quality control standard Product are added separately in the PCR reaction tubes of the 50 μ L systems containing PCR reaction solutions and archaeal dna polymerase, according to after optimization PCR reaction conditions are expanded, and specific reaction 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；

The PCR reaction solutions by identify brucella A19 vaccine strains and S2 vaccine strains primer pair and containing dNTPs, Mg²⁺, distilled water PCR buffer solutions composition；

The primer pair is respectively SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, SEQ ID No.4, is distinguished A19-F, A19-R and S2-F, S2-R are named as, sequence is as follows：

Forward primer A19-F：5'-TTCACCGTCATCGGATAATAGGTGCCA-3'

Reverse primer A19-R：5'-GGCGGGCTGCATTCTTTACTCTGC-3'

Forward primer S2-F：5'-CCTGCTGAGCGATGACCACCA-3'

Reverse primer S2-R：5'-CCGCCAGAACATGCGATTTGA-3'

Described PCR reaction systems are 50 μ L, specifically include following component：

(3) 50 μ L PCR primers are taken, are detected with 1.5% agarose gel electrophoresis, observation differentiates B. abortus A19 strain primer pair amplifies go out band corresponding to 562bp and therewith size, differentiate pig kind brucella S2 vaccine strain primer pair amplifies Go out band corresponding to 539bp and therewith size；Those skilled in the art will be appreciated that, when agarose gel electrophoresis detects, display Band corresponding with purpose band size, it is intended that pcr amplification product size is close, because sample source or target kind are different, PCR Primer size has smaller difference, but shows that band is corresponding, such as the band in the range of difference 30bp/20bp shows size pair Should.

(4) if above-mentioned PCR testing results are not inconsistent with purpose band, pattern of descriptive parts is that brucella is negative, if PCR Test strip size is correct, then carries out gel extraction purifying to positive band, then carry out sequencing again.

(5) result judgement：

The judgement of A19 vaccine strains identification result, the therewith 562bp purified using A19-F primer pairs or product corresponding to size It is sequenced, if sequencing result peak figure is simple spike, is matched completely after being compared with SEQ ID No.5, then the sample is A19 epidemic diseases Miao Zhu；If sequencing result peak figure is simple spike, 7 nucleotides sequences are had more at the 344th site after being compared with SEQ ID No.5 Arrange (AGATTTC or TTTCAGA), then the sample is other brucella street strains in addition to A19 vaccine strains or vaccine strain；Such as Fruit sequencing result compares with SEQ ID No.5, in the sequence the 354th of peak figure, i.e. occurs set peak after AGATTTCAGA, then the sample This is mixed for A19 vaccine strains and other brucella strains.

S2 vaccine strains identification result judges, the 539bp that is purified using S2-F primer pairs and product corresponding to size enters therewith Row sequencing, if sequencing result peak figure is simple spike, matched completely after being compared with SEQ ID No.6, then the sample is S2 vaccines Strain；If sequencing result peak figure is simple spike, 25 nucleotides sequences are had more at the 369th site after being compared with SEQ ID No.6 Arrange (TCCCTCATAAATGGCTTTCTTCATA), then the sample in addition to S2 vaccine strains other brucella street strains or Vaccine strain；If sequencing result compares with SEQ ID No.5, occurs set peak behind the site of peak figure sequence the 369th, then the sample is S2 vaccine strains and other brucella strains mix.

It should be understood by those skilled in the art that above-mentioned discrimination method, its direct purpose is strain identification species, and non-acquisition Medical diagnosis on disease result.

The present invention achieves following effect：

(1) new technological means is provided for the discriminating of China's A19 and S2 vaccine strain；

(2) primer pair of the present invention has higher specificity, by amplification-electrophoresis detection, effectively can be distributed in area Shandong Salmonella and other Conventional bacteria bacterial strains；

(3) discrimination method of the present invention, effectively other brucella street strains is made a distinction with vaccine strain, had High accuracy rate.

Brief description of the drawings

Upstream and downstream sequence PCR is expanded at Fig. 1 A19 vaccine strain deletion segments ,+：Template is A19 vaccine strain positive quality control marks The pcr amplification product 562bp of quasi- product ,-：Template is negative quality control standard product, 1-5：Template is respectively B. abortus vaccine Strain A19 strains, pig kind brucella vaccine strain S2 strains, brucella melitensis vaccine strain M5-90 strains, Br.ovis 63/290 Strain, Brucellacanis RM6/66 pnca gene groups DNA pcr amplification product 562bp；6-10：Template is respectively Escherichia coli O 157: H7, YE O:9th, salmonella, staphylococcus aureus, cow mycobacteria genomic DNA.

Upstream and downstream sequence PCR is expanded at Fig. 2 S2 vaccine strain deletion segments ,+：Template is S2 vaccine strain positive quality control standards The pcr amplification product 539bp of product ,-：Template is negative quality control standard product, 1-5：Template is respectively pig kind brucella vaccine strain S2 strains, B. abortus vaccine strain A19 strains, brucella melitensis vaccine strain M5-90 strains, Br.ovis 63/290 Strain, Brucellacanis RM6/66 pnca gene groups DNA pcr amplification product 539bp；6-10：Template is respectively Escherichia coli O 157: H7, YE O:9th, salmonella, staphylococcus aureus, cow mycobacteria genomic DNA.

Sequencing peak figure at Fig. 3 A19 vaccine strain deletion segments, A：Sequencing peak figure is simple spike, with SEQID No.5 ratios To rear matching completely, detection template is A19 vaccine strains.B：Sequencing peak figure is simple spike, 344 after being compared with SEQ ID No.5 7 nucleotide sequences (AGATTTC or TTTCAGA) are had more at site, detection template is other cloth Shandongs in addition to A19 vaccine strains Salmonella street strain or vaccine strain.C：Sequencing result compares with SEQ ID No.5, and peak figure is behind the site of sequence the 354th, i.e., Occurs set peak after AGATTTCAGA sequences, detection template is A19 vaccine strains and the mixing of other brucella strains.

Sequencing peak figure at Fig. 4 S2 vaccine strain deletion segments, A：Sequencing peak figure is simple spike, with SEQ ID No.6 ratios To rear matching completely, detection template is S2 vaccine strains.B：Sequencing peak figure is simple spike, after being compared with SEQ ID No.6 at 369 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA) are had more, detection template is other in addition to S2 vaccine strains Brucella street strain or vaccine strain.C：Sequencing result compares with SEQ ID No.6, and peak figure occurs after sequence site 369 Peak is covered, detection template is S2 vaccine strains and the mixing of other brucella strains.

Embodiment

Following embodiments are used to further illustrate the present invention, but are not limited to the scope of the present invention.

Following examples are carried out according to normal test conditions and method, or according to the test bar proposed by manufacturer Part.

1. test material

B. abortus vaccine strain A19 strains (B.abortus A19), pig kind brucella vaccine strain S2 strains (B.suis S2), brucella melitensis vaccine strain M5-90 strains (B.melitensis M5-90), 63/290 plant of Br.ovis (B.ovis 63/290), Brucellacanis RM6/66 strains (B.canis RM6/66), Escherichia coli O 157:H7, enterocolitis Yersinia O:9th, salmonella, staphylococcus aureus (CMCC25623), cow mycobacteria (M.bovis ATCC 19210), be clinically diagnosed as the DNA samples such as brucella positive blood, milk sample, aborted fetus tissue and aerosol by Cow Research Center, Shandong Academy of Agricultural Sciences's disease research room preserves.

Archaeal dna polymerase (TaKaRa LA Taq) is purchased from precious bioengineering (Dalian) Co., Ltd, and agarose is complete purchased from Beijing Shi Jin Bioisystech Co., Ltd, Fast DNA extraction kit, DNA of bacteria extracts kit, Ago-Gel QIAquick Gel Extraction Kit Etc. being purchased from TIANGEN Biotech (Beijing) Co., Ltd., other biochemical reagents are that import packing or domestic analysis are pure.PCR draws Thing is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and center is sequenced by Shandong Academy of Agricultural Sciences in determined dna sequence Complete.

2. laboratory apparatus

Grads PCR instrument (TaKaRa TP-600), electrophoresis apparatus (Beijing Liuyi Instrument Factory's DYY-6C types), Alpha human-like Gel imaging system red (ProteinSimple companies of the U.S.), DNA sequencer (ABI3730XL DNA Analyzer).

Embodiment one, differentiate brucella street strain and the application method of vaccine strain A19 and S2 PCR kit

(1) genomic DNA of sample to be tested is extracted：Using Fast DNA extraction kit to blood to be detected, milk sample, Tissue samples, aerosol equal samples carry out the extraction of genomic DNA.

(2) using the sample genomic dna of extraction as template, entered respectively using A19-F, A19-R and S2-F, S2-R primer pairs Performing PCR expands, while using positive quality control standard items and negative quality control standard product as positive and negative control, successively by 50 The each component of μ L reaction systems is added in PCR reaction tubes, respectively 10 × PCR Buffer：5.0 μ L, dNTP Mixture (2.5mM)：8.0 μ L, 10 μM of forward primer：2.0 μ L, 10 μM of reverse primer：2.0 μ L, sample to be tested DNA：5.0 μ L, DNA Polymerase：0.5 μ L, finally add 27.5 μ L distilled waters (ddH₂O) supply to 50 μ L.PCR expanding effect directly influences detection As a result specificity and sensitivity, it is therefore desirable to optimized to the relevant parameter in PCR reaction conditions, including primer is dense Degree, Mg²⁺Concentration, annealing temperature, annealing time, cycle-index and extension of time etc..The optimization of PCR reaction conditions is using existing Technology, the PCR reaction conditions after final optimization pass are：Primer concentration is 0.4 μm of oL/L, Mg²⁺Concentration is 0.2mmoL/L, and PCR reacts 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.With above-mentioned optimization well Reaction condition enter performing PCR amplification.

(3) after reaction terminates, 50 μ L PCR primers are taken, electrophoresis (120V25min) inspection is carried out with 1.5% Ago-Gel Survey, observation differentiates that B. abortus A19 strain primer pair amplifies go out 562bp band, differentiates pig kind brucella S2 vaccine strains Amplify 539bp band.

(4) if above-mentioned PCR testing results are not inconsistent with purpose band, pattern of descriptive parts is that brucella is negative, if PCR Test strip size is correct, then carries out gel extraction purifying to positive band, then carry out sequencing with DNA sequencer.

(5) result judgement：

A19 vaccine strains identification result judges that the 562bp products purified using A19-F primer pairs are sequenced, if sequencing As a result peak figure is simple spike, is matched completely after being compared with SEQ ID No.5, then the sample is A19 vaccine strains；If sequencing result Peak figure is simple spike, had more after being compared with SEQ ID No.5 at the 344th site 7 nucleotide sequences (AGATTTC or TTTCAGA), then the sample is other brucella street strains in addition to A19 vaccine strains or vaccine strain；If sequencing result with SEQ ID No.5 are compared, and in the sequence the 354th of peak figure, i.e. occur set peak after AGATTTCAGA, then the sample is A19 vaccines Strain and other brucella strains mixing.

S2 vaccine strains identification result judges that the 539bp products purified using S2-F primer pairs are sequenced, if sequencing knot Fruit peak figure is simple spike, is matched completely after being compared with SEQ ID No.6, then the sample is S2 vaccine strains；If sequencing result peak Figure is simple spike, and 25 nucleotide sequences are had more at the 369th site after being compared with SEQ ID No.6 (TCCCTCATAAATGGCTTTCTTCATA), then the sample is other brucella street strains in addition to S2 vaccine strains or epidemic disease Miao Zhu；If sequencing result compares with SEQ ID No.6, occurs set peak behind the site of peak figure sequence the 369th, then the sample is S2 Vaccine strain and other brucella strains mix.

The assembling of embodiment two, PCR kit

(1) PCR reaction solutions are prepared：

1. separately design the primer of 2 pairs of identification Brucella abortus A19 vaccine strains and pig kind brucella S2 vaccine strains：For SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, SEQ ID No.4, are named as A19-F, A19-R and S2-F, S2- R, sequence are as follows：

A19-F：5'-TTCACCGTCATCGGATAATAGGTGCCA-3'

A19-R：5'-GGCGGGCTGCATTCTTTACTCTGC-3'

S2-F：5'-CCTGCTGAGCGATGACCACCA-3'

S2-R：5'-CCGCCAGAACATGCGATTTGA-3'

2. the preparation of PCR reaction solutions：PCR reaction solutions are by B. abortus A19 vaccine strains or pig kind brucella S2 epidemic diseases The primer of seedling strain and contain dNTPs, Mg²⁺, distilled water (ddH₂O PCR buffer solutions composition), the cumulative volume of PCR reaction solutions are 44.5 μ L, the amount of every primer is 2 μ L (primer concentration is 10 μm of oL/L), contains dNTPs, Mg²⁺, distilled water (ddH₂O PCR) The total amount of buffer is 40.5 μ L, wherein distilled water (ddH₂O) it is 27.5 μ L.

(2) preparation of positive quality control standard items：

1. the extraction of template DNA：Respectively to B. abortus vaccine strain A19 and pig kind brucella vaccine strain S2 bacterium Liquid application DNA of bacteria extracts kit extracts DNA.

2. PCR is expanded：Using the DNA of extraction as template, according to PCR reaction systems：μ L of DNA profiling 5, the μ of archaeal dna polymerase 0.5 L, μ L, the PCR reaction conditions of PCR reaction solutions 44.5 are：94 DEG C of 3min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 are followed Ring, 72 DEG C of 10min, expands purpose fragment in PCR instrument, and PCR is identified with 1.5% agarose gel electrophoresis (120V 25min) Purpose product.

3. the foundation of the structure and positive quality control standard items of recombinant plasmid：PCR primer is subjected to gel-purified recovery, then PEASY-T3 carriers are connected, conversion, double digestion is identified after extracting plasmid, positive recombinant plasmid is sequenced, and carry out sequence Compare.The correct recombinant plasmid of sequence is the positive quality control standard items obtained.

Described positive quality control standard items include B. abortus A19 vaccine strain positive quality control standard items and pig kind cloth Shandong Salmonella S2 vaccine strain positive quality control standard items.

Described B. abortus A19 vaccine strain positive quality control standard items are by the nucleotide fragments containing 562 bases Be connected to the recombinant plasmid after pEASY-T3 carriers composition, described pig kind brucella S2 vaccine strain positive quality control standard items by Nucleotide fragments containing 539 bases are connected to the recombinant plasmid composition after pEASY-T3 carriers.

(3) negative quality control standard product：Negative quality control standard product are aseptic double-distilled water, and volume is 5 μ L.

Embodiment three, brucella vaccine strain identification reagent box specific test

Using the PCR kit of the present invention respectively to B. abortus A19, pig kind brucella S2, sheep kind cloth Lu Shi 63/290 plant of bacterium M5-90, Br.ovis, Brucellacanis RM6/66 strains, Escherichia coli O 157:H7, enterocolitis Yersinia O:9th, salmonella, staphylococcus aureus, cow mycobacteria genomic DNA are expanded, and are set positive and cloudy Property control, the specificity of checking PCR reactions, PCR reaction conditions are：94 DEG C of 5min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C 30sec, 35 circulations, 72 DEG C of 10min.Reaction carries out the purifying and sequencing of PCR primer again after terminating.

B. abortus A19 vaccine strains differentiate specific outcome as shown in figure 1, in agarose gel electrophoresis result：+ be Positive quality control standard items, 1-5：Respectively B. abortus A19, pig kind brucella S2, brucella melitensis M5-90, silk floss 63/290 plant of brucella melitensis, Brucellacanis RM6/66 strains amplify 562bp band, and-, 6-10：It is respectively cloudy Property control standard items, Escherichia coli O 157:H7, YE O:9th, salmonella, staphylococcus aureus and Cow mycobacteria is without band.Sequence and peak figure comparison result are as shown in figure 3, only at the point of A19 vaccine strains 344 after sequencing In the presence of the missing (Fig. 3-A) of 7 nucleotide sequences (AGATTTC), other brucella strains and vaccine strain result are Fig. 3-B institutes Show.

Pig kind brucella S2 vaccine strains differentiate specific outcome as shown in Fig. 2 in agarose gel electrophoresis result：+ be Positive quality control standard items, 1-5：Respectively pig kind brucella S2, B. abortus A19, brucella melitensis M5-90, silk floss 63/290 plant of brucella melitensis, Brucellacanis RM6/66 strains amplify 539bp band, and-, 6-10：It is respectively cloudy Property control standard items, Escherichia coli O 157:H7, YE O:9th, salmonella, staphylococcus aureus and Cow mycobacteria is without band.Sequence and peak figure comparison result are as shown in figure 4, only S2 vaccine strains are at 369 after sequencing The missing (Fig. 4, A) of 25 nucleotide sequences (TCCCTCATAAATGGCTTTCTTCATA), other brucella strains at point be present It is shown in Fig. 4-B with vaccine strain result.

The discriminating of example IV, brucella positive clinical sample vaccine strain

The brucella positive sample (having already passed through PCR amplifications sequence verification) preserved respectively to this laboratory, including 2 parts Blood sample, 2 parts of milk samples, the cowshed aerosol sample of 2 parts of aborted fetus tissue samples and 2 parts of different dairy cow farms are carried out The discriminating of vaccine strain, it is as a result as shown in the table.

Claims

A kind of 1. primer sets for being used to differentiate brucella wild mushroom and vaccine strain A19 and S2, it is characterised in that including：For Differentiate brucella vaccine strain A19 primer SEQ ID No.1 and SEQ ID No.2, for differentiating brucella vaccine strain S2 Primer SEQ ID No.3 and SEQ ID No.4.
2. the primer sets described in claim 1 are used for the application for preparing the kit, chip of discriminating and/detection brucella.
3. include the kit of the primer sets described in claim 1.
4. kit according to claim 3, it is characterised in that also include：PCR reaction solutions, archaeal dna polymerase, positive matter Control standard items and negative quality control standard product.
5. kit according to claim 4, it is characterised in that the positive quality control standard items include B. abortus A19 vaccine strain positive quality control standard items and pig kind brucella S2 vaccine strain positive quality control standard items.
6. kit according to claim 5, it is characterised in that the B. abortus A19 vaccine strain positive quality controls Standard items nucleotide fragments shown in SEQ ID NO.5 form with the recombinant plasmid that pEASY-T3 is formed, the pig kind cloth Lu Shi The recombinant plasmid group that bacterium S2 vaccine strain positive quality control standard items nucleotide fragments shown in SEQ ID NO.6 are formed with pEASY-T3 Into.
7. a kind of method of discriminating brucella for non-disease diagnostic purpose, it is characterised in that comprise the following steps：

（1）Extract the genomic DNA of sample to be tested；

（2）Using the sample genomic dna of extraction as template, enter performing PCR amplification, used primer is discriminating brucella vaccine Strain A19 primer SEQ ID No.1 and SEQ ID No.2, and/discriminating brucella vaccine strain S2 primer SEQ ID No.3 With SEQ ID No.4；

（3）PCR primer is taken, is detected with agarose gel electrophoresis, sees whether that B. abortus vaccine strain A19 occur draws Thing is to 562 bp and band corresponding to size, and 539 bp that/pig kind brucella vaccine strain S2 is amplified therewith that amplify And band corresponding to size therewith；

（4）If above-mentioned PCR testing results are not inconsistent with purpose band, pattern of descriptive parts is that brucella is negative, purpose band phase Symbol, then be brucella.
8. a kind of non-disease diagnostic purpose differentiates brucella street strain and vaccine strain A19 and S2 method, it is characterised in that

（1）Extract the genomic DNA of sample to be tested

（2）Using the sample genomic dna of extraction as template, enter performing PCR amplification, used primer is described in claim 1 Primer sets；

（3）PCR primer is taken, is detected with agarose gel electrophoresis, sees whether that B. abortus vaccine strain A19 occur draws Thing band corresponding to size to 562 bp that amplify and therewith, and 539 bp that amplify of pig kind brucella vaccine strain S2 and Band corresponding to size therewith；

（4）If above-mentioned PCR testing results are not inconsistent with purpose band, pattern of descriptive parts is that brucella is negative, if PCR is detected Stripe size is correct, then carries out gel extraction purifying to positive band, then carry out sequencing again；

（5）Result judgement：A19 vaccine strains identification result judges, to using SEQ ID NO:1 and SEQ ID NO:2 primer pairs are pure Change 562 bp or product corresponding to size is sequenced therewith, if sequencing result peak figure is simple spike, with SEQ ID No.5 Matched completely after comparison, then the sample is A19 vaccine strains；If sequencing result peak figure is simple spike, compared with SEQ ID No.5 7 nucleotide sequences are had more at the 344th site afterwards, then the sample is the wild poison of other brucella in addition to A19 vaccine strains Strain or vaccine strain；If sequencing result compares with SEQ ID No.5, after the sequence the 354th of peak figure, i.e. AGATTTCAGA Afterwards, there is set peak, then the sample is that A19 vaccine strains and other brucella strains mix；

S2 vaccine strains identification result judges, to using SEQ ID NO:3 and SEQ ID NO:4 primer pairs purifying 539 bp and with Size corresponding to product be sequenced, if sequencing result peak figure is simple spike, latter complete is compared with SEQ ID No.6 Match somebody with somebody, then the sample is S2 vaccine strains；If sequencing result peak figure is simple spike, in the 369th site after being compared with SEQ ID No.6 Place has more 25 nucleotide sequences, then the sample is other brucella street strains in addition to S2 vaccine strains or vaccine strain；Such as Fruit sequencing result compares with SEQ ID No.6, occurs set peak behind the site of peak figure sequence the 369th, then the sample is S2 vaccine strains Mixed with other brucella strains.
9. the methods described of claim 7 or 8, it is characterised in that the sample to be tested is blood, milk sample, tissue samples, aerosol Sample.
10. the methods described of claim 7 or 8, it is characterised in that PCR amplification conditions are：94 DEG C of 3min, 94 DEG C of 30sec, 60 DEG C 30sec, 72 DEG C of 30sec, 35 circulations, 72 DEG C of 10min.