CN101041862A - Method for sifting Holstein cow BLAD carrier and special primer and reagent case - Google Patents

Abstract

The invention discloses a sieving method of hesitant cow BLAD carrier and specific primer and agent box, wherein the primer is composed of primer couple with nucleic acid sequence in the list 1 and list 2. The sieving method comprises the following steps: adopting detected cow genome DNA as mold; proceeding PCR augumentation guided by primer; recycling PCR augumented segment; using restriction enzyme TaqI to cut the segment; detecting the segment at 193bp and 131bp as normal cow and 324bp, 193bp and 131bp DNA as BLAD carrier and 324bp DNA as BLAD ill cow; fitting for selecting superior cow and detected one with BLAD gene effectively.

Description

Examination holstein cow BLAD carrier's method and primer special and test kit

Technical field

The present invention relates to utilize PCR-RFLP technology examination holstein cow BLAD carrier's method and primer special and test kit.

Background technology

Bovine leukocyte adhesion defects disease (Bovine leukocyte adhesion deficiency, BLAD) be by the recessive inheritance disease of single-gene control on a kind of euchromosome of holstein cow, be (the Shuster D.E. that a point mutation by the CD18 gene of coding neutrophilic granulocyte surface glycoprotein causes, Bosworth B.T., Kehrli M.E.Jr.Sequence of the bovine CD18-encoding cDNA:comparison with the human andmurine glycoproteins.Gene.1992,114 (2): 267-271.).The CD18 gene has been positioned on No. 1 karyomit(e).Shuster etc. analyze 1 CD18 gene cDNA sequence of suffering from the He Sitanniu of BLAD, found that sport G from 5 ' end the 383rd bit base by A, this sudden change is missense mutation, cause 128 aspartic acid to sport glycine (D128G), cause the β of leukocyte surface ₂Integrating plain the expression obviously reduces or lacks and cause clinical onset.The physiological foundation of BLAD is the defective of leukocyte adhesion and correlation function (comprise and engulfing and chemotaxis).The BLAD pilosity is born in the holstein cow at 1-14 monthly age, and milk cow shows as morbidity when recessive mutation CD18 gene pure, and milk cow shows as normally when the recessive mutation genetic heterozygosis, but this ox is the carrier of BLAD.

The ill ox of BLAD is a typical clinical symptom with the repeatability infection, mainly shows as appetite and descends, and asoscope swelling has bate pits, the submandibular lymph nodes enlargement, and body weight obviously descends.Oral mucosa, gum and periglottis ulcer often take place in the ill ox of BLAD, symptoms such as gingival atrophy, and premolar tooth also can take place and come off in course of disease elder, and repeatability takes place and infects and general property lymphoid hyperplasia in soft tissues such as mucous membrane and enteron aisle.Ill ox is the easy infection pneumonia also; need frequent or life-time service antibiotic therapy (the Ackermann M.R. that just can survive; Kehrli M.E..Jr.and Morfitt D.C.Ventraldermatitis and vasculitis in a calf with bovine leukocyte adhesion deficiency.J.Am.Vet.Med.Assoc.1993,202:413-415.).Male calf also hematoma of scrotum can occur; recurrence after 5 days; continuous injection microbiotic 10 talentes can temporarily recover (Andrews A.H.; FishwicK J.and WatersR.J.Bovine leukocyte adhesion deficirncy (BLAD) in a one year and HolsteinFriesian bull-the first report in the united kingdom.Br.Vet.J.1996,152:347-351.).After BLAD suffers from the ox birth, the extreme difference that grows, wherein the overwhelming majority will be dead in 1 year, has only only a few can survive about 2 years, and these ill oxen not tool breeding and nurture ability, thereby caused enormous economic loss to dairy.

Find through pedigree analysis, the ill calf of the BLAD that has reported is all from a common ancestors OsborndaleIvanhoe (being born in nineteen fifty-two) (Kehrli M.E.Jr., Schimalistieg F.C., Anderson D.C., VanDer Maaten M.J., Hughes B.J., Ackermann M.R., Wilhelmsen C.L., Brown G.B., Stevens M.G.and Whetstone C.A.Molecular definition of the bovinegranulocytopathy syndrome:identification of deficiency of the Mac-1 (CD11b/CD18) glycoprotein.Am.J.Vet.Res.1990,51:1826-1836.), the production performance record of this He Sitanniu is very outstanding, frequency of utilization is high in the artificial insemination system, and OsborndaleIvanhoe, Pennstate Ivanhoe Star, be the grandfather between the Carlin-M Ivanhoe Bell three, father and son's relation, they all are the BLAD carrier, the frequency of utilization of these three outstanding breeding oxens is all very high, and it is extremely necessary therefore the He Sitan cows being cooked large-scale BLAD generaI investigation.Also in other kind ox, do not find the report of this disease at present.

Nineteen ninety, Kehrli et al finds after deliberation, this disease is to express defective (the Kehrli M.E.Jr. of institute by a kind of cell surface glycoprotein relevant with leukocyte adhesion-integration plain (CD11/CD18), Schimalistieg F.C., Anderson D.C., Van Der Maaten M.J., Hughes B.J., Ackermann M.R., WilhelmsenC.L., Brown G.B., Stevens M.G.and Whetstone C.A.Molecular definition of thebovine granulocytopathy syndrome:identification of deficiency of the Mac-1 (CD11b/CD18) glycoprotein.Am.J.Vet.Res.1990,51:1826-1836.).Integrating plain beta 2 subunit unit is CD18, and molecular mass is 95kDa.CD18 can form the plain dimer LFA-1 of different integration (Lymphocyte Function-associated antigen-1 with CD11a～CD11d (α L, α M, α X, α D) respectively, α L β 2, CD11a/CD18), Mac-1 (α M β 2, CD11b/CD18), p150,95 (α X β 2, CD11c/CD18) and α D β 2 (CD11d/CD18).These albumen all are to be made of wherein a kind of alpha subunit (CD11) and common β subunit.CD18 is expressed in all white corpuscles.The CD18 cytoplasmic domain can interact with the various kinds of cell skeleton.Integrate plain on neutrophil leucocyte, working and need Ca ²⁺Ion participates in cell second messenger's form.When stimulating with chemical substance, the Ca in the BLAD ox neutrophil leucocyte ²⁺ANOMALOUS VARIATIONS can take place in concentration.When stimulating white corpuscle with zymosan and concanavalin A, can make Ca in the healthy neutrophilic granulocyte as Nagahata et al ²⁺Concentration increases, has of short durationly to increase stage and stable increasing the stage, and the Ca in the BLAD ox neutrophil leucocyte ²⁺Concentration has only of short duration increasing the stage, lack stable (the Nagahata H. that increases the stage, Higuchi H., Noda H.and Tamoto K.Adhesiveness for extracellular matricex and Lysosomal Enzyme release fromnormal and β 2 integrin-deficient bovine neutrophils.J.Vet.Med.Sci.1996,40 (10): 783-786.).Normal of short duration to increase the stage be cell when being upset, the Ca that stores in the cell ²⁺Due to the release, stablizing and increasing the stage is to be combined with iC3b by Mac-1 (CD11b/CD18), activates Ca ²⁺Enter that the intracellular signal pathway produces, and then the outer Ca of born of the same parents ²⁺Enter in the born of the same parents.Because the CD18 function of the neutrophil leucocyte of BLAD ox morphs, and makes Ca ²⁺It is weakened and lack stable increasing the stage to enter the intracellular signal pathway.1992, Shusteret al has carried out cloning and sequencing to the CD18 gene cDNA sequence of the He Sitanniu that suffers from BLAD, and its CD18 sequence with human and rat carried out sequence alignment, found that, the base origination point sudden change that the CD18 gene of BLAD ox is the 383rd, VITAMIN B4 is replaced (A383G) by guanine, thereby cause outer 128 aspartic acids of high conservative region glycoprotein of born of the same parents to be replaced (D128G) by glycine, cause plain expression of integration of leukocyte surface obviously to be reduced or shortage, cause clinical onset.

Whether but only can't accurately locate the milk cow individuality according to pedigree analysis is the BLAD carrier, and the just deduction on the population level, and having only combines pedigree analysis with Molecular Detection can thoroughly get rid of the suspicious carrier of BLAD.Be as the criterion and make a definite diagnosis disconnected ill ox (BLAD) and carrier (BL), reduce it and be the financial loss that cattle breeding causes, be necessary to set up a kind of fast, examination BLAD carrier's method accurately and efficiently.

(Restriction Fragment Length Polymorphism RFLP) is the molecular marking technique that grows up the earliest to restriction fragment length polymorphism.After RFLP is meant the genomic dna of using the digestion with restriction enzyme Different Individual, the dna fragmentation that generation varies in size, transfer on the supporting film by electrophoresis and Southern trace, utilize the clone or the sequence of mark (probe) carry out molecular hybridization, thereby show the difference of endonuclease bamhi on length with the probe DNA sequence homologous.RFLP is marked with following characteristics: it is a kind of codominant marker for (1), to the direct somatotype of genotype, can provide information more complete on the single site; (2) no phenotypic effect, the influence that not done mutually by gene and environment; (3) stability and good reproducibility.But early stage low, the complicated operating process of RFLP technical intelligence content, and generally need radio isotope inspection, thereby this technology is restricted.

After round pcr produced, new PCR-RFLP technology was arisen at the historic moment.It is with round pcr and rflp analysis combined utilization, the method for rapid detection dna polymorphism and variation.The PCR-RFLP method adopts round pcr earlier; again with the amplified production of Disease-causing gene behind digestion with restriction enzyme, produce the fragment different with the expanding fragment length of normal gene, distinguish genotype by electrophoresis; can point out this species diversity, judge whether to exist Disease-causing gene in view of the above.

The advantage of PCR-RFLP has been to overcome the defective of a large amount of DNA of standard rflp analysis needs, can study the DNA sample of trace, and through the product of pcr amplification behind digestion with restriction enzyme, can directly detect with the EB staining, fast and convenient, unique shortcoming is the primer that needs to select and design in advance extension increasing sequence.Long for amplified fragments, have only one or the fragment that is no more than 2 point of contacts to make in this way.The PCR-RFLP technology is used comparatively extensive in inherited disease detects, for example old or the like people once with the PCR-RFLP methods analyst difference that the lipophorin gene state property of multiple dementia patient and normal population distributes in the Chinese han population (old etc., Zhang Junwu, Zhang Zhen is fragrant, Zhao Hualu, Li Xiaoqing, Wu Yaning, Qu Qiuming. apolipoprotein E gene polymorphism and alzheimer's disease. Acta Genetica Sinica .2003,30 (12): 1167-1170).

Summary of the invention

Restriction fragment length polymorphism (RFLP) the examination holstein cow BLAD carrier's method and the primer special thereof that the purpose of this invention is to provide a pair of CD18 of utilization gene.

The primer that is used for examination holstein cow BLAD carrier provided by the present invention, right by the primer that the nucleotide sequence of sequence in the sequence table 1 and sequence 2 is formed.

The primer that to be made up of the nucleotide sequence of sequence in the sequence table 1 and sequence 2 is to called after F/R, and the sequence 1 in the sequence table is by 18 based compositions, and the sequence 2 in the sequence table is by 19 based compositions.

Second purpose of the present invention provides a kind of examination holstein cow BLAD carrier's method.Examination holstein cow BLAD carrier's provided by the present invention method, be that genomic dna with ox to be measured is a template, carry out pcr amplification under the right guiding of the primer that the nucleotide sequence of sequence 1 and sequence 2 is formed in by sequence table, after amplification finishes, reclaim pcr amplified fragment, then it being carried out enzyme with restriction enzyme Taq I cuts, endonuclease bamhi is detected, cutting the ox to be measured that obtains big or small 193bp of being and 131bp dna fragmentation through enzyme is normal ox, cut the acquisition size through enzyme and be 324bp, the ox to be measured of 193bp and 131bp dna fragmentation is the BLAD carrier, and cutting the acquisition size through enzyme is the sick ox of BLAD for the ox to be measured of 324bp dna fragmentation.

In above-mentioned screening method, the source of the genomic dna of described ox to be measured is widely, can or contain the ox hair sample etc. of hair follicle from blood (fresh or freezing), seminal fluid (fresh or freezing), tissue sample (as ear tissue etc.) to extract.

Wherein, with 25 μ l cumulative volumes is example, the reaction system of pcr amplification comprises: template DNA (100ng/ μ l) 1 μ l, each 0.5 μ l of upstream and downstream primer (25pmol/L), 10 * pcr amplification damping fluid, 2.5 μ l, 4 kinds of dNTP mixtures (dNTPs, 2.5mmol/L) 0.5 μ l, Taq archaeal dna polymerase (2.5U/uL) 0.5 μ l is with redistilled water postreaction system to 25 μ l.The composition of 10 * pcr amplification damping fluid is: TrisHCl (pH8.0) 100mM, KCl 500mM, MgCl ₂20mM, 0.1% gelatin.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of insulations.

With 10 μ l cumulative volumes is example, and Taq I endonuclease reaction system comprises: pcr amplification product 8.5 μ l, Taq I restriction endonuclease damping fluid 1 μ l, Taq I restriction endonuclease (10U/ μ l) 0.5 μ l.The endonuclease reaction condition can be: digested 2-3 hour down 60-70 ℃ (being preferably 65 ℃).Available 3.0-4.0% (being preferably 3.5%) agarose gel electrophoresis detects endonuclease bamhi.

The 3rd purpose of the present invention provides a kind of examination holstein cow BLAD carrier's test kit.

Test kit provided by the present invention comprises the above-mentioned primer special that is used for examination holstein cow BLAD carrier.

According to practical situation, described test kit can comprise that also Taq archaeal dna polymerase, 10 * pcr amplification damping fluid (contain Mg ²⁺), one or more reagent in dNTPs, Taq I enzyme, Taq I restriction endonuclease damping fluid, redistilled water, 50 * TAE, bromination second pyridine stock solution and 6 * gel loading buffer etc.; The solvent of described various solution is water.

The invention provides a kind of examination holstein cow leukocyte adhesion deficiency disease (BLAD) carrier's method and primer special thereof.This method is the A/G sudden change according to the ill ox CD18 of BLAD genes encoding region sequence the 383rd site, thus the principle that Taq I restriction enzyme site is disappeared, a kind of PCR-RFLP method that detects milk cow BLAD of foundation.By a pair of primer at the design of A383G mutational site, but specific amplification goes out the fragment that contains above-mentioned point mutation that length is the milk cow CD18 gene of 324bp, again after restriction enzyme TaqI enzyme is cut, normal individual can produce the DNA band of 193bp and 131bp, the BLAD carrier then produces the DNA band of 193bp, 131bp and 324bp, and the recessive individuality that isozygotys still is 324bp.Screening method of the present invention is simple to operate, and quick, expense is cheap, accuracy height (can reach 100%), and can realize automatic direct detection.In addition, can develop the relevant detection test kit according to the bright method of we, be used to screen healthy individual, reject the BLAD carrier, this test kit has that using method is easy, quick, the sensitive advantage, does not need special instrument, is fit to the needs that Routine Test Lab detects, and detected result is reliable, stable, accurate, for the breeding work of kind of ox is provided convenience.The present invention will play a significant role in the molecule seed selection of detrimental detection of BLAD and excellent breeding cattle, have a extensive future.

Below in conjunction with specific embodiment the present invention is described in further detail.

Description of drawings

Fig. 1 is 2% agarose gel electrophoresis detected result of 60 ℃ pcr amplification product for annealing temperature

Fig. 2 is the allelotrope collection of illustrative plates and the Taq I restriction enzyme site synoptic diagram of the CD18 gene of normal individual, BLAD carrier and BLAD diseased individuals

Fig. 3 is the PCR-RFLP analytical results of the CD18 gene 324bp pcr amplified fragment of 116 china holstein cowses

Fig. 4 is the nucleotide sequence comparison result of the CD18 gene among BLAD carrier and the GenBank

Fig. 5 is the sequencing result of BLAD carrier's CD18 gene 324bp pcr amplified fragment

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.The primer synthesizes and examining order is finished by the living worker's biotechnology in Shanghai Services Co., Ltd.

The preparation of embodiment 1, examination holstein cow BLAD carrier and dedicated kit thereof

One, the design of primers that is used for examination holstein cow BLAD carrier

Ill ox of BLAD or carrier's CD18 gene sports G from 5 ' end the 1200th bit base A; Taq I restriction enzyme specific recognition T*CGA; when undergo mutation in this site; sequence becomes TCGG by T*CGA; Taq I recognition site disappears; according to this principle; with reference to Kriegesmann; portion gene group sequence (the Kriegesmann B. of the milk cow CD18 gene of B. delivering; Jansen S.; Baumgartner B.G.and Brenig B.Partialgenomic structure of the bovine CD18 gene and the refinement of test of bovineleukocyte adhesion deficiency[J] .J.Dairy.Sci.1997; 80:2547-2549.); adopt the upstream and downstream of Oligo6.0 software to design the primer that the one couple of PCR amplification comprises the nucleotide fragments in this mutational site that causes BLAD in its A/G point mutation site of the 1200th; the expection expanding fragment length is 324bp, and primer sequence is as follows:

F (upstream primer): 5 '-CCTGCATCATATCCACAG-3 ' (sequence 1 in the sequence table)

R (downstream primer): 5 '-GTTTCAGGGGAAGATGGAG-3 ' (sequence 2 in the sequence table)

New synthetic primer is solid powdery, it is concentrated at the bottom of the centrifuge tube in centrifugal 1 minute earlier before dissolving, is diluted to 25umol/L with distilled water again.

Two, the optimization of PCR reaction conditions

The bigger factor of PCR influence is comprised: the magnesium ion concentration in annealing temperature, the 10 * amplification buffer and the quality of template DNA etc.The purpose of optimizing the PCR reaction is to find and can makes the highest annealing temperature of amplification efficiency and magnesium ion concentration, and reduces the consumption of other composition in the PCR reaction system under the prerequisite that guarantees amplification efficiency as far as possible.When determining concrete annealing temperature and magnesium ion concentration, respectively the two is set a series of gradients, finally obtain optimized combination.Wherein, the optimization method of annealing temperature is as follows:

Selecting the He Sitan bull at breeding oxen station, Beijing is experiment material, according to document (Chen Huiyong. utilize Chinese He Sitan location BTA6 to go up the QTLs[D that influences milk production trait]. Beijing: China Agricultural University, 2005) (genomic dna can be from blood (fresh or freezing) for the genomic dna of the DNA extraction method of the 27th page of description extraction seminal fluid (fresh or freezing) in, tissue sample (as ear tissue etc.) or contain in the ox hair sample of hair follicle extracts), then as template, under the guiding of primer, utilize the grads PCR instrument to carry out pcr amplification to F/R, 25 μ l PCR reaction systems are: template DNA (100ng/ μ l) 1 μ l, on, each 0.5 μ l of downstream primer (25pmol/L), 10 * pcr amplification damping fluid (TrisHCl (pH8.0) 100mM, KCl 500mM, MgCl ₂20mM, 0.1% gelatin) 2.5 μ l, 4 kinds of dNTP mixtures (dNTPs, 2.5mmol/L) 0.5 μ l, Taq archaeal dna polymerase (2.5U/ul, TIANGEN Biotech (Beijing) Co., Ltd.) 0.5 μ l, ddH ₂O 20 μ l; PCR reaction conditions initial setting is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ± 10 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of insulations.After reaction finishes, the pcr amplification product of different annealing temperature is respectively got 3ul carry out the detection of 2% agarose gel electrophoresis, wherein annealing temperature is 2% agarose gel electrophoresis detected result (the swimming lane 1-6:PCR amplified production as shown in Figure 1 of 60 ℃ pcr amplification product, swimming lane M:100bp ladder Marker dna molecular amount standard), pcr amplification product length is 324bp, consistent with expected results, and amplified band is single, concentration is higher, can be used for PCR-RFLP detects, therefore, the PCR reaction conditions of optimizing is decided to be: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations; 72 ℃ are extended 7min.

Three, with PCR-RFLP method examination holstein cow BLAD carrier

1, pcr amplification and PCR product concentrates

According to pedigree, gather totally 116 parts in china holstein cows sample, wherein 68 portions of bulls freeze essence, respectively from Beijing Milk Cow Center (38), milk cow centre of development, Tianjin (18), Hebei province's herding breeding workstation (12), be stored in-80 ℃ standby; And 48 parts of cow blood samples, respectively from three in the north suburb, one in western suburb, two in western suburb, four in western suburb, eastern suburb hotly foretell, Chaoyang Nan Chang and south mouthfuls two, be stored in after the collection-80 ℃ standby.Extract that above-mentioned bull is frozen the genomic dna of essence and cow blood and as template, carry out pcr amplification at Auele Specific Primer that step 1 obtains under to the guiding of F/R, the PCR reaction system is identical with embodiment 1, and reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations; 72 ℃ are extended 7min.After reaction finishes, pcr amplification product is carried out 2% agarose gel electrophoresis detect, the result all obtains the band of 324bp size, conforms to expected results, and pcr amplification product is concentrated, and concrete grammar may further comprise the steps:

1) 2 pipes, 25 μ l PCR products is placed same 0.5mL centrifuge tube;

2) dehydrated alcohol of adding 5 μ l NaAc (3M, pH 5.2) and 2 times of volumes;

3) ice bath is 10 minutes;

4) 12000rpm is centrifugal 2 minutes;

5) discard dehydrated alcohol, vacuum is drained;

6) the DNA precipitation is dissolved in 10 μ l aqua sterilisas.

2, rflp analysis

With TaqI the pcr amplification product that step 1 obtains is carried out single endonuclease digestion, 10 μ l endonuclease reaction systems are: pcr amplification product 8.5 μ l, Taq I restriction endonuclease damping fluid 1 μ l, Taq I restriction endonuclease (10U/ μ l) 0.5 μ l, in the total reaction system, add a mineral oil, prevent volatilization.The endonuclease reaction condition is: 65 ℃ digested 2-3 hour.After endonuclease reaction finishes, enzyme is cut product carry out the detection of 3.5% agarose gel electrophoresis, method is: the pcr amplification product 6 μ l that the enzyme of learning from else's experience is cut, add 1 μ l, 6 * gel sample-loading buffer (0.25 tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene green grass or young crops, 40% (W/V) aqueous sucrose solution), be splined on 3.5% sepharose (containing the EB staining fluid), simultaneously add 5 μ l DNA 100ladder Marker in the sample hole on one point and do reference, constant voltage (8-10V/cm) electrophoresis 20-30min, on ultraviolet transilluminator, observe, take a picture with the gel imaging system of Alpha Innotech company.

According to the pcr amplification product length of CD18 gene and the position of A/G point mutation, behind the TaqI digestion with restriction enzyme, the result should show three kinds of genotype (banding pattern): 1) 193bp, 131bp (normal individual); 2) 324bp, 193bp, 131bp (BLAD carrier); 3) 324bp (BLAD diseased individuals), called after AA, AB and BB respectively.The allelotrope collection of illustrative plates of the CD18 gene of normal individual, BLAD carrier and BLAD diseased individuals and Taq I restriction enzyme site synoptic diagram be ((1) is normal individual, and (2) are the BLAD carrier, and (3) are the BLAD diseased individuals) as shown in Figure 2.The Taq I enzyme that above-mentioned 68 Chinese He Sitan bulls and 48 cows are amounted to the CD18 gene PCR amplified production of 116 china holstein cowses is cut the agarose gel electrophoresis detected result of product, and (swimming lane M is dna molecular amount standard (100bp ladder Marker) as shown in Figure 3; Swimming lane 1 is a blank; Swimming lane 2 is the pcr amplification product of cutting without enzyme as positive control; Swimming lane 3-4 is AB genotype (a BLAD heterozygote); Swimming lane 5-7 is AA genotype (a normal gene homozygote); the result has only found AA and two kinds of genotype of AB; be dominance homozygote (106 normal individuals) and heterozygote (10 BLAD carrier), do not detect allozygote (the ill ox of BLAD), i.e. the BB genotype.Calculate colony's genotype frequency and gene frequency formula and see formula 1, gene frequency and genotype frequency statistics see Table 1.

Gene frequency: p=P+R/2

q＝Q+R/2

(wherein, p:BLAD ^NGene frequency; Q:BLAD ⁿGene frequency; P:BLAD ^NNFrequency; R:BLAD ^NnFrequency; Q:BLAD ^NnFrequency; N represents normal gene, and n represents the recessive mutation gene, and NN represents the normal gene homozygote, and Nn represents heterozygote, and nn represents recessive mutation gene pure)

The genotype frequency of table 1 BLAD heterozygote and BLAD gene frequency

The sample classification	Detect number N	Carrier's number	The carrier leads (%)	Gene frequency
					It is average to freeze the essence and blood sample	68 48	2 8	2.94 16.67 8.62	0.0147 0.0834 0.0431

Four, order-checking is identified

The pcr amplification product of the 324bp of the suspicious He Sitan milk of 10 BLAD bull that step 3 is measured checks order, (the GenBank accession number is Y12672 to utilize the nucleotide sequence of the ox CD18 gene that DNAMAN3.0 software announces the dna sequence dna and the NCBI of the CD18 gene fragment that obtains then, this CD18 gene order is the normal individual sequence) carry out homology comparison, the result (goes up the behavior extension increasing sequence as shown in Figure 4, following behavior NCBI announces sequence, the arrow indication is a mutating alkali yl), homology reaches 98.33%, carry out sequential analysis with CHROMAS software again, the result as shown in Figure 4, find that amplified fragments is from 5 ' end the 193rd bit base origination point sudden change (arrow indication base N), occur bimodally, be judged as the A/G heterozygote from peak figure color.This sequencing result is consistent with the PCR-RFLP detected result of step 3, illustrates that the result is accurate.All individual bulls are repeated PCR-RFLP to be analyzed, twice experimental result is in full accord, proves that BLAD carrier's of the present invention detection method is stable, reliable results, molecular detecting method of the present invention is combined with pedigree analysis, can thoroughly get rid of the suspicious carrier of BLAD.

Five, preparation examination holstein cow BLAD carrier's test kit

The test kit that the present invention is used for examination holstein cow BLAD carrier comprises following reagent:

(1) 2.5U/ul Taq archaeal dna polymerase;

(2) 10 * pcr amplification damping fluids;

(3)2.5mmol/L dNTPs；

(4) Taq I restriction endonuclease damping fluid;

(5) 10U/ μ l Taq I restriction endonuclease;

(6) 6 * gel sample-loading buffers;

(7) primer special that is used for examination holstein cow BLAD carrier of step 1 acquisition, 25umol/L;

(8)3M NaAc(pH 5.2)；

(9)ddH ₂O。

The specification of test kit is 100 a times/box, the amount of each component is in every box: 1 bottle of 2.5U/ul Taq archaeal dna polymerase (100ul/ bottle), 1 bottle of 10 * pcr amplification damping fluid (500ul/ bottle), 2.5mmol/L 1 bottle of dNTPs (100ul/ bottle), 1 bottle of Taq I restriction endonuclease damping fluid (200ul/ bottle), 1 bottle of 6 * gel sample-loading buffer (200ul/ pipe), upstream and downstream primer each 1 bottle (100ul/ bottle), 3M NaAc (pH 5.2) 1 bottle of (1000ul/ bottle) and ddH ₂1 bottle of O (1000ul/ bottle).By above-mentioned dosage each component in the test kit is carried out packing, obtain being used for examination holstein cow BLAD carrier's test kit after the packing.

Sequence table

<160>2

<210>1

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<230>

<400>1

cctgcatcat atccacag 18

<210>2

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<230>

<400>2

gtttcagggg aagatggag 19

Claims (9)

1, the primer that is used for examination holstein cow BLAD carrier, right by the primer that the nucleotide sequence of sequence in the sequence table 1 and sequence 2 is formed.

2, a kind of examination holstein cow BLAD carrier's method, be that genomic dna with ox to be measured is a template, carrying out pcr amplification by claim 1 under the right guiding of described primer, after amplification finishes, reclaim pcr amplified fragment, then it being carried out enzyme with restriction enzyme Taq I cuts, endonuclease bamhi is detected, cutting the ox to be measured that obtains big or small 193bp of being and 131bp dna fragmentation through enzyme is normal ox, cut the acquisition size through enzyme and be 324bp, the ox to be measured of 193bp and 131bp dna fragmentation is the BLAD carrier, and cutting the acquisition size through enzyme is the sick ox of BLAD for the ox to be measured of 324bp dna fragmentation.

3, screening method according to claim 2, it is characterized in that: described 25 μ l pcr amplification reaction systems are: template DNA 1 μ l, each 0.5 μ l of upstream and downstream primer, 10 * pcr amplification damping fluid, 2.5 μ l, 4 kinds of dNTP mixture 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l is with redistilled water postreaction system to 25 μ l.

4, screening method according to claim 2 is characterized in that: described PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations; Last 72 ℃ of 7min.

5, screening method according to claim 2 is characterized in that: described 10 μ l Taq I endonuclease reaction systems are: pcr amplification product 8.5 μ l, Taq I restriction endonuclease damping fluid 1 μ l, Taq I restriction endonuclease 0.5 μ l.

6, screening method according to claim 2 is characterized in that: described endonuclease reaction condition is: digested 2-3 hour down at 60-70 ℃.

7, according to each described screening method of claim 2-6, it is characterized in that: the described method that endonuclease bamhi is detected is the 3.0-4.0% agarose gel electrophoresis.

8, a kind of examination holstein cow BLAD carrier's test kit comprises the described primer that is used for examination holstein cow BLAD carrier of claim 1.

9, test kit according to claim 8 is characterized in that: described test kit also comprises one or more reagent in Taq archaeal dna polymerase, 10 * pcr amplification damping fluid, dNTPs, Taq I enzyme, Taq I restriction endonuclease damping fluid, redistilled water, 50 * TAE, bromination second pyridine stock solution and the 6 * gel loading buffer; The solvent of described various solution is water.

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