CN107099607A - It is a set of while detecting primer combination and the kit of 93 kinds of ox genetic defect genes and lethal haplotype - Google Patents

Abstract

The invention discloses a set of while detecting 93 kinds of ox genetic defect genes and the primer combination of lethal haplotype and kit.Pass through the set primer and kit, 93 kinds of genetic defect genes (such as PIRM syndromes can disposably be detected, bend tail syndrome, epidermolysis bollosa etc.) cause and effect mutational site (SNP, the insertion of short-movie section or missing) and lethal haplotype (HH1, HH3, HH4, HH5, JH1), filter out the ox of carrier and lethal haplotype carrier with monogenic inheritance dcc gene, so as to the seed selection for kind of ox and genetic evaluation, the seed selection and selective pairing of cows, population genetic is improved, rearing new variety, the protection of genetic resources is provided and accurately instructed.The features such as present invention has high flux, low cost, high accuracy, it is adaptable to beef cattle and dairy bread, can ox breeding and breeding field extensive use.

Description

It is a set of while detecting the primer combination of 93 kinds of ox genetic defect genes and lethal haplotype And kit

Technical field

The present invention relates to technical field of animal husbandry, it is related to a kind of for determining whether ox carries 93 kinds of ox genetic defect genes With the detection method of lethal haplotype, specifically, that is, DNA extractions, PCR multiplex amplifications technology, Single base extension technology are utilized With flight mass spectrum technology, Genotyping is carried out simultaneously to 93 kinds of genetic defect genes and lethal haplotype, determines whether the ox is taken Band recessive inheritance dcc gene and lethal haplotype, be related to cows recessive inheritance dcc gene and lethal haplotype rejecting and The screening technique of body mark, the seed selection and selective pairing of cows, the milk cow of beta-casein ad hoc type milk etc..

Background technology

So far, genetic defect known to ox has 506 kinds, and belong to Mendelian inheritance has 233 kinds, it is known that cause and effect is prominent What is become has 133 kinds of (http://omia.angis.org.au/home/), most numerical mutation is harmful.Some cause heredity to lack Sunken monogenic (Mendelian) mutation has been revealed.Because carrier is easy to identified and is excluded from breeding group, show Property mutation be easy to from colony remove.And the management for recessive mutation is then more difficult, sub-fraction carrier will necessarily It is stored in colony.In the case of removing homozygote (defect phenotype) according to Phenotypic Selection (natural selection or artificial selection), take Frequency with person will level off to zero, and the probability for genetic defect occur is extremely low.When top bull is the carrier of genetic defect, defect Mutation will keep higher frequency in colony, related genetic defect will turn into one it is serious the problem of, to not being pair Special dairy cattle varietal population is just more dangerous, because they have special group structure, for example, height inbreeding, less Founder, effective population size continuous decrease, artificial insemination widely use, these factors cause modern ox to genetic defect More susceptible (Pausch et al, 2015).For recessive detrimental mutation, by identifying carrier and being rejected from colony, or Person offsets the diffusion of detrimental mutation using effective seed selection and selective pairing strategy, its frequency is maintained at relatively low level.

Genetic defect can have a strong impact on the production performance and economic value of ox, and gene occurs even resulting in early stage during homozygosis extremely Die, cause great economic loss.The carrier of some genetic defect gene and lethal haplotype will influence ox production performance, Great economic loss is caused to cattle-raising.For example, the fat in the breast of deformity of spine syndrome (HHC) influence holstein cow Amount, its somatic cell score of dactylion (Mulefoot) carrying ox is higher, and breeding age and genomic breeding value are lower.He Sitan (HH0) carrier of haplotype 0 of ox is relevant with the conception rate of fat and blue or green cow, and the lethal haplotypes 1 (HH1) of He Sitan are with giving milk Amount, fat, albumen, somatic cell score, unisexuality shape breeding age are relevant with reproductive capacity, the lethal haplotype 2 (HH2) of Holstein cow with Albumen, breeding age, young ox conception rate, cow conception rate are relevant, and the lethal haplotype 4 (HH4) of Holstein cow is with giving milk, albumen Relevant with reproductive capacity, He Sitan haplotypes 5 are relevant with protein content, daughter's pregnancy rate.Jersey haplotype 1 (JH1) is imitated with phenotype Should be unrelated, but fat, albumen, breeding age, daughter's pregnancy rate, the genome of Grown cow conception rate and heifer conception rate are educated Plant value and there is significant difference；Jersey haplotype 2 (JH2) is only relevant with lower Grown cow conception rate, in milk cow colony of the U.S. In (about livestock on hand 9,000,000), every year caused by genetic defect reproductive capacity decline and calf Deaths produce economic loss About 11,000,000 dollars, wherein, about 200 dollars of gestation loss caused by every ox, the dead loss of calf getting up early is about 342 Dollar (assuming that in death in the 21st day), often extends 1 day, the loss of 2 dollars of increase, in addition, causing after calf birth or raising early The genetic defect of phase death, the economic loss that it is caused is especially serious, because its economic loss, which is far longer than, causes embryo's early stage Dead genetic defect.

The carriers of mutation of genetic defect gene is identified, the inbreeding and heredity that special control ox can be formulated in breeding lack Sunken matingplan plan, instructs seed selection and plan apolegamy, to reduce frequency of the carrier in colony, it is to avoid carrier hands over Match somebody with somebody.We can also filter out the colony for not carrying specific genetic defect gene according to testing result.For as A2 types β-junket egg White gene (beta-casein has an A1, A2, A3, B, C, D, E, F, H1, H2, I, the genetic variation type such as G), can also be filtered out specific Mutant type milk cow, sets up the milk cow colony of term single gene or combination gene homozygosis, and then cultivate new strain, kind.And Lactoglobulin AA types have high milk yield and a protein content of whey, and BB types have high casein and fat content, its BB Type is more conducive to prepare cheese product.In addition to breeding, the information of genetic defect also has important value for conservation work, it Carrier can be avoided to mate, particularly some endangered breeds, because the degree of its inbreeding is bigger, the risk of genetic defect occur Also big (Williams et al, 2016).

Carrier's specific mutation or the identification tradition of lethal haplotype are the test in laboratory by individual, for example, SNP is marked Note, microsatellite marker, casein isomers, CVM specific gene are detected and completed, it is known that genetic defect genetic test it is general It is to be detected for specific dcc gene using methods such as sequencing, round pcrs.These methods once can only typically be detected Single dcc gene, detection quantity is few, cost is high and bothersome.And the chip of low-density can disposably detect up to a hundred Kind genetic defect genotype and haplotype type, the species of the dcc gene of one-time detection is more, cost is lower, more efficiently, just Just it is and practical.

China authorizes patent of invention, CN201010225605.2, the side of detecting bovine leukocyte adhesion deficiency by applying allele specific polymerase chain reaction (AS-PCR) Method, the invention is related to the method for detecting bovine leukocyte adhesion defects in vitro using AS-PCR methods, is prepared for a kind of for ox The Specific PCR primers of adhesion defects gene C D18 detection method are used for the Disease-causing gene for carrying out ox adhesion defects in vitro Screening, the invention can only detect single genetic defect gene.

Chinese invention applies for a patent CN201510569964.2, and a kind of examination holstein cow leukocyte adhesion deficiency disease is taken Molecular detecting method with person, the competitive allele spy whether Holstein cow carries BLAD is identified the invention provides a kind of Different in nature PCR primer sets, the invention utilizes KASP technologies, by designing one group of primer for being directed to BLAD mutational sites, carries out anti- Should, analysis is easily recognized by supporting software progress after about 1.5 hours, you can accurately obtain the base of all testing samples Because of type, so as to realize the purpose of examination BLAD carrier, the invention can only detect single BLAD genetic defect genes.

Chinese invention patent application CN201610262293.X, a kind of nucleic acid mass spectrum for being used to detect four kinds of hereditary diseases of milk cow Detection product and application, disclosure of the invention one kind can detect simultaneously milk cow whether have spine malformation syndrome (CVM), Leukocyte adhesion deficiency disease (BLAD), uridine monophosphate synthase deficiency disease (DUMPS), the primer of citrullinemia (CN) four kinds of hereditary diseases System, based on the product prepared by the primer system, can only be realized simultaneously to milk cow CVM, BLAD, DUMPS, CN4 kind hereditary disease Pleomorphism site is detected at the 4 of gene-correlation.

China authorizes patent of invention, CN200610150317.9, a kind of method of detection CVM deleterious gene of oxen, the invention There is provided a kind of method of detection ox spine malformation syndrome (CVM) carrier, this method designs synthetic primer to ox first DNA sample carries out PCR (PCR), and then carrying out single-strand conformation polymorphism (SSCP) to PCR primer analyzes, according to Polyacrylamide gel electrophoresis result judges whether ox carries CVM deleterious genes, and its weak point is can only to detect single CVM Carriage, and repeatability is poor.

China authorizes patent of invention, CN201310617444.5, a kind of bovine uridine monophosphate synthase deficiency disease deleterious gene detection Primer composition and its kit and application, a kind of bovine uridine monophosphate synthase deficiency disease deleterious gene detection of the disclosure of the invention Primer composition and its kit and application, the method for the detection of the invention is complete group of DNA conduct in extraction bovine blood first Template, carries out nest-type PRC (NestedPCR) amplification, and gained PCR primer is sequenced, can be directly acquainted with according to sequencing result prominent Become site base change, be characterized in, it is necessary to twice expand and product is sequenced, be easily introduced mistake, and can only detect Single dcc gene.

China authorizes patent of invention, and CN200910272954.7 has invented a kind of application AS-PCR detections bovine uridine monophosphate and closed The method of enzyme deficiency disease, the invention belongs to Animal diseases detection technique field, and in particular to application AS-PCR detects bovine uridine monophosphate The method of synthase deficiency disease, is prepared for a kind of Specific PCR primers for bovine uridine monophosphate synthase deficiency disease detection method, is expanding Increase production the middle introducing base mismatch of thing, it is necessary to carry out Genotyping using electrophoresis, because product length is shorter, be easily caused misjudgement, And unsuitable high flux is obtained, the detection of substantial amounts of cows.

China authorizes patent of invention CN201110440150.0, discloses a kind of bovine degenerative axonopathy carrier screening Method and its kit, the invention is using the genomic DNA of ox to be measured as template, and design sense primer and anti-sense primer are carried out to it Pcr amplification reaction, then reclaims pcr amplified fragment, digestion is carried out with FastDigest restriction enzymes TspRI, in endonuclease reaction body It is to carry out gel detection after digestion, the screening method is simple to operate, quick, low-cost but equally exists and can only detect single The deficiency of genetic defect gene.

It there is no relevant other genetic defect genes of detection and lethal haplotype technique study both at home and abroad at present, it is more same without energy When detect the high-flux detection method research of hundreds of known ox genetic defect gene cause and effect mutation.

The content of the invention

The problem of existing for prior art, the present inventor incorporates 93 kinds of ox genetic deficiency diseases by performing creative labour Cause and effect mutational site (SNP, the insertion of short-movie section or missing) information, obtained can be used at the same detect Niu Butong heredity lack Fall into primer combination and its kit of disease.The high specificity of primer combination and its kit, sensitivity are high, can quickly, Accurately 93 kinds of ox genetic deficiency diseases of detection, and can once testing while nearly hundred parts of milk cow samples are detected, with quick inspection Survey, the characteristics of flux is high.

Specifically, the present invention relates to following technical scheme：

First, the invention discloses a set of while detecting drawing for 93 kinds of the ox related cause-effect mutational site of genetic defect 101 Thing, the primer is divided into four groups, specifically as shown in table 1- tables 4.

The present invention will detect drawing needed for 93 kinds of genetic defect genes, 101 cause and effect mutational sites related to lethal haplotype Thing combination is divided into 4 detection holes, while being detected to different genetic deficiency diseases.Each hole (1,2,3,4) described primer Composite sequence is as follows：

1 hole primer combination：For the PIRM syndromes of detection ox, spherocytosis, β caseins 8, β caseins simultaneously 4th, I type leukocyte adhesion deficiency levy, Qi-east syndrome, recessive red hair, β caseins 9, double fleshes, β caseins 1, congenital fish scale Sick, lethal haplotype (HH1), lethal haplotype (HH3), lethal haplotype (HH4), dyschondroplasia nanism, backbone are abnormal Shape syndrome, achondrogenesis, citrullinemia, Angus nanism, acrodermatitis enteropathica, miscarriage SLC37A2, life Long anhormonia type nanism, cardiomyopathy and the long defect of continuous shape, the nanism genetic defect base with inflammatory lesion perspec-tive The primer combination of cause.Its sequence and relevant information are as shown in table 1 below：

Table 1

2 hole primer combinations：For detecting that a variety of eye defects, bending tail syndrome, the α type mannosides of ox are stored up simultaneously Disease, miscarriage (JH1), degeneration aixs cylinder disease, male sterility, the abnormal low expression of beta lactoglobulin, lethal haplotype (HH5), β junket eggs White 11, anhidrotic ectodermal dysplasia, yellow fat disease, lactoglobulin, the SHBG that miscarries, perinatal period weak calf syndrome, congenital It is myodystony I types, zinc deficiency sample syndrome, dactylion, proteoglycan type nanism, the depauperation of lethal multiple organ, dominant In vain with bilateral deafness, trimethyl aminuria disease, Mao Shaose shallow, spider legs syndrome, congenital myasthenia, recessive red hair 373, β junket The primer combination of the genetic defect gene of albumen 6.Its sequence and relevant information are as shown in table 2 below：

Table 2

3 hole primer combinations：For the epidermolysis bollosa of detection ox, β types mannosidosis, spinal cord marrow simultaneously It is sheath formation obstacle, thrombopathia, mucopolysaccharidosis type ⅢB, acerous/to have angle, recessive red hair EBR, polymelia, horse all comprehensive Simulator sickness, recessive red hair 358, protoporphyria, β caseins 12, goitre, α types mannosidosis, maple syrup urine disease-H, egg White glycan nanism, congenital pseudomyotonia, two class lithoxidurias, dominant red hair, forelimb are to waist muscle is abnormal, nutrition Not good figure epidermolysis bollosa, five type glycogen storage diseases, the primer combination of Duchenne-Arandisease genetic defect gene.Its sequence Row and relevant information are as shown in table 3 below：

Table 3

4 hole primer combinations：β caseins 3, recessive red hair, congenital pseudomyotonia, Ai Lai for detecting ox simultaneously Er-when Lip river syndrome, light hypotrichosis, two type glycogen storage diseases (Pompe), haemophilia A, β caseins 7, gum paramnesia Knurl type osteopetrosis, Kappa caseins, dilated cardiomyopathy, two type glycogen storage diseases (Pompe), congenital Muscle tensility barrier Hinder II type, β caseins 2, hypotrichosis, uridine phosphate synthase deficiency disease, F11 factors shortage, the heredity of β caseins 5, dermatosparaxis Dcc gene primer is combined.Its sequence and relevant information are as shown in table 4 below：

Table 4

The present invention mentality of designing be：Covering comprehensively as far as possible known to ox genetic defect gene and lethal haplotype On the premise of cause and effect is mutated, design it is a kind of efficiently, can apply and cost alap ox genetic defect gene cause and effect mutation High-flux detection method.The present invention is based on the corresponding cause and effect mutational site sequence information of Niu Butong genetic deficiency diseases, by uniqueness Design and Combinatorial Optimization, obtain pcr amplification reaction primer and single base extension primer, then in the enterprising performing PCR reaction of 384 orifice plates The purpose fragment in sample mutational site to be checked is amplified, then purification reaction is carried out to PCR primer with shrimp alkaline phosphotase, is finally used The product of single base extension primer pair PCR purifying carries out single base extension on 384 orifice plates and carries out Mass Spectrometer Method, so that Obtain the genotype in mutational site.

High flux detection primer of the present invention and the design of packet mainly include the control point of three aspects.

(1) for the selection of ox genetic defect gene and lethal haplotype, the ox that inventor's early stage is mutated to known cause and effect Genetic defect gene and lethal haplotype are analyzed, and find to be directed to osteopetrosis, short vertebra syndrome (HH0), gonad development The cause and effect mutational site information of the genetic deficiency diseases of ox such as bad, renal dysplasia, miscarriage, Rome Nola's beef cattle cataract, Insertion or the missing of the cause and effect mutation of this several ox genetic deficiency diseases are found during assessment design design of primers, leads to not set Count primer and carry out Classification Identification, the detection primer design and 93 kinds of ox genetic deficiency diseases of the invention of preliminary experiment 6 kinds of genetic defectes Design of primers conformability extreme difference, through analyzing 6 kinds of genetic defectes or the insertion for large fragment or missing, or detection primer designs simultaneous Capacitive is too poor (positive-sense strand and antisense strand base design can not meet requirement), in contrast, muscle hypertrophy (double fleshes), cardiomyopathy and The continuous long defect of shape, anhidrotic ectodermal dysplasia, Mao Shaose shallow, spider legs syndrome, congenital myasthenia, recessive red hair 373rd, several fragments such as two class lithoxidurias, two type glycogen storage diseases, Pompe, F11 factors shortage, dermatosparaxis is slotting Enter or deleting genetic dcc gene design of primers can effectively with many SNP site multi-primerses design integrations of the invention and simultaneous Hold.The present invention realizes many insertions or deletion mutation detection combines the design of primers that many SNP are detected.

(2) for 93 kinds of ox genetic deficiency diseases of the invention, 101 cause and effect mutational sites is related to altogether, are mainly faced It is the integration difficulty of the limit.Usual multiplex PCR combines fewer its of number of loci in multiple single base Amplification and mutually infected Possibility is lower, and bit number of points of the present invention is more, therefore screening combination and optimization from 101 sites, through preliminary screening, every group About 25 weights are more excellent up and down, and the investigation of primer is extended further combined with multiple single base, the design of 4 hole detection primers has been primarily determined that Target, and carry out packet and trial and error analysis for dcc gene；Big because integrating difficulty, inventor's early stage arranges its related locus letter After breath, issue Beijing biotech firm and carry out primer assessment and design using software, feedback analysis finds that the biotech firm can set Count out 87 mutational sites of detection primer combination (another 14 sites can not be integrated, including dactylion, trimethyl aminuria disease, Recessive red hair 373, mucopolysaccharidosis type ⅢB, congenital ichthyosis, zinc deficiency sample syndrome, dystrophic epidermolysis table Skin loosen disease, gum hamartoma type osteopetrosis, β caseins 3, β caseins 11, β caseins 12, β types mannosidosis, Anhidrotic ectodermal dysplasia, haemophilia A), and need to be divided into 5 holes and detected；On this basis, inventor enters Packet and integration (specific packet situation is shown in Table 1-4) that the unique strategy of row is carried out, redesign out covering 101 of the present invention The 4 hole detection primers in individual cause and effect mutational site.

(3) because covering, site is more, detection difficulty big, specificity and the detection accuracy progress of the present invention emphatically to primer Investigate, the present invention uses AssayDesigner3.1 Software for Design to multiple PCR primer, by the primer to synthesis uses matter (because site is too many, the present invention is to there is the primer pair of impurity to carry out to ensure to expand accurate and free from admixture for spectrum detection molecules amount Reject), after the present invention is detected to the primer of Software for Design, wherein trial and error is analyzed, most of primer pair composite theory molecule Amount, 12 pairs have impurity, and 2 pairs of molecular weight mistakes, inventor is redesigned and detected to it, obtain multiple PCR primer.Herein On the basis of, inventor is further designed and optimized to Single base extension primer, and feedback adjustment multiple PCR primer pair, most Primer sets of the present invention (table 1- tables 4) are formd eventually.

Secondly, the invention discloses the detection kit for including above-mentioned primer sets.

In preferred embodiment, the kit also include pcr amplification reaction reagent, alkaline phosphatase treatment reagent, Single base extension.

Specifically, pcr amplification reaction reagent includes dNTP (25mM each), MgCl₂(25mM)、HotStar Taq(5U/ μl)、PCR Bμffer、ddH₂O etc.；

Alkaline phosphatase treatment reagent includes ddH₂O, shrimp alkaline phosphotase Buffer, shrimp alkaline phosphotase (1U/ μ l)；

Single base extension includes：iPLEX Bμffer Plus、iPLEX Termination mix、iPLEX Enzyme、ddH₂O。

In addition, the invention discloses a kind of while the method for 93 kinds of ox genetic defect genes of detection, comprises the following steps：

(1) ox DNA is extracted；In preferred embodiment DNA is extracted from bovine blood, seminal fluid or hair follicle；

(2) multi-PRC reaction：Pcr amplification reaction is carried out using using above-mentioned primer and step (1) DNA；Specifically , pcr amplification reaction is carried out in 384 holes using the primer and DNA that have diluted, pcr amplification reaction uses 5.000 μ l systems, Constitute and be：1 μ l DNA (20ng/ μ l), 1.000 μ l amplimers mixtures (500nM each), 0.100 μ l dNTP (25mM each)、0.325μl MgCl2(25mM)、0.100μl HotStar Taq(5U/μl)、0.625μl PCR Bμffer with 15mM MgCl2、1.850μl ddH2O；

In preferred embodiment, pcr amplification reaction program is：First 94 DEG C of 5min；Then 94 DEG C of 20sec, 56 DEG C 30sec, 72 DEG C of 1min, 45 circulations of this step；Last 72 DEG C of 3min；

(3) product alkaline phosphatase treatment, removes remaining dNTPs, specifically, in upper step each hole 5.000 μ l PCR primer respectively adds 2.00 μ l shrimp alkaline phosphotases to handle, and removes remaining dNTPs,

It is preferred that, wherein, 2.00 μ l shrimp alkaline phosphotases processing reaction solution composition is：1.53μl ddH₂O, 0.17 μ l shrimps Alkaline phosphatase Buffer, 0.30 μ l shrimp alkaline phosphotases (1U/ μ l)；

Wherein, 7 μ l product alkaline phosphatase treatment liquid response procedures are：37℃20min；85℃5min；4℃∞；

(4) single base extension, single base extension is carried out using above-mentioned primer, specific as follows：Shrimp alkaline phosphatase Product after ferment treatment, carries out single base extension, the reaction system and program of optimization are as follows：

Wherein, single base extension uses 9 μ l reaction systems, constitutes and is：7 μ l product alkaline phosphatase treatments products, 0.200 μ l iPLEX B μ ffer Plus, 0.200 μ l iPLEX Termination mix, 0.940 μ l extension primer mixtures (7μM：14μM)、0.041μl iPLEX Enzyme、0.619μl ddH2O；

Wherein, the program of single base extension is：First 94 DEG C of 30sec；Then 94 DEG C of 5sec, (52 DEG C of 5sec, 80 DEG C 5sec, 5 circulations), 40 circulations of this step；72℃3min；

(5) purifying resin reaction is carried out after single base extension, it is specific as follows：Uniformly filled in 384Dimple plates Resin simultaneously places 10min, and in the μ l water of each Kong Zhongjia 16 of 384 sample planes, rapping Dimple makes resin fall into the every of sample plane In individual hole, sample version room temperature is then rotated into 30min.

(6) Mass Spectrometer Method and data output are utilized, concrete operations are as follows：With point sample instrument by the extension products after purifying resin Move on 384 hole SpectroCHIP chips, then it is analyzed using MALDI-TOF mass spectrographs, it is as a result soft using TYPER4.0 Part carries out data processing, according to the size of wild different extension products molecular weight corresponding with mutational site, obtains mutational site Parting information.

The present invention is also public to have opened a kind of method of cattle genetics and breeding, and genetic defect gene is removed using above method screening Homozygosis and/carrier, supporting seed selection and selective pairing technology optimize ox breeding.

The present invention achieves following beneficial effect：

(1) present invention is directed to a variety of genetic defect genes of ox and lethal haplotype technique study first, and one is proposed first Plant the high-flux detection method for detecting that ox genetic defect gene cause and effect known to nearly hundred kinds is mutated simultaneously.

(2) detection method of the invention can detect 93 kinds of ox genetic deficiency diseases, 101 cause and effect mutational sites, and 4 holes simultaneously Detection can cover 101 cause and effect mutational sites, and the disposable detection sample throughput of the present invention is high, expense is low, and develops inspection Test agent box, makes operation easier, quick.

(3) ox of the invention to pass through the genetic deficiency diseases carrier that direct Sequencing is identified, can be accurate as detection object Ox genetic deficiency diseases genotype (Fig. 1-Fig. 2) really is detected, shows primer, reaction system, condition and the detecting step of the present invention Strong etc. accurate and effective, repeated high, stability, this invention can carry out early stage (birth) detection to ox genetic deficiency diseases, or Person is predicted to the genetic defect situation of biparental progeny, eliminates the economic loss that deleterious gene type is brought to cattle-raising, can Produce significant economic and social benefit.

Brief description of the drawings

Fig. 1：The ox of the genetic defect carrier of deformity of spine syndrome (HHC) direct Sequencing and the identification of the inventive method As a result, left figure is sequencing result, and right figure is analysis result of the present invention；

Fig. 2：The ox of the genetic defect carrier of β casein A1/A2 heterozygous direct Sequencings and the identification of the inventive method As a result, left figure is sequencing result, and right figure is analysis result of the present invention；

Fig. 3：The testing result of the lethal haplotype of ox (HH1) carrier

Fig. 4：The testing result of the recessive red carrier of hair 373 of ox

Fig. 5：The testing result of the lethal haplotype of ox (HH5) carrier

Fig. 6：The testing result of cow-bezoar fat disease carrier

Fig. 7：The testing result of the recessive red hair EBR carrier of ox

Fig. 8：The recessive red carrier of hair 358 of ox and the testing result of homozygous mutation, upper figure is carrier, and figure below is prominent for homozygosis Become

Fig. 9：The testing result of ox maple syrup urine disease homozygous mutation

Figure 10：The testing result of cow's milk globulin different shaped, upper figure is AA types, and middle figure is AB types, and figure below is BB types

Embodiment

With reference to specific embodiment, the present invention is further illustrated.

Embodiment 1：The method of 93 kinds of ox genetic defect genes of detection is built simultaneously

1.PCR amplification primers are designed and condition optimizing

SNP sequence informations are arranged into reference format, carrying out 93 cause and effect mutational sites, (SNP, the insertion of short-movie section lack Lose) design of pcr amplification reaction primer and Single base extension primer, to influence detection reaction system condition and PCR procedure conditions Optimize, including reagent concentration, temperature, time, reaction cycle number etc., then carry out the PCR reactions in 384 holes (93 causes and effects are dashed forward Become site and be divided into 4 PCR holes detection simultaneously).Pcr amplification reaction is specific as follows：

(1) design of primers of each mutational site sequence, analysis optimization examination are carried out using AssayDesigner3.1 softwares Primer after testing, mixes the PCR primer in each hole needed for detection mutational site with deionized water respectively so that final each hole In primer mixture concentration be 0.5 μM.

(2) DNA is extracted from bovine blood, seminal fluid or hair follicle, with its OD value of Instrument measuring and 1.25% Ago-Gel electricity After swimming detection is qualified, 96 orifice plates are transferred to, -20 DEG C store for future use.

(3) pcr amplification reaction is carried out in 384 holes using the primer and sample DNA that have diluted, it is as follows：

Pcr amplification reaction uses 5.000 μ l systems, constitutes and is：1 μ l DNA (20ng/ μ l), 1.000 μ l amplimers are mixed Compound (500nM each), 0.100 μ l dNTP (25mM each), 0.325 μ l MgCl₂(25mM)、0.100μl HotStar Taq(5U/μl)、0.625μl PCR Bμffer with 15mM MgCl₂、1.850μl ddH₂O。

Pcr amplification reaction program is：First 94 DEG C of 5min；Then 94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min, this step 45 Individual circulation；Last 72 DEG C of 3min.

2. product alkaline phosphatase treatment, as follows：

The 5.000 μ l PCR primers in each hole respectively add 2.00 μ l shrimp alkaline phosphotases to handle in upper step, remove remaining DNTPs, it is specific as follows：

Wherein, 2.00 μ l shrimp alkaline phosphotases processing reaction solution composition is：1.53μl ddH₂O, 0.17 μ l shrimp alkaline phosphatases Enzyme Buffer, 0.30 μ l shrimp alkaline phosphotases (1U/ μ l).

Wherein, 7 μ l product alkaline phosphatase treatment liquid response procedures are：37℃20min；85℃5min；4℃∞.

3. single base extension, as follows：

Product after shrimp alkaline phosphotase processing, progress single base extension, the reaction system and program of optimization, such as Under：

Wherein, single base extension uses 9 μ l reaction systems, constitutes and is：7 μ l product alkaline phosphatase treatments products, 0.200 μ l iPLEX B μ ffer Plus, 0.200 μ l iPLEX Termination mix, 0.940 μ l extension primer mixtures (7μM：14μM)、0.041μl iPLEX Enzyme、0.619μl ddH₂O。

Wherein, the program of single base extension is：First 94 DEG C of 30sec；Then 94 DEG C of 5sec, (52 DEG C of 5sec, 80 DEG C 5sec, 5 circulations), 40 circulations of this step；72℃3min.

4. purifying resin reacts, as follows：

(1) uniform potting resin and 10min is placed in 384Dimple plates.

(2) in the μ l water of each Kong Zhongjia 16 of 384 sample planes.

(3) rapping Dimple makes resin fall into each hole of sample plane, and sample version room temperature then is rotated into 30min.

5. Mass Spectrometer Method and data output, as follows：

The extension products after purifying resin are moved on 384 hole SpectroCHIP chips with point sample instrument, then used MALDI-TOF mass spectrographs are analyzed it, as a result carry out data processing using TYPER4.0 softwares, according to wild and mutational site pair The size for the different extension products molecular weight answered, obtains mutational site parting information.

Embodiment 2：Kit for detecting 93 kinds of ox genetic defect genes

The composition of kit includes：1-4 holes amplimer mixture (500nM each), 1-4 holes extension primer mixture (7μM：14μM)、dNTP(25mM each)、MgCl₂(25mM)、0.100μl HotStar Taq(5U/μl)、PCR Bμffer with 15mM MgCl₂, shrimp alkaline phosphotase Buffer, shrimp alkaline phosphotase (1U/ μ l), iPLEX B μ ffer Plus, iPLEX Termination mix、iPLEX Enzyme、ddH₂O, 384Dimple plate, 384 sample planes, resin.

Wherein, the amplimer mixture and extension primer mixture described in kit, for example above-mentioned content of the invention of its sequence Part is described.Use the kit carry out ox tissue samples detecting step for：

(1) performing PCR amplification is entered using the DNA in the reagent in kit bovine blood, seminal fluid or the hair follicle qualified to Quality Control Reaction, 93 kinds of ox genetic defect cause and effect mutational sites of each sample are divided into 4 holes, detected in 384 holes, reacting hole Condition is specific as follows：

Pcr amplification reaction uses 5.000 μ l systems, constitutes and is：1 μ l DNA (20ng/ μ l), 1.000 μ l amplimers are mixed Compound (500nM each), 0.100 μ l dNTP (25mM each), 0.325 μ l MgCl₂(25mM)、0.100μl HotStar Taq(5U/μl)、0.625μl PCR Bμffer with 15mM MgCl₂、1.850μl ddH₂O。

Pcr amplification reaction program is：First 94 DEG C of 5min；Then 94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min, this step 45 Individual circulation；Last 72 DEG C of 3min.

(2) the 5.000 μ l PCR primers in each hole respectively add the processing of 2.00 μ l shrimp alkaline phosphotases reaction solutions (composition are： 1.53μl ddH₂O, 0.17 μ l shrimp alkaline phosphotases Buffer, 0.30 μ l shrimp alkaline phosphotases (1U/ μ l)), remove remaining DNTPs, 7 μ l product alkaline phosphatase treatment liquid response procedures are：37℃20min；85℃5min；4℃∞.

(3) after shrimp alkaline phosphotase processing, single base extension is carried out, it is as follows：

The μ l reaction systems of single base extension 9, constitute and are：7 μ l product alkaline phosphatase treatments liquid, 0.200 μ l IPLEX B μ ffer Plus, 0.200 μ l iPLEX Termination mix, 0.940 (7 μM of μ l extension primers mixture：14μ M)、0.041μl iPLEX Enzyme、0.619μl ddH₂O。

The program of single base extension is：First 94 DEG C of 30sec；Then 94 DEG C of 5sec, (52 DEG C of 5sec, 80 DEG C of 5sec, 5 Individual circulation), 40 circulations of this step；72℃3min.

(4) uniform potting resin and 10min is placed in 384 Dimple plates；In the μ of each Kong Zhongjia 16 of 384 sample planes L water；Rapping Dimple makes resin fall into each hole of sample plane, and sample version room temperature then is rotated into 30min.

(5) extension products after purifying resin are moved on 384 hole SpectroCHIP chips with point sample instrument, then used MALDI-TOF mass spectrographs are analyzed it, as a result carry out data processing using TYPER4.0 softwares, according to wild and mutational site pair The size for the different extension products molecular weight answered, obtains site parting information.

Embodiment 3：The pattern detection of ox difference genetic defect gene and characteristic milk gene

The blood sample of 96 holstein cows is gathered, the reagent and its phase in the kit of the embodiment of the present invention 2 is utilized Step is closed, the cause and effect mutational site (SNP, the insertion of short-movie section or missing) of the genetic deficiency diseases of milk cow sample is detected.

As a result find that part ox carries (heterozygosis) genetic defect gene and lethal haplotype (Fig. 3-Figure 10), and tested through sequencing Card.The genotype call results of specific sample are as shown in table 5 below：

5. Ns of genetic defect genes of table and lethal haplotype (phenotypic character) testing result

Genetic defect (phenotypic character)	Sample number (genotype)
		Lethal haplotype (HH1)	3 carryings
Lethal haplotype (HH3)	7 carryings
		Deformity of spine syndrome (HHC)	4 carryings
Recessive red hair 373	2 carryings
		Lethal haplotype (HH5)	4 carryings
Yellow fat disease	3 carryings
		Recessive red hair EBR	4 carryings
Recessive red hair 358	12 carryings, 1 homozygous mutation
		Maple syrup urine disease	1 homozygous mutation
Light hypotrichosis	8 carryings, 1 homozygous mutation
		Lactoglobulin	23 AA, 25 BB, 48 carryings
β caseins	35 A2,11 A1,49 carryings

SEQUENCE LISTING

<110>Cow Research Center, Shandong Academy of Agricultural Sciences

<120>It is a set of while detecting primer combination and the kit of 93 kinds of ox genetic defect genes and lethal haplotype

<130>

<160> 303

<170> PatentIn version 3.5

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acgttggatg actggaaata gaaaccccgc 30

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acgttggatg acgtcaccca catcaccatc 30

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cgacaagctc ctcac 15

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acgttggatg aggcctctct gtatctaacc 30

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acgttggatg gcaaacatca tccagatggg 30

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tctaacccca cggag 15

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acgttggatg aaaatctgca ccttcctctg 30

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acgttggatg gactgaggag gaaacatgac 30

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cactcaccag cctcttc 17

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acgttggatg tccctcctct tactcaaacc 30

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acgttggatg ccatagcctc cttcactttg 30

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ttgtggtgcc gcctttc 17

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acgttggatg ttgacgttga cgaggtcatc 30

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acgttggatg ttcaatgtga ccttccggag 30

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ccatcaggta gtacagg 17

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acgttggatg gctgctgtgt acagagtatg 30

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acgttggatg ctcaagggta atctgcactg 30

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gagtatgtgt gtgtggc 17

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acgttggatg atgtcatcga cgtgctcatc 30

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acgttggatg agaagatgga gatgtagcgg 30

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gcttcctggg tgccattg 18

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acgttggatg agaggaggga tgttttgtgg 30

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acgttggatg taaaatccac ccctttgccc 30

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ttggtgggag gctgttag 18

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acgttggatg agttagaggg taacgacagc 30

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acgttggatg agtcaaggta acagacacac 30

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cacgacagca tcgagattc 19

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acgttggatg caaattacct ctgtttgctg 30

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acgttggatg ataaacagca tttctttatg 30

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ttcggtttgc tgctgttcct 20

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acgttggatg acatgttaac tcgcaggagg 30

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acgttggatg acaggtatgg tatgatccag 30

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tgatgtaagc tggaagggag 20

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acgttggatg tgaaggtgtc ctctatgctg 30

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acgttggatg ttactcactt ggcactctgg 30

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gacccaaagt tctggaattt a 21

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acgttggatg atattgtgca actgggcctc 30

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acgttggatg attccgttat cgacctcctg 30

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gaaacttcag aggtttatcg g 21

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acgttggatg tctgaggctc tttttggttc 30

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acgttggatg gccctggatc tttctcatac 30

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ggtcttacct gagaatgtgt g 21

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acgttggatg tacagggtca caaaggtgtc 30

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acgttggatg accctagttt gagagaggag 30

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ttgactcttc tagcacgaca ct 22

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acgttggatg aagtaaaccc cagcaaagcc 30

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acgttggatg aaggaacttt cagctggctc 30

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gggtaaaaac atgctgtgag aa 22

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acgttggatg cctttgtacc ccttcctcag 30

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acgttggatg tggaccccct ggcgagaag 29

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ctttacagaa ggaccatcat ctc 23

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acgttggatg ttgcttggtg cacaagatgg 30

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acgttggatg taaggccaat tactgctctg 30

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acttttgcaa aaatacaaat tca 23

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acgttggatg tgggcgatct ccacctgctt 30

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acgttggatg atccagtcca gcgcactgta 30

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gcgcgagagg tgcccaggag gta 23

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acgttggatg acaatctgat tctcaaaggc 30

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acgttggatg attctcacct gcaaagcctc 30

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tgcccagaaa gataacaaga cgac 24

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acgttggatg agccacaccc agaccccag 29

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acgttggatg gtgttctgcc acgaggtgc 29

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ggggatggaa ccccgcaagg ctca 24

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acgttggatg actctctgct aacccagatg 30

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acgttggatg acatgtgata gcaggtgtag 30

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gatgaccacc ttcccctgca ggtac 25

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acgttggatg tccggaagga cctgcataag 30

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acgttggatg agatggctgg caactagaag 30

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aggacggacc tgcataagac ggaga 25

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acgttggatg taatgattcc cgcctcacag 30

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acgttggatg tgctctctct ccagggtaac 30

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ccgtatggat gtcttcccgc ggcctg 26

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acgttggatg gaacaaaaac ttaagcaag 29

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acgttggatg agccccatga tgagtttgag 30

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taacaaaaac ttaagcaaga tacatg 26

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acgttggatg cgtcgatgac attgtccagc 30

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acgttggatg gtcagcaacg tgctggaga 29

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acgtagatga cattgtccag ctgctgc 27

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acgttggatg aggtaggcgg ctcccggca 29

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acgttggatg ctggcagcgt gcagcctgg 29

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cgctcgctgc ccccc 15

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acgttggatg aacagcacca gagagcagca 30

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acgttggatg ctactcccta ccttgttcag 30

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agacctgccg cccac 15

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acgttggatg ccaaacacac tgtgatgacc 30

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acgttggatg ccaaacacac tgtgatgacc 30

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accatgggct cagac 15

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acgttggatg agtgactttg ccttgtcttg 30

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acgttggatg caactctctc ctgaagtcac 30

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gcccctgaag aggtt 15

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acgttggatg tattttccct gctaggagtc 30

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acgttggatg aggtgtgttt ggatcccaag 30

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gctaggagtc gctgtg 16

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acgttggatg ttcccctcat ccgtaccttc 30

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acgttggatg aagggtctct ctactcactc 30

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tccgtacctt ctctgtt 17

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acgttggatg ccatctcctc caaaccatgt 30

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acgttggatg tgaaccccat gtcaccctc 29

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ccaaaccatg tgactgt 17

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ccaaaccatg tgactgt 17

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acgttggatg aagtaaaccc cagcaaagcc 30

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gtgtaggtct catggca 17

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acgttggatg taaaatccac ccctttgccc 30

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acgttggatg agaggaggga tgttttgtgg 30

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cccgtccctg gacccatcc 19

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acgttggatg ggagctgagt cccgcttca 29

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acgttggatg tgatgggacc gttggggtcg 30

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cttaaccctg agcagcccc 19

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acgttggatg tggtgaagta aagccattcc 30

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acgttggatg tgaccccatg ttttccttcc 30

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gtaaagccat tccatcaaa 19

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acgttggatg atggagaaca gtgctgagcc 30

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acgttggatg aggaccacac agctggtctc 30

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cagcccgagc aaagcctgg 19

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acgttggatg cagacaactg aagactctcc 30

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acgttggatg gatcttggtg aggttaaagg 30

<210> 117

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gacgcagcag tggtcctgga 20

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acgttggatg acaatcccag ccaaatcttc 30

<210> 119

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acgttggatg ggacatatac ttgcagggac 30

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acgttggatg ggacatatac ttgcagggac 30

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acgttggatg gtcggtgatc aaagagtggg 30

<210> 122

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acgttggatg gtcggtgatc aaagagtggg 30

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cgtcctggcc gggacaccct g 21

<210> 124

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acgttggatg gcgttttgca ctccaagttc 30

<210> 125

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acgttggatg tcggggactc acctgtgtca 30

<210> 126

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ccgccgtgcc aacatggact g 21

<210> 127

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acgttggatg acacacaaag tccgaggttc 30

<210> 128

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acgttggatg ctttgctcat agggaccaac 30

<210> 129

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gggccggtga gtgcatccgc c 21

<210> 130

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acgttggatg tgacgatggg gtagcggag 29

<210> 131

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acgttggatg accacaggcc agctctatg 29

<210> 132

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<212> DNA

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gggaggaggc tgccgtcagc ca 22

<210> 133

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<400> 133

acgttggatg aagagaggtt ctgggacagg 30

<210> 134

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<212> DNA

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<400> 134

acgttggatg catgacagct aaaacctggc 30

<210> 135

<211> 23

<212> DNA

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tacctttttc ctgaaaagct tat 23

<210> 136

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<212> DNA

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<400> 136

acgttggatg cctagttcct taatgcggtc 30

<210> 137

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<212> DNA

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acgttggatg ggcaccatta ctaatagccc 30

<210> 138

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<212> DNA

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<400> 138

atgcggtcat ttatgttaaa tctt 24

<210> 139

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<212> DNA

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<400> 139

acgttggatg atgaaggcta tccatgggac 30

<210> 140

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<212> DNA

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acgttggatg ccagttagaa atgactgtcg 30

<210> 141

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<212> DNA

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<400> 141

ggagaggaca tgctgtttat cact 24

<210> 142

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<212> DNA

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acgttggatg gagtctttgg ttgctggaag 30

<210> 143

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<212> DNA

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acgttggatg cagaagaaca cccatcagag 30

<210> 144

<211> 24

<212> DNA

<213>Artificial sequence

<400> 144

ggatatggtg ctgagaaaat acct 24

<210> 145

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<212> DNA

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<400> 145

acgttggatg ggagaaactc actctgtgtc 30

<210> 146

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<212> DNA

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<400> 146

acgttggatg tttgatgcgc caagattccc 30

<210> 147

<211> 25

<212> DNA

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<400> 147

gcacctcact ctgtgtctca gaacc 25

<210> 148

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<212> DNA

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<400> 148

acgttggatg ccatctaccg cagtacctg 29

<210> 149

<211> 30

<212> DNA

<213>Artificial sequence

<400> 149

acgttggatg acaagtaagt cttcccaccg 30

<210> 150

<211> 26

<212> DNA

<213>Artificial sequence

<400> 150

ggcggggagg tcacctactt cccttt 26

<210> 151

<211> 29

<212> DNA

<213>Artificial sequence

<400> 151

acgttggatg tcagcaacgt gctggagac 29

<210> 152

<211> 30

<212> DNA

<213>Artificial sequence

<400> 152

acgttggatg acgtcgatga cattgtccag 30

<210> 153

<211> 26

<212> DNA

<213>Artificial sequence

<400> 153

aggccgtcat gcgctgctgg aggccg 26

<210> 154

<211> 30

<212> DNA

<213>Artificial sequence

<400> 154

acgttggatg ctctggcttt cagtaaaggg 30

<210> 155

<211> 30

<212> DNA

<213>Artificial sequence

<400> 155

acgttggatg agtctccaaa gtgaaggagg 30

<210> 156

<211> 26

<212> DNA

<213>Artificial sequence

<400> 156

gggtgtttag ggaagggcat ttcttt 26

<210> 157

<211> 30

<212> DNA

<213>Artificial sequence

<400> 157

acgttggatg tatcaaacgt gaggccactg 30

<210> 158

<211> 30

<212> DNA

<213>Artificial sequence

<400> 158

acgttggatg agatcgccac ctacaggaag 30

<210> 159

<211> 15

<212> DNA

<213>Artificial sequence

<400> 159

cccacctgca ctcct 15

<210> 160

<211> 30

<212> DNA

<213>Artificial sequence

<400> 160

acgttggatg gctccaattc actcttgctg 30

<210> 161

<211> 30

<212> DNA

<213>Artificial sequence

<400> 161

acgttggatg gggagattca gtgacaatgg 30

<210> 162

<211> 15

<212> DNA

<213>Artificial sequence

<400> 162

cttgctggtg ggttt 15

<210> 163

<211> 30

<212> DNA

<213>Artificial sequence

<400> 163

acgttggatg tgtttcaagc tctggaactc 30

<210> 164

<211> 30

<212> DNA

<213>Artificial sequence

<400> 164

acgttggatg atgaccaacg gatactcagg 30

<210> 165

<211> 16

<212> DNA

<213>Artificial sequence

<400> 165

tctggaactc ctacct 16

<210> 166

<211> 30

<212> DNA

<213>Artificial sequence

<400> 166

acgttggatg aagctgggcc aaattctggg 30

<210> 167

<211> 30

<212> DNA

<213>Artificial sequence

<400> 167

acgttggatg gcgtgttgaa attctgcagg 30

<210> 168

<211> 16

<212> DNA

<213>Artificial sequence

<400> 168

attgtccccc aggaac 16

<210> 169

<211> 30

<212> DNA

<213>Artificial sequence

<400> 169

acgttggatg aactccacca tggtaggcac 30

<210> 170

<211> 30

<212> DNA

<213>Artificial sequence

<400> 170

acgttggatg tccttctgcc agcccagctc 30

<210> 171

<211> 16

<212> DNA

<213>Artificial sequence

<400> 171

gggcatcggc cagaac 16

<210> 172

<211> 30

<212> DNA

<213>Artificial sequence

<400> 172

acgttggatg ttgaccatca cacccatctg 30

<210> 173

<211> 30

<212> DNA

<213>Artificial sequence

<400> 173

acgttggatg ctttgctctt tgctaagcgg 30

<210> 174

<211> 16

<212> DNA

<213>Artificial sequence

<400> 174

gggcaggact taggcg 16

<210> 175

<211> 30

<212> DNA

<213>Artificial sequence

<400> 175

acgttggatg ttggcaatgg cagccactac 30

<210> 176

<211> 29

<212> DNA

<213>Artificial sequence

<400> 176

acgttggatg agtgcctgga ggtgtccat 29

<210> 177

<211> 17

<212> DNA

<213>Artificial sequence

<400> 177

gggctgagaa agagccc 17

<210> 178

<211> 30

<212> DNA

<213>Artificial sequence

<400> 178

acgttggatg tcatctcctt tagctccacc 30

<210> 179

<211> 30

<212> DNA

<213>Artificial sequence

<400> 179

acgttggatg atagattgac ctagaagctg 30

<210> 180

<211> 18

<212> DNA

<213>Artificial sequence

<400> 180

cctccagcca cagtgctc 18

<210> 181

<211> 18

<212> DNA

<213>Artificial sequence

<400> 181

cctccagcca cagtgctc 18

<210> 182

<211> 30

<212> DNA

<213>Artificial sequence

<400> 182

acgttggatg caaacatggg ccatcatcac 30

<210> 183

<211> 18

<212> DNA

<213>Artificial sequence

<400> 183

cgttcattat gctgcatt 18

<210> 184

<211> 29

<212> DNA

<213>Artificial sequence

<400> 184

acgttggatg tcagcaacgt gctggagac 29

<210> 185

<211> 29

<212> DNA

<213>Artificial sequence

<400> 185

acgttggatg tcagcaacgt gctggagac 29

<210> 186

<211> 18

<212> DNA

<213>Artificial sequence

<400> 186

gggagacggc agtcatgc 18

<210> 187

<211> 30

<212> DNA

<213>Artificial sequence

<400> 187

acgttggatg ccaaatcctt cttcaccagc 30

<210> 188

<211> 29

<212> DNA

<213>Artificial sequence

<400> 188

acgttggatg tcctcggagg tgtcgggag 29

<210> 189

<211> 19

<212> DNA

<213>Artificial sequence

<400> 189

atccccagcc agcagctgt 19

<210> 190

<211> 30

<212> DNA

<213>Artificial sequence

<400> 190

acgttggatg ggaaggtgca gattttcaac 30

<210> 191

<211> 30

<212> DNA

<213>Artificial sequence

<400> 191

acgttggatg cacaaagaaa tgcccttccc 30

<210> 192

<211> 19

<212> DNA

<213>Artificial sequence

<400> 192

tagtgagagt caggctctg 19

<210> 193

<211> 30

<212> DNA

<213>Artificial sequence

<400> 193

acgttggatg agcctgtttg tccctgcttg 30

<210> 194

<211> 30

<212> DNA

<213>Artificial sequence

<400> 194

acgttggatg gtgtccacac agtagcactc 30

<210> 195

<211> 20

<212> DNA

<213>Artificial sequence

<400> 195

ccctttctcc ctgtccagtg 20

<210> 196

<211> 30

<212> DNA

<213>Artificial sequence

<400> 196

acgttggatg ccatctgcag cgtctttttc 30

<210> 197

<211> 30

<212> DNA

<213>Artificial sequence

<400> 197

acgttggatg ccatctccag atgggttttg 30

<210> 198

<211> 20

<212> DNA

<213>Artificial sequence

<400> 198

tcttgtcttg ataatccagg 20

<210> 199

<211> 30

<212> DNA

<213>Artificial sequence

<400> 199

acgttggatg ggagttcata gacaagctcg 30

<210> 200

<211> 29

<212> DNA

<213>Artificial sequence

<400> 200

acgttggatg gttgatgatc tggccctgc 29

<210> 201

<211> 21

<212> DNA

<213>Artificial sequence

<400> 201

cctcctccag cccaatgtca t 21

<210> 202

<211> 30

<212> DNA

<213>Artificial sequence

<400> 202

acgttggatg acggctgtct tctcaacatc 30

<210> 203

<211> 30

<212> DNA

<213>Artificial sequence

<400> 203

acgttggatg ttcacacgtt gccatatgtc 30

<210> 204

<211> 21

<212> DNA

<213>Artificial sequence

<400> 204

ttgcttctat ctcaggttga t 21

<210> 205

<211> 30

<212> DNA

<213>Artificial sequence

<400> 205

acgttggatg tgcttccaag cccatgtcac 30

<210> 206

<211> 30

<212> DNA

<213>Artificial sequence

<400> 206

acgttggatg tttgaaattg ggcctgatcc 30

<210> 207

<211> 21

<212> DNA

<213>Artificial sequence

<400> 207

aatggggagg acttctggat a 21

<210> 208

<211> 30

<212> DNA

<213>Artificial sequence

<400> 208

acgttggatg tgcctcttgg ggtagttgag 30

<210> 209

<211> 30

<212> DNA

<213>Artificial sequence

<400> 209

acgttggatg acctatggtg aacactaccc 30

<210> 210

<211> 22

<212> DNA

<213>Artificial sequence

<400> 210

cgtcgttgag gcactacccc aa 22

<210> 211

<211> 30

<212> DNA

<213>Artificial sequence

<400> 211

acgttggatg tcccctgcta cttcatcgac 30

<210> 212

<211> 30

<212> DNA

<213>Artificial sequence

<400> 212

acgttggatg aatgcggctc cacttgattc 30

<210> 213

<211> 23

<212> DNA

<213>Artificial sequence

<400> 213

accgggacca ccgccccctc cac 23

<210> 214

<211> 30

<212> DNA

<213>Artificial sequence

<400> 214

acgttggatg gggtggtaga cttgatggtg 30

<210> 215

<211> 30

<212> DNA

<213>Artificial sequence

<400> 215

acgttggatg ttctctgctg acttcatgcc 30

<210> 216

<211> 22

<212> DNA

<213>Artificial sequence

<400> 216

aggcgacttg atggtgagga tg 22

<210> 217

<211> 30

<212> DNA

<213>Artificial sequence

<400> 217

acgttggatg actctctcgg gacgtagaag 30

<210> 218

<211> 30

<212> DNA

<213>Artificial sequence

<400> 218

acgttggatg ataaccgttt ggctgctctg 30

<210> 219

<211> 23

<212> DNA

<213>Artificial sequence

<400> 219

cagaggccgt gcctcctccg aag 23

<210> 220

<211> 30

<212> DNA

<213>Artificial sequence

<400> 220

acgttggatg cagctcacca gaaatatccc 30

<210> 221

<211> 30

<212> DNA

<213>Artificial sequence

<400> 221

acgttggatg attacgtcat gtgcgctcag 30

<210> 222

<211> 24

<212> DNA

<213>Artificial sequence

<400> 222

tctaaccaga aatatcccaa acgc 24

<210> 223

<211> 30

<212> DNA

<213>Artificial sequence

<400> 223

acgttggatg aaagcagaga ggaggcaatg 30

<210> 224

<211> 30

<212> DNA

<213>Artificial sequence

<400> 224

acgttggatg tctctctgcc tttttcctcc 30

<210> 225

<211> 24

<212> DNA

<213>Artificial sequence

<400> 225

gagcggaggc aatgagctta cctt 24

<210> 226

<211> 30

<212> DNA

<213>Artificial sequence

<400> 226

acgttggatg tatgatgtcc tgctggttcc 30

<210> 227

<211> 30

<212> DNA

<213>Artificial sequence

<400> 227

acgttggatg aaaaggcacc cagtctgtag 30

<210> 228

<211> 24

<212> DNA

<213>Artificial sequence

<400> 228

ctggtatatt agagagttag ggtt 24

<210> 229

<211> 30

<212> DNA

<213>Artificial sequence

<400> 229

acgttggatg tcaaggactt ctacgagctg 30

<210> 230

<211> 30

<212> DNA

<213>Artificial sequence

<400> 230

acgttggatg cagggttaca catcaccaac 30

<210> 231

<211> 25

<212> DNA

<213>Artificial sequence

<400> 231

caatcagacc aatgggatca cccct 25

<210> 232

<211> 30

<212> DNA

<213>Artificial sequence

<400> 232

acgttggatg gatcgccacc atgaaggaac 30

<210> 233

<211> 30

<212> DNA

<213>Artificial sequence

<400> 233

acgttggatg atgctgtgta accaaacagg 30

<210> 234

<211> 26

<212> DNA

<213>Artificial sequence

<400> 234

gaagtgtcgt cttcgtgtct tcccag 26

<210> 235

<211> 30

<212> DNA

<213>Artificial sequence

<400> 235

acgttggatg taaaatccac ccctttgccc 30

<210> 236

<211> 30

<212> DNA

<213>Artificial sequence

<400> 236

acgttggatg agaggaggga tgttttgtgg 30

<210> 237

<211> 15

<212> DNA

<213>Artificial sequence

<400> 237

tccctggacc catcc 15

<210> 238

<211> 30

<212> DNA

<213>Artificial sequence

<400> 238

acgttggatg accgctacat ctccatcttc 30

<210> 239

<211> 30

<212> DNA

<213>Artificial sequence

<400> 239

acgttggatg agatggccgc aatgatcctc 30

<210> 240

<211> 15

<212> DNA

<213>Artificial sequence

<400> 240

gccctgcggt accac 15

<210> 241

<211> 30

<212> DNA

<213>Artificial sequence

<400> 241

acgttggatg ccacggcgat cttaaagtag 30

<210> 242

<211> 30

<212> DNA

<213>Artificial sequence

<400> 242

acgttggatg tctggctcat caacattggc 30

<210> 243

<211> 15

<212> DNA

<213>Artificial sequence

<400> 243

ccacggatcc aggag 15

<210> 244

<211> 30

<212> DNA

<213>Artificial sequence

<400> 244

acgttggatg gggaagaacc atcgatcagc 30

<210> 245

<211> 30

<212> DNA

<213>Artificial sequence

<400> 245

acgttggatg aaacagagag ctcctcaccc 30

<210> 246

<211> 15

<212> DNA

<213>Artificial sequence

<400> 246

gcctgggggt agact 15

<210> 247

<211> 15

<212> DNA

<213>Artificial sequence

<400> 247

gcctgggggt agact 15

<210> 248

<211> 30

<212> DNA

<213>Artificial sequence

<400> 248

acgttggatg agacgtagcc actgggttct 30

<210> 249

<211> 16

<212> DNA

<213>Artificial sequence

<400> 249

atgaagcaag gctcag 16

<210> 250

<211> 30

<212> DNA

<213>Artificial sequence

<400> 250

acgttggatg tggcttggga cagtgatgtg 30

<210> 251

<211> 29

<212> DNA

<213>Artificial sequence

<400> 251

acgttggatg cagggccatg tgctgcttg 29

<210> 252

<211> 17

<212> DNA

<213>Artificial sequence

<400> 252

tcctgccctc acgacca 17

<210> 253

<211> 30

<212> DNA

<213>Artificial sequence

<400> 253

acgttggatg tgggagaagg aagtacatgg 30

<210> 254

<211> 30

<212> DNA

<213>Artificial sequence

<400> 254

acgttggatg caactcatta cagcatccgc 30

<210> 255

<211> 17

<212> DNA

<213>Artificial sequence

<400> 255

ccatcaactc catgcga 17

<210> 256

<211> 30

<212> DNA

<213>Artificial sequence

<400> 256

acgttggatg agaggaaggt gcagattttc 30

<210> 257

<211> 30

<212> DNA

<213>Artificial sequence

<400> 257

acgttggatg gcccttccct aaatatccag 30

<210> 258

<211> 17

<212> DNA

<213>Artificial sequence

<400> 258

gtgagagtca ggctctg 17

<210> 259

<211> 29

<212> DNA

<213>Artificial sequence

<400> 259

acgttggatg atacccccca gcagtacct 29

<210> 260

<211> 30

<212> DNA

<213>Artificial sequence

<400> 260

acgttggatg tccacgtgtc acaggatgag 30

<210> 261

<211> 18

<212> DNA

<213>Artificial sequence

<400> 261

ccgtacctgg ggcaccgt 18

<210> 262

<211> 30

<212> DNA

<213>Artificial sequence

<400> 262

acgttggatg tgatcttctg ctccaagtcg 30

<210> 263

<211> 30

<212> DNA

<213>Artificial sequence

<400> 263

acgttggatg gagaaggtga ccatgcagaa 30

<210> 264

<211> 18

<212> DNA

<213>Artificial sequence

<400> 264

ggaggaggcc aggcggtc 18

<210> 265

<211> 30

<212> DNA

<213>Artificial sequence

<400> 265

acgttggatg tccacgtgtc acaggatgag 30

<210> 266

<211> 29

<212> DNA

<213>Artificial sequence

<400> 266

acgttggatg atacccccca gcagtacct 29

<210> 267

<211> 19

<212> DNA

<213>Artificial sequence

<400> 267

agtaatgaac ccctcgccc 19

<210> 268

<211> 30

<212> DNA

<213>Artificial sequence

<400> 268

acgttggatg aatactgtgc ctgccaagtc 30

<210> 269

<211> 30

<212> DNA

<213>Artificial sequence

<400> 269

acgttggatg tggtggaatg gccataaatg 30

<210> 270

<211> 19

<212> DNA

<213>Artificial sequence

<400> 270

cagccaacta ccatggcac 19

<210> 271

<211> 29

<212> DNA

<213>Artificial sequence

<400> 271

acgttggatg gtgagcgtca gcaacgtgc 29

<210> 272

<211> 30

<212> DNA

<213>Artificial sequence

<400> 272

acgttggatg tgacattgtc cagctgctgc 30

<210> 273

<211> 19

<212> DNA

<213>Artificial sequence

<400> 273

atccgcaacg tgctggaga 19

<210> 274

<211> 30

<212> DNA

<213>Artificial sequence

<400> 274

acgttggatg tcatcttctc acagtagccc 30

<210> 275

<211> 30

<212> DNA

<213>Artificial sequence

<400> 275

acgttggatg gaaagctgtc tagactctgg 30

<210> 276

<211> 20

<212> DNA

<213>Artificial sequence

<400> 276

ggggtgccct tcggatctca 20

<210> 277

<211> 30

<212> DNA

<213>Artificial sequence

<400> 277

acgttggatg gatcttccca atctcggtgc 30

<210> 278

<211> 29

<212> DNA

<213>Artificial sequence

<400> 278

acgttggatg cctgcctctt cttcccctt 29

<210> 279

<211> 20

<212> DNA

<213>Artificial sequence

<400> 279

cgcaggctgc gatgttggtg 20

<210> 280

<211> 29

<212> DNA

<213>Artificial sequence

<400> 280

acgttggatg cactcacggt caccttggc 29

<210> 281

<211> 30

<212> DNA

<213>Artificial sequence

<400> 281

acgttggatg tgatggcaga ggtggagtag 30

<210> 282

<211> 21

<212> DNA

<213>Artificial sequence

<400> 282

gctccccgtt catgccgccg t 21

<210> 283

<211> 30

<212> DNA

<213>Artificial sequence

<400> 283

acgttggatg gtatatggct attaggacgg 30

<210> 284

<211> 30

<212> DNA

<213>Artificial sequence

<400> 284

acgttggatg gtgtctgtgt ggaaggccat 30

<210> 285

<211> 21

<212> DNA

<213>Artificial sequence

<400> 285

gctattagga cggagatgat c 21

<210> 286

<211> 31

<212> DNA

<213>Artificial sequence

<400> 286

acgttggatg gaacaaatta cctctgtttg c 31

<210> 287

<211> 30

<212> DNA

<213>Artificial sequence

<400> 287

acgttggatg ataaacagca tttctttatg 30

<210> 288

<211> 22

<212> DNA

<213>Artificial sequence

<400> 288

actaactctg tttgctgctg tt 22

<210> 289

<211> 30

<212> DNA

<213>Artificial sequence

<400> 289

acgttggatg actcctggag tcaagtgaag 30

<210> 290

<211> 30

<212> DNA

<213>Artificial sequence

<400> 290

acgttggatg ggtgcaaatg gctgaagaac 30

<210> 291

<211> 22

<212> DNA

<213>Artificial sequence

<400> 291

aatgctggtt tcatgcttac tc 22

<210> 292

<211> 30

<212> DNA

<213>Artificial sequence

<400> 292

acgttggatg caatatgtgc ttctgctgtg 30

<210> 293

<211> 30

<212> DNA

<213>Artificial sequence

<400> 293

acgttggatg tgtccagttc ttctcccaag 30

<210> 294

<211> 22

<212> DNA

<213>Artificial sequence

<400> 294

ggggattctg ctgtgcagtg tt 22

<210> 295

<211> 30

<212> DNA

<213>Artificial sequence

<400> 295

acgttggatg tttgcttagg agccatagcc 30

<210> 296

<211> 30

<212> DNA

<213>Artificial sequence

<400> 296

acgttggatg ttactcaaac ccctgtggtg 30

<210> 297

<211> 23

<212> DNA

<213>Artificial sequence

<400> 297

gggcgtcact ttggagactc cca 23

<210> 298

<211> 30

<212> DNA

<213>Artificial sequence

<400> 298

acgttggatg tcgcatcagc attgagaacg 30

<210> 299

<211> 30

<212> DNA

<213>Artificial sequence

<400> 299

acgttggatg tctcagccgt tgaaccaatc 30

<210> 300

<211> 30

<212> DNA

<213>Artificial sequence

<400> 300

acgttggatg tctcagccgt tgaaccaatc 30

<210> 301

<211> 30

<212> DNA

<213>Artificial sequence

<400> 301

acgttggatg tctcagccgt tgaaccaatc 30

<210> 302

<211> 30

<212> DNA

<213>Artificial sequence

<400> 302

acgttggatg tctcagccgt tgaaccaatc 30

<210> 303

<211> 24

<212> DNA

<213>Artificial sequence

<400> 303

ggtgactcta cgtcggagac gtcg 24

Claims (10)

1. it is a set of while detecting the primer sets in 93 kinds of genetic defectes of ox, 101 related cause-effect mutational sites, it is characterised in that described Primer is divided into four groups, and every group of primer includes multiplexed PCR amplification primer and Single base extension primer, first group such as sequence table SEQ ID NO:Shown in 1-78, second group such as sequence table SEQ ID NO:Shown in 79-156, the 3rd group such as sequence table SEQ ID NO:157- Shown in 234, the 4th group such as sequence table SEQ ID NO:Shown in 235-303.

2. it is a set of while detecting the detection kit in 93 kinds of genetic defectes of ox, 101 related cause-effect mutational sites, it is characterised in that The kit includes the primer sets described in claim 1.

3. detection kit according to claim 2, it is characterised in that the kit, which also includes pcr amplification reaction, to be tried Agent, alkaline phosphatase treatment reagent, single base extension.

4. kit according to claim 3, it is characterised in that the pcr amplification reaction reagent includes dNTP, MgCl₂、 HotStar Taq、PCR Bμffer、ddH₂O；

Or, alkaline phosphatase treatment reagent includes ddH₂O, shrimp alkaline phosphotase Buffer, shrimp alkaline phosphotase；

Or, single base extension includes：iPLEX Bμffer Plus、iPLEX Termination mix、iPLEX Enzyme、ddH₂O。

5. it is a kind of while detecting the method for 93 kinds of ox genetic defectes, it is characterised in that to comprise the following steps：

(1) ox DNA is extracted；

(2) multiplexed PCR amplification is carried out using using multiplexed PCR amplification primer described in claim 1 and step (1) DNA Reaction；

(3) alkaline phosphatase treatment is carried out to step (2) amplified production, removes dNTPs；

(4) product obtained using step (3), Single base extension primer carries out Single base extension described in usage right requirement 1 Reaction；

(5) resin is added to step (4) reaction product to purify；

(6) genetic defect genotype is obtained using Mass Spectrometer Method analysis.

6. method according to claim 5, it is characterised in that

Step extracts DNA in (1) from bovine blood, seminal fluid or hair follicle；

Or, step (2) carries out pcr amplification reaction using the primer and DNA diluted in 384 holes；

It is preferred that, pcr amplification reaction uses 5.000 μ l systems, constitutes and is：1 μ l DNA, 1.000 μ l amplimers mixtures, 0.100μl dNTP、0.325μl MgCl₂、0.100μl HotStar Taq、0.625μl PCR Bμffer、1.850μl ddH2O；

It is preferred that, pcr amplification reaction program is：First 94 DEG C of 5min；Then 94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min, this step 45 circulations；Last 72 DEG C of 3min；

Or, every 5.000 μ l PCR primers add 2.00 μ l shrimp alkaline phosphotases to handle in step (3), remove remaining dNTPs；

It is preferred that, 2.00 μ l shrimp alkaline phosphotases processing reaction solution composition is：1.53μl ddH₂O, 0.17 μ l shrimp alkaline phosphotases Buffer, 0.30 μ l shrimp alkaline phosphotases；

It is preferred that, step (2) amplified production carries out alkaline phosphatase treatment response procedures and is：37℃20min；85℃5min；4℃ ∞；

Or, single base extension uses 9 μ l reaction systems in step (4), constitutes and is：7 μ l product alkaline phosphatase treatments Product, 0.200 μ l iPLEX B μ ffer Plus, 0.200 μ l iPLEX Termination mix, 0.940 μ l extension primers (7 μM of mixture：14μM)、0.041μl iPLEX Enzyme、0.619μl ddH2O；

It is preferred that, the program of single base extension is：First 94 DEG C of 30sec；Then 94 DEG C of 5sec, (52 DEG C of 5sec, 80 DEG C 5sec, 5 circulations), 40 circulations of this step；72℃3min；

Or, in step (5), adding resin progress purifying concrete operations is：Uniform potting resin and put in 384Dimple plates 10min is put, in the μ l water of each Kong Zhongjia 16 of 384 sample planes, rapping Dimple makes resin fall into each hole of sample plane, so Sample version room temperature is rotated into 30min afterwards；

Or, Mass Spectrometer Method process is in step (6)：The extension products after purifying resin are moved into 384 holes with point sample instrument On SpectroCHIP chips, then it is analyzed using MALDI-TOF mass spectrographs, as a result enters line number using TYPER4.0 softwares According to processing, according to the size of wild different extension products molecular weight corresponding with mutational site, mutational site parting information is obtained.

7. a kind of be used to detect the PIRM syndromes of ox, spherocytosis, β caseins 8, β caseins 4, I type leucocyte simultaneously Adhesion defects are levied, Qi-east syndrome, recessive red hair, β caseins 9, double fleshes, β caseins 1, congenital ichthyosis, lethal list times Type HH1, lethal haplotype HH3, lethal haplotype HH4, dyschondroplasia nanism, deformity of spine syndrome, cartilage growth Incomplete, citrullinemia, Angus nanism, acrodermatitis enteropathica, miscarriage SLC37A2, growth hormone deficiency type dwarf Disease, cardiomyopathy and the continuous long defect of shape, combine with the primer of the nanism genetic defect gene of inflammatory lesion perspec-tive, it is special Levy and be, the primer includes multiplexed PCR amplification primer and Single base extension primer, specific primer such as sequence table SEQ ID NO: Shown in 1-78.

8. it is used to detect a variety of eye defects of ox simultaneously, bends tail syndrome, α types mannosidosis, the JH1 that miscarries, moves back Row aixs cylinder disease, male sterility, the abnormal low expression of beta lactoglobulin, lethal haplotype HH5, β casein 11, anhidrotic ectodermal Depauperation, yellow fat disease, lactoglobulin, miscarriage SHBG, perinatal period weak calf syndrome, congenital myodystony I types, zinc lack Weary sample syndrome, dactylion, proteoglycan type nanism, the depauperation of lethal multiple organ, it is dominant in vain with bilateral is deaf, trimethyl Aminuria disease, Mao Shaose shallow, spider legs syndrome, congenital myasthenia, recessive red hair 373, β caseins 6 genetic defect genes Primer is combined, it is characterised in that the primer includes multiplexed PCR amplification primer and Single base extension primer, specific primer such as sequence List SEQ ID NO:Shown in 79-156.

9. for the epidermolysis bollosa of detection ox, β types mannosidosis, spinal cord dysmyelination, blood simultaneously It is platelet disorder, mucopolysaccharidosis type ⅢB, acerous/to have angle, recessive red hair EBR, polymelia, marfan's syndrome, recessive red hair 358th, protoporphyria, β caseins 12, goitre, α types mannosidosis, maple syrup urine disease-H, proteoglycan type dwarf Disease, congenital pseudomyotonia, two class lithoxidurias, dominant red hair, forelimb to waist muscle exception, dystrophic epidermolysis Epidermolysis, five type glycogen storage diseases, the primer combination of Duchenne-Arandisease genetic defect gene, it is characterised in that described Primer includes multiplexed PCR amplification primer and Single base extension primer, specific primer such as sequence table SEQ ID NO:157-234 institutes Show.

10. for detect simultaneously the β caseins 3 of ox, recessive red hair, congenital pseudomyotonia, Ai Laier-when Lip river syndrome, Light hypotrichosis, two type glycogen storage diseases, haemophilia A, β caseins 7, gum hamartoma type osteopetrosis, Kappa junket eggs In vain, dilated cardiomyopathy, the type of congenital myodystony II, β caseins 2, hypotrichosis, uridine phosphate synthase deficiency disease, F11 because Sub- shortage, β caseins 5, the combination of dermatosparaxis genetic defect gene primer, it is characterised in that the primer includes multiplex PCR Amplimer and Single base extension primer, specific primer such as sequence table SEQ ID NO:Shown in 235-303.