CN104630365A - Detection marker and primer for detecting milch cow tuberculosis susceptibility - Google Patents
Detection marker and primer for detecting milch cow tuberculosis susceptibility Download PDFInfo
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Abstract
The invention relates to a detection marker and a primer for detecting milch cow tuberculosis susceptibility, and belongs to the technical field of biomedicine. According to the invention, polymorphism of BOLA-DRB3.2 gene in a PB (Peripheral Blood) genome of a milch cow is detected through the primer, an SNP locus is taken as the marker to design the primer: upstream: 5'-CTCTGTCTGTTGAGCGAAGATG-3', downstream: 5'-AGTCACGCATTGACAAAATCAC-3', the genotype of a sample is determined, the risk of the tuberculosis is evaluated through determining the genotype of the gene in the sample, so that a scheme is provided for evaluating risk of early warning of tuberculosis infection of an individual, and a method is provided for milch cow tuberculosis elimination and antituberculous healthy milch cow population breeding.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to a kind of the detection target and the primer that detect milk cow tuberculosis susceptibility, being specifically related to judge the susceptibility of milk cow to tuberculosis by detecting gene SNP site polymorphism.
Background technology
Bovine tuberculosis is the chronic debilitating transmissible disease of the important infecting both domestic animals and human caused by mycobacterium bovis, is once called as " white pestilence ".Due to latent period long (1-10), lack effectively preventing means, both effective vaccine prevention had not been had, specific pharmacological agent is not had yet, detect tuberculosis ox both at home and abroad, the method adopted is all isolation, slaughters and purify, financial loss is huge, and brings serious public health security hidden danger, brings great threat to public health security.
Announce according to the World Health Organization and food and agricultural organization, about 10% people's paratuberculosis is infected by milk prapes, in recent years, along with the appearance of Mycobacterium tuberculosis drug-resistant bacterial strain, tuberculosis is reviving again in recent years, the new cases about 800 ten thousand that whole world tuberculosis is annual, about has 3,000,000 people to die from tuberculosis every year, namely has approximately every 10 seconds a people to die from tuberculosis; China has the incidence of tuberculosis far above world average level, and be that 22 tuberculosis height bear one of country in the world, tuberculosis patient quantity occupies second place of the world.Along with the development of China's dairy in recent years, milk prapes is also on the rise.
Because the current pathogenesis to mycobacterium tuberculosis var bovis is understood very few, prevention and control fundamental research lungy receives serious restriction, carry out bovine tuberculosis resistibility and susceptibility research, the screening bovine tuberculosis susceptible excessive risk factor, for distinguishing susceptible cows and healthy cows, select healthy cows significant.
BoLA and bovine leukocyte antigen, namely major histocompatibility complex (the major histocompatibility complex of ox, MHC), also known as ajor histocompatibility complex gene, comprise the locus of a series of close linkage, height polymorphism, closely related with the function of immune system of body.BoLA-DRB3.2 gene is the exons 1 of bovine leukocyte antigen gene, is positioned on No. 23 karyomit(e)s, and coded MHC-II molecule plays very important effect in immunity system.Research shows, disease resistance and the susceptibility of BoLA-DRB3.2 gene and domestic animal have close relationship, and has now found that BoLA-DRB3.2 gene and the susceptibility of milk cow to diseases such as mastitises exist dependency.Finding the HLA gene of people and tuberculosis correlation research, there is great dependency in HLA-DRB gene and human tuberculosis's resistibility.But little for the research of BoLA-DRB3.2 gene and prapes susceptibility dependency, need further experiment to determine.
Summary of the invention
Because the current method for the detection of milk cow tuberculosis susceptibility is little, so seek a kind of newly, effective milk cow to cultivate bovine tuberculosis purification and healthy population in conjunction with the detection method of susceptibility and has a very big significance, for this problem, the invention provides the primer of a pair detection milk cow tuberculosis susceptibility.
The technical solution used in the present invention is as follows:
A first aspect of the present invention is for providing a kind of detection target for detecting milk cow tuberculosis susceptibility, i.e. BoLA-DRB3.2 gene.
Second aspect of the present invention is for providing a kind of primer for detecting milk cow tuberculosis susceptibility, namely with the rs137786474 SNP site of BOLA-DRB3.2 gene for detect target, according to this SNP site and apart from primers within the scope of end points 1-1000bp, designed primer length is between 20-25bp, and can measure the genotype of BOLA-DRB3.2 gene.
Described primer is that this primer can measure the genotype of the rs137786474 SNP site of BOLA-DRB3.2 gene with the rs137786474 SNP site of BOLA-DRB3.2 gene for detecting target design;
Described with BoLA-DRB3.2 gene r rs137786474 SNP site for the primer detecting the design of target is:
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3 SEQ ID No.1;
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3 SEQ ID No.2;
The described sequencing primer with BoLA-DRB3.2 gene rs137786474 SNP site is:
5’- CTCTGTCTGTTGAGCGAAGATG-3 SEQ ID No.1。
A third aspect of the present invention to provide primer to apply in the detection of milk cow tuberculosis susceptibility.
Specifically comprise the steps:
Step (1), the preparation of template: the genomic dna extracting tested milk cow;
Step (2), design of primers: described primer is:
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3;
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3.
Step (3), pcr amplification and agarose gel electrophoresis detect;
Step (4), PCR primer direct Sequencing without non-specific amplification band is detected to agarose gel electrophoresis, the PCR primer order-checking that agarose gel electrophoresis detects containing non-specific band is checked order after cutting glue purification again, judges according to the genotype of sequencing result to the rs137786474 SNP site of BOLA-DRB3.2 gene;
Step (5), judges the tuberculate risk of tested milk cow.
In technique scheme, described PCR amplification system is:
EasyTaq enzyme, 5 U/ μ L, 0.5 μ l;
10 × Buffer(is containing Mg
2+) 5 μ l;
Upstream and downstream primer, 10 pM, each 1 μ l;
dNTPs，2.5 mM，4μl；
Template DNA, 50 ng/ μ L, 4 μ l;
ddH
2O 34.5μl；
Cumulative volume 50 μ l.
In technique scheme, described pcr amplification program is 95 DEG C of denaturation 3min, 95 DEG C of sex change 45s, and 58 DEG C of annealing 45s, 72 DEG C are prolonged raw 1min, and carry out 35 circulations (sex change-annealing-Yan Sheng), 72 DEG C are prolonged raw 8min again, last 4 DEG C of preservations.
In technique scheme, the concrete grammar that described step (3) agarose gel electrophoresis detects is as follows: join 1%(w/v) sepharose, the product and the 1 μ L 10xloading buffer that get 5 μ L amplifications mix rear loading, and electrophoresis 25min under 120V, ultraviolet judges PCR result.(as occurred, a specificity band indicates without non-specific amplification, occurs non-specific amplification as occurred that two or more band represent)
In technique scheme, the genotypic concrete determination methods of the described rs137786474 SNP site to BOLA-DRB3.2 gene is: contrast by the base of SeqMan program in DNASTAR software to the rs137786474 site of sequence, draw the genotype in this site, genotype is divided into A/A, A/G and G/G type
In technique scheme; it is described that to judge that the concrete steps of the tuberculate risk of tested milk cow only suffer from tubercular risk as the genotype of: BoLA-DRB3.2 gene rs137786474 SNP site as the genotypic ox of A/A the highest; the tuberculous risk of G/G genotype is the genotypic 0.5-0.55 of A/A times; the tuberculate risk of A/G genotype is minimum, then according to the genotype of BoLA-DRB3.2 gene rs137786474 SNP site by cows Classification Management.
compared with prior art, its beneficial effect is in the present invention:
Designed, designed primer of the present invention; with rs137786474 SNP site for target designs primer; the BoLA-DRB3.2 gene polynorphisms in milk cow peripheral blood genomic dna is detected by this primer; measure the genotype of each sample; and assess the tuberculous risk of milk cow by the genotype measured, provide realizing individual assessment of suffering from tuberculosis early warning risk with a kind of new scheme.
accompanying drawing illustrates:
Fig. 1 is the electrophoresis detection figure of 18 tested milk cow DNA sample; Wherein M is DL2000 molecular weight marker;
Fig. 2 is the electrophoresis detection figure after 10 tested milk cow DNA sample pcr amplifications; Wherein M is DL2000 molecular weight marker.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by buying the conventional products obtained.
Contriver studies and finds that the polymorphism of BoLA-DRB3.2 gene rs137786474 SNP site is relevant to the susceptibility lungy of milk cow, can assess tuberculosis risk milk cow individuality, present invention achieves by BoLA-DRB3.2 gene rs137786474 SNP site polymorphic detection milk cow tuberculosis susceptibility.
DNA sequencing method, Restrictive fragment length polymorphism, Manganic pyrophosphate complex initiation method, Taqman probe method etc. can be adopted to BoLA-DRB3.2 gene rs137786474 SNP site genotype detection method.
Below by DNA sequencing method, the utilization of primer of the present invention is described in detail.
One. experiment material and method
1. experiment material
1.1 laboratory sample
With the milk cow of Kunming, Yunnan Province, Yuxi and Dali for research object, through the positive sample 126 parts of PPD intracutaneous transformation reactions testing inspection, negative sample 165 parts, totally 291 parts.
1.2 experiment reagent
It is complete that formula gold whole blood DNA extracts test kit, TaKaRa company DNA glue reclaims test kit.Easy Taq enzyme Gou Quanshi capital company limited, DL 2000Mark is purchased from precious biotechnology (Dalian) company limited.Gel imaging instrument, PCR amplification instrument are purchased from BIO-RAD company, and electrophoresis apparatus is purchased from Liuyi Instruments Plant, Beijing.
2. experimental technique
2.1 sample collecting
To the Niu Jinhang blood specimen collection after PPD detects.Positive is that twice PPD detects the ox blood being the positive.Negative ox is that twice PPD test is negative and cattle farm same as positive ox, same colony house, age ± the ox blood sample only of 8 years old.Sample is all anticoagulated whole blood, and in-20 DEG C of preservations.Put into ice chest and transport the storage of-80 DEG C, laboratory back.
2.2 design of primers
PCR detects BoLA-DRB3.2 gene rs137786474 SNP site polymorphism primer;
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3;
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3.
Sequencing primer for BoLA-DRB3.2 gene rs137786474 SNP site:
5’- CTCTGTCTGTTGAGCGAAGATG-3。
2.3 whole blood genomes extract
Extract test kit according to full formula gold whole blood DNA and extract DNA specification sheets requirement extraction DNA, its method is:
1. in 1.5 mL EP pipes, add 200 μ L whole bloods.
2. add 20 μ L Proteinase K solution, mixing.
3. add 220 μ l BB3, fully put upside down mixing, place 10 minutes for 56 DEG C, for several times, solution strain is limpid (as solution does not thoroughly become limpid, please extend pyrolysis time to solution is limpid) in period mixing.May white precipitate be produced when adding BB3, can disappear during general 56 DEG C of placements, can not subsequent experimental be affected.As solution does not become limpid, illustrate that lysis is thorough, may cause extracting DNA amount less and the DNA extracted impure.
4. add 220 μ L dehydrated alcohols, fully put upside down mixing, now may occur flocks.
5. all add in an adsorption column by previous step gained solution and flocks, centrifugal 30 seconds of 12000 rpm, discard effluent liquid.
6. please first check whether before adding 500 μ L CB3(uses and added dehydrated alcohol), centrifugal 30 seconds of 12000 rpm, discard effluent liquid.
7. please first check whether before adding 500 μ L WB3(uses and added dehydrated alcohol), centrifugal 30 seconds of 12000 rpm, discard effluent liquid.
8. repeating step 7 once.
9. adsorption column is put back in collection tube, centrifugal 2 minutes of 12000 rpm, thoroughly remove WB3 residual in adsorption column.
10. adsorption column is placed in a clean centrifuge tube, 100-200 μ L EB or deionized water (pH>7.0) is added in the central authorities of post, (EB or deionized water are 60-70 DEG C of water-bath preheating, better effects if), room temperature leaves standstill 1 minute, centrifugal 2 minutes of 12000 rpm, eluted dna.
11. for obtaining more DNA, carry out second time wash-out, 100-200 μ L EB or deionized water (pH>7.0) is added in the central authorities of post, (EB or deionized water are 60-70 DEG C of water-bath preheating, better effects if), room temperature leaves standstill 1 minute, centrifugal 2 minutes of 12000 rpm, eluted dna.The DNA eluted is in-20 DEG C of preservations.
12. get 5 μ L extraction DNA detects for agarose gel electrophoresis, checks extraction effect and DNA concentration; Part electrophoresis detection the results are shown in Figure 1.
2.4 pcr amplification object fragments
Object fragment is obtained according to following reaction system and reaction conditions.
PCR reaction system: EasyTaq enzyme (5 U/ μ L) 0.5 μ l, 10 × Buffer(are containing Mg
2+) 5 μ l, each 1 μ l (10 pM) of upstream and downstream primer, 2.5 mM dNTPs 4 μ l, template DNA (50 ng/ μ L) 4 μ l, ddH2O 34.5 μ l, cumulative volume 50 μ l, altogether 50 μ l.PCR reaction conditions: 95 DEG C of denaturation 3min, 95 DEG C of sex change 45s, 58 DEG C of annealing 45s, 72 DEG C are prolonged raw 1min, and carry out 35 circulations (sex change-annealing-Yan Sheng), 72 DEG C extend 8min again, last 4 DEG C of preservations; Reaction terminates rear taking-up PCR primer, and 1% agarose gel electrophoresis detects pcr amplification effect, and partial detection is shown in Fig. 2.Test strip becomes clear, can direct Sequencing without the sample of non-specific amplification band, check order again after the sample that test strip contains non-specific band should cut glue purification.
The concrete grammar that agarose gel electrophoresis detects is as follows: join 1%(w/v) sepharose, the product and the 1 μ L 6xloading buffer that get 5 μ L amplifications mix rear loading, electrophoresis 25min under 120V, ultraviolet judges PCR result, as occurred, a specificity band indicates without non-specific amplification, occurs non-specific amplification as occurred that two or more band represent.
2.5 glue recovery methods
Use TaKaRa company DNA glue to reclaim test kit and purifying is carried out to PCR primer.Operation steps is as follows.
1. to PCR reaction solution (or other enzymatic reaction solution) if in add 5 times amount Buffer DC(need add Buffer DC quantity not sufficient 100 μ l time should add 100 μ l), then Homogeneous phase mixing.
2. the Spin Column in test kit is placed on Collection Tube.
3. be transferred in Spin Column by the solution of aforesaid operations 1., centrifugal 1 minute of room temperature 12000rpm, abandons filtrate.(note) is as once centrifugal in filtrate added in Spin Column again, can improve the rate of recovery of DNA.
4. the Buffer WB of 700 μ l is added in Spin Column, room temperature 12000 rpm centrifugal 30 seconds, abandon filtrate.100% ethanol of designated volume has been added in (note) PLSCONFM Buffer WB.
5. repetitive operation step 4.
6. Spin Column is placed on Collection Tube, centrifugal 1 minute of room temperature 12000 rpm.
7. be placed in by Spin Column on the centrifuge tube of 1.5 new ml, add aqua sterilisa or the Elution Buffer of 30 μ l in the centre of Spin Column film, room temperature leaves standstill 1 minute.(note) is conducive to when aqua sterilisa or Elution Buffer being heated to 60 DEG C of uses improving elution efficiency.
8. room temperature 12000 rpm centrifugal 1 minute eluted dna.
2.6 PCR primer order-checkings
PCR primer glue is reclaimed result and carries out two-way order-checking.SeqMan program in application DNAstar software checks sequence, checks peak figure with Chromas software.Order-checking is completed by Hua Da genome company.
2.7 find SNP site and site and tuberculosis susceptibility analysis
Contrast by the base of SeqMan program in DNASTAR software to the rs137786474 site of sequence, draw the genotype in this site, genotype is divided into A/A, A/G and G/G type.The dependency of this site and tuberculosis susceptibility is analyzed by on-line analysis software SNPstats.It is the highest that BoLA-DRB3.2 gene rs137786474 SNP site suffers from tuberculosis risk.
Table 1.BoLA-DRB3.2 gene pleiomorphism and trouble tuberculosis risk
As shown in table 1, gene type is carried out by the positive ox of 126 tuberculosis and 165 negative ox contrasts of tuberculosis, the probability of each genotype relative to the positive ox of tuberculosis and the negative ox of tuberculosis is found: it is the highest that the genotypic ox of BoLA-DRB3.2 gene rs137786474 A/A only suffers from tubercular risk, the tuberculous risk of G/G genotype is genotypic 0.52 times of A/A, it is very low that risk have dropped the tuberculate risk of 48%, A/G genotype.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof
.
Sequence in the present invention:
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3 (SEQ ID No.1);
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3 (SEQ ID No.2).
Sequence in the present invention:
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3 (SEQ ID No.1);
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3 (SEQ ID No.2).
Claims (10)
1. for detecting a detection target for bovine tuberculosis, it is characterized in that: described is BOLA-DRB3.2 gene for detecting the detection target of bovine tuberculosis.
2. one kind for detecting the primer of bovine tuberculosis susceptibility, it is characterized in that: described primer with the rs137786474 SNP site of BOLA-DRB3.2 gene for detect target, according to this SNP site and apart from primers within the scope of end points 1-1000bp, designed primer length is between 20-25bp, and can measure the genotype of BOLA-DRB3.2 gene.
3. the primer for detecting bovine tuberculosis susceptibility according to claim 2, is characterized in that:
Described primer is that this primer can measure the genotype of the rs137786474 SNP site of BOLA-DRB3.2 gene with the rs137786474 SNP site of BOLA-DRB3.2 gene for detecting target design;
Described with BOLA-DRB3.2 gene rs137786474 SNP site for the primer detecting target design is:
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3;
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3.
4. the primer described in Claims 2 or 3 is detecting the application in bovine tuberculosis susceptibility.
5. primer according to claim 4 is detecting the application in bovine tuberculosis susceptibility, it is characterized in that, comprises the steps:
Step (1), the preparation of template: the genomic dna extracting tested milk cow;
Step (2), design of primers: described primer is:
Upstream primer: 5 '-CTCTGTCTGTTGAGCGAAGATG-3;
Downstream primer: 5 '-AGTCACGCATTGACAAAATCAC-3;
Step (3), pcr amplification and agarose gel electrophoresis detect;
Step (4), PCR primer direct Sequencing without non-specific amplification band is detected to agarose gel electrophoresis, the PCR primer order-checking that agarose gel electrophoresis detects containing non-specific band is checked order after cutting glue purification again, judges according to the genotype of sequencing result to the rs137786474 SNP site of BOLA-DRB3.2 gene;
Step (5), judges the tuberculate risk of tested milk cow.
6. primer according to claim 5 is detecting the application in bovine tuberculosis susceptibility, and it is characterized in that, described PCR amplification system is:
EasyTaq enzyme, 5 U/ μ L, 0.5 μ l;
10 × Buffer(is containing Mg
2+) 5 μ l;
Upstream and downstream primer, 10 pM, each 1 μ l;
dNTPs，2.5 mM，4μl；
Template DNA, 50 ng/ μ L, 4 μ l;
ddH
2O 34.5μl；
Cumulative volume 50 μ l.
7. primer according to claim 5 is detecting the application in bovine tuberculosis susceptibility, it is characterized in that, described pcr amplification program is 95 DEG C of denaturation 3min, 95 DEG C of sex change 45s, 58 DEG C of annealing 45s, 72 DEG C are prolonged raw 1min, and sex change-annealing-Yan Sheng carries out 35 circulations, 72 DEG C extend 8min again, last 4 DEG C of preservations.
8. primer according to claim 5 is detecting the application in bovine tuberculosis susceptibility, it is characterized in that, the concrete grammar that described step (3) agarose gel electrophoresis detects is as follows: join 1%(w/v) sepharose, the product and the 1 μ L 6xloading buffer that get 5 μ L amplifications mix rear loading, electrophoresis 25min under 120V, ultraviolet judges PCR result, as occurred, a specificity band indicates without non-specific amplification, occurs non-specific amplification as occurred that two or more band represent.
9. primer according to claim 5 is detecting the application in bovine tuberculosis susceptibility, it is characterized in that, the genotypic concrete determination methods of the described rs137786474 SNP site to BOLA-DRB3.2 gene is: contrast by the base of SeqMan program in DNASTAR software to the rs137786474 site of sequence, draw the genotype in this site, genotype is divided into A/A, A/G and G/G type.
10. primer according to claim 5 is detecting the application in bovine tuberculosis susceptibility; it is characterized in that; it is described that to judge that the concrete steps of the tuberculate risk of tested milk cow only suffer from tubercular risk as the genotype of: BoLA-DRB3.2 gene rs137786474 SNP site as the genotypic ox of A/A the highest; the tuberculous risk of G/G genotype is the genotypic 0.5-0.55 of A/A times; the tuberculate risk of A/G genotype is minimum, then according to the genotype of BoLA-DRB3.2 gene rs137786474 SNP site by cows Classification Management.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868113A (en) * | 2017-01-24 | 2017-06-20 | 中国疾病预防控制中心传染病预防控制所 | SNP marker and its application for identifying mycobacterium bovis |
| CN106929581A (en) * | 2017-03-21 | 2017-07-07 | 中国人民解放军第三〇九医院 | Applications of the SNP rs7576984 and rs2066802 in tuberculosis susceptibility is detected |
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| CN106868113A (en) * | 2017-01-24 | 2017-06-20 | 中国疾病预防控制中心传染病预防控制所 | SNP marker and its application for identifying mycobacterium bovis |
| CN106929581A (en) * | 2017-03-21 | 2017-07-07 | 中国人民解放军第三〇九医院 | Applications of the SNP rs7576984 and rs2066802 in tuberculosis susceptibility is detected |
| CN106929581B (en) * | 2017-03-21 | 2019-08-20 | 中国人民解放军第三〇九医院 | Application of the single nucleotide polymorphism rs7576984 and rs2066802 in detection tuberculosis susceptibility |
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