CN103740816B - The Primer composition that a kind of ox citrullinemia deleterious gene detects and test kit thereof and application - Google Patents

The Primer composition that a kind of ox citrullinemia deleterious gene detects and test kit thereof and application Download PDF

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CN103740816B
CN103740816B CN201310708022.9A CN201310708022A CN103740816B CN 103740816 B CN103740816 B CN 103740816B CN 201310708022 A CN201310708022 A CN 201310708022A CN 103740816 B CN103740816 B CN 103740816B
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primer
citrullinemia
test kit
deleterious gene
gene
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CN103740816A (en
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郭霄峰
吴晓薇
洪洁心
代元元
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South China Agricultural University
Inspection and Quarantine Technology Center of Guangdong Entry Exit Inspection and Quarantine Bureau
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Inspection and Quarantine Technology Center of Guangdong Entry Exit Inspection and Quarantine Bureau
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

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Abstract

The invention discloses Primer composition and test kit and application that a kind of ox citrullinemia deleterious gene detects.Primer composition of the present invention is made up of primer sets A and primer sets B, and described primer sets A is made up of primer 1 and primer 2, and described primer sets B is made up of primer 3 and primer 4; The nucleotide sequence of primer 1 ~ primer 4 is respectively as shown in SEQ ID NO.1 ~ 4.Present invention also offers the test kit comprising described combination of primers, the method that test kit of the present invention is applied to the detection of ox citrullinemia deleterious gene is as follows: complete group of DNA in extraction bovine blood is as template, carry out nest-type PRC (Nested PCR) amplification, gained PCR primer checks order, the base change in mutational site can be directly acquainted with according to sequencing result, ensure that the accuracy of result, meet fast, precisely, the demand of the detection technique of the feature such as high-throughput.

Description

The Primer composition that a kind of ox citrullinemia deleterious gene detects and test kit thereof and application
Technical field
The present invention relates to biological technical field.More specifically, Primer composition and test kit and application that a kind of ox citrullinemia deleterious gene detects is related to.
Background technology
Along with the promotion and application of artificial insemination and embryo transfer technology, milk cow outstanding kind of matter (embryo, seminal fluid, kind ox) widespread use in the world, while significantly improving heritability, accelerates the harm of ox recessive genetic disorder to cows.Particularly an outstanding breeding oxen can produce hundreds of thousands of agent even up to a million doses of frozen semens all one's life, if carry recessive inheritance dcc gene, likely spreads fast in worldwide, brings huge financial loss to the production of livestock industry.China is mainly from a large amount of milk cow of state's import outstanding kind of matter such as the U.S., Canada, Germany, Australia, New Zealand and Uruguays, during import, quarantine focuses on preventing importing into of zoonosis and parasitosis, but the quarantine request preventing ox hereditary defect is not comprised, so the ox hereditary defect carrier existed enters the risk of China.
Genetic deficiency diseases is that genetic material producer sudden change in sexual cell or zygote and chromosome aberration cause; thus make growth individuality occur the exception in anatomical structure or the infringement on physiological function; usually cause the infringement of some organ; form deformity; affect production performance, even cause death.There is congenital and feature that is familial.Hereditary defect venereal disease is divided into autosomal inheritance defect disease and allosomal inheritance defect disease, wherein autosomal inheritance defect old complaint is divided into again monogenic inheritance defect disease, polygenic inheritance defect disease and chromosome aberration genetic deficiency diseases three kinds according to pathogenesis, and allosomal inheritance defect is more rare.Euchromosome monogenic inheritance defect disease is controlled by pair of alleles, and recessive deleterious alleles carrier phenotype is normal, when two parents are carrier, offspring has 1/4 may occur allozygote, namely phenotype is ill, and 1/2 may be recessive heterozygote, is deleterious gene carrier.
Citrullinemia (Citrullinemia, CN) is that a kind of euchromosome single-gene recessive inheritance defect of Holstein cow ornithine cycle generation metabolism disorder is sick.This disease found (Harper etc., 1986) early than 1986 in the Holstein cow group of Australia.Act normally during sick ox birth, but occur that spirit is depressed in the near future, gait disturbance, convulsions, the symptom such as blind, after general birth, 5 d are dead, mortality ratio 100%.Citrullinemia (CN) genetic base is by ASS gene (the Argininosuccinate synthetase of the encode arginine succsinic acid synthetic enzyme on milk cow o.11 karyomit(e) (BTA11), ASS) there is the point mutation of C → T in the 5th exon, codon CGA is made to change terminator codon TGA into, thus its translation product argininosuccinate synthase becomes 85 amino acid whose protein (being normally 412 amino acid) of brachymemma, and lose argininosuccinate synthase activity.Cause citrulline can not change argininosuccinate acid into and put aside in a large number in tissue and blood, thus cause ornithine cycle generation metabolic disturbance, and then cause serum amylase too high.Ill ox cardinal symptom is that in blood, citrulline content raises, and ornithine cycle is obstructed and is caused hyperammonemia, causes birth interior death in latter one week.
According to this mutational site, mainly contain PCR-RFLP and AS-PCR two kinds of methods at present and detect citrullinemia (CN).PCR-RFLP method designs at catastrophe point place avaIIrestriction enzyme site, carries out enzyme to PCR primer and cuts, and cuts result judge whether to have according to enzyme avaIIrestriction enzyme site is in order to judge whether containing catastrophe point, but after the method introducing base mismatch, the amplification efficiency of PCR can be affected, and there is false-positive possibility in addition.AS-PCR method designs special primer according to mutational site, wherein a kind of base state in a chain and mutational site is complementary, another chain designs according to a conventional method, special primer has amplified production in a kind of genotype, amplified production is not had in another kind of genotype, according to the presence or absence that amplification is produced, thus determine whether the sudden change of base, but the method cause sometimes when the base of the 3' end of primer and the base of template not complementary, extend and still can carry out, easily cause the erroneous judgement of result.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiency that existing ox citrullinemia deleterious gene detects detection technique, provides the Primer composition that a kind of ox citrullinemia deleterious gene detects.
Another technical problem that the present invention will solve is to provide the test kit that a kind of ox citrullinemia deleterious gene detects.
The present invention also has a technical problem that will solve to be to provide the application of described Primer composition and test kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The invention provides a kind of new Primer composition, be the Primer composition that ox citrullinemia deleterious gene detects, be made up of primer sets A or primer sets B; Described primer sets A is made up of primer 1 and primer 2; Described primer sets B is made up of primer 3 and primer 4; The nucleotide sequence of primer 1 ~ 4 is respectively as shown in SEQ ID NO.1 ~ 4.
Described primer sets A is outer primer, and primer sets B is inner primer, and inner primer designs in the fragment of external primer amplification.
Preferably, described ox citrullinemia deleterious gene is the gene of the 86th amino acid codes generation nonsense mutation of argininosuccinate synthase gene.
Preferably, the mol ratio of primer 1 and primer 2 is 1: 1, and the mol ratio of primer 3 and primer 4 is 1: 1.
The invention provides the application of Primer composition in employing nested PCR method detection ox citrullinemia deleterious gene that described ox citrullinemia deleterious gene detects.
The present invention provides a kind of test kit of the Primer composition containing described ox citrullinemia deleterious gene detection simultaneously.
In described test kit, preferably, described primer sets A or primer sets B is complete independent packaging.
Preferably, described test kit, includes following component: the Primer composition that ox citrullinemia deleterious gene of the present invention detects, archaeal dna polymerase, PCR reaction buffer, dNTP and aqua sterilisa.
Preferably, the Primer composition that described test kit detects except comprising described ox citrullinemia deleterious gene, also includes Pfu DNA Polymerase enzyme (2.5U/ul) and 10 × Pfu Buffer, dNTP Mixture(2.5 mmol/L), and ddH 20.
The present invention provides described test kit detecting the application in ox citrullinemia deleterious gene simultaneously.
Ox citrullinemia deleterious gene detection method of the present invention utilizes nested PCR method to carry out twice PCR reaction, and the template of first time PCR reaction is complete group of DNA in bovine blood, and primer is primer sets A; The template of second time PCR reaction is the diluent (sterilized water dilution) of first time PCR reaction product, and primer is primer sets B; PCR primer is checked order, detects ox citrullinemia deleterious gene according to sequencing result.
Preferably, described first time PCR reacts the fragment that amplification obtains a 420bp of argininosuccinate synthase gene, as shown in SEQ ID NO.5; Second time PCR reaction amplification obtains the fragment of a 162bp of argininosuccinate synthase gene, as shown in SEQ ID NO.6; Detecting ox citrullinemia deleterious gene concrete grammar according to sequencing result is check whether the 86th amino acid codes of argininosuccinate synthase gene nonsense mutation occurs according to sequencer map.If this site is unimodal C in sequencer map, then it is non-citrullinemia deleterious gene; This site is bimodal, simultaneously containing C and T, is then ox citrullinemia deleterious gene; This site is unimodal T, be then ox citrullinemia patient.
Preferably, the ratio of each component in the amplification system that reacts of described first time PCR: primer 1: primer 2: PCR reaction buffer: dNTP: archaeal dna polymerase: aqua sterilisa=2: 2: 10: 10: 1: 73;
Preferably, the ratio of each component in the amplification system of second time PCR reaction: primer 3: primer 4: PCR reaction buffer: dNTP: archaeal dna polymerase: aqua sterilisa=2: 2: 10: 10: 1: 73.
The present invention has following beneficial effect:
The Primer composition that scientific design of the present invention a group is new, have excellent specific amplification, amplification efficiency is high, effectively avoids false-negative result, is applied to detection ox citrullinemia (CN) deleterious gene accuracy high.
Invention also provides and detect ox citrullinemia (CN) detrimental method, creatively study the gene of ox, the feature larger according to the genome of ox, sum up the nested PCR detection method of twice, by twice PCR, avoid prior art only by a PCR, due to the concentration of DNA profiling and the relation of purity, amplification efficiency is not high, and the problem of the fragment that easily makes a mistake.And second time PCR reaction occurs that the probability that primer pairing is also increased in false segments is extremely low.
The judgement of net result of the present invention is undertaken by order-checking, directly can measure the base change in mutational site, further ensure that the accuracy of result.
Instant invention overcomes the troublesome operation such as the gel electrophoresis required for prior art, the present invention has quick, accurate, high-throughout advantage, provide strong technical foundation for detecting ox citrullinemia (CN) deleterious gene, and be specially adapted in foreign trade the detrimental detection technique demand of ox citrullinemia (CN).
Accompanying drawing explanation
Fig. 1 is non-citrullinemia deleterious gene carrier (normal type) milk cow ASS gene sequencing figure, and arrow place is mutational site.
Fig. 2 is citrullinemia deleterious gene carrier milk cow ASS gene sequencing figure, and arrow place is mutational site.
Fig. 3 is the HRM temperature variation of CN gene homogenization.
Fig. 4 is the HRM difference view of CN gene.
Embodiment
Further illustrate the present invention below in conjunction with the drawings and specific embodiments, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, equipment and method are the art conventional reagent, equipment and method.
Embodiment of the present invention Pfu DNA used Polymerase enzyme (2.5U/ul) and 10 × Pfu Buffer, dNTP Mixture (2.5 mmol/L), all purchased from Tian Gen bio tech ltd.
embodiment 1 prepares the nest-type PRC reaction kit of examination ox citrullinemia
1, design of primers
Constantly analyze, sum up and adjust the design of primer in conjunction with Integral Thought of the present invention according to ASS gene order (GenBank:AC_000168) on NCBI, finally according to ASS gene (the Argininosuccinate synthetase of the encode arginine succsinic acid synthetic enzyme on ox o.11 karyomit(e) (BTA11), ASS) there is the point mutation of C → T in the 5th exon, devise the detrimental nested PCR method of a kind of detection ox citrullinemia, obtain primer 1, primer 2, primer 3 and primer 4.Sequence is as shown in SEQ ID NO.1 ~ 4:
Primer 1:SEQ ID NO.1:actgcatttt caagaccacc ctg;
Primer 2: SEQ ID NO.2:tctgggcggg aaccaacgat tgtc;
Primer 3:SEQ ID NO.3:attgaggaca tcagcaagga g;
Primer 4:SEQ ID NO.4:cacatacttg gctccttctc.
Wherein, primer 1 and primer 2 are primer sets A(outer primer), primer 3 and primer 4 are primer sets B(inner primer).
All primers can adopt the ordinary methods such as synthesis to obtain.The present embodiment primer is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd.
Test kit is become with primer sets of the present invention reagent set relevant with PCR reaction.Described primer sets is primer sets A and primer sets B; It is archaeal dna polymerase, PCR reaction buffer, dNTP and aqua sterilisa that described PCR reacts related reagent.
The present embodiment uses (nest-type PRC reaction kit) for primer sets A and primer sets B simultaneously, and the preparation detecting the detrimental test kit of ox citrullinemia is described.
2, the nest-type PRC reaction kit that prepared by the present embodiment comprises two parts: first time PCR(outer primer) test kit and second time PCR(inner primer) test kit.
(1) first time PCR(outer primer) test kit
Test kit, except comprising outer primer (primer 1 and primer 2), also comprises PCR reaction reagent: Pfu DNA Polymerase enzyme (2.5U/ul), 10 × Pfu Buffer, dNTP Mixture(2.5 mmol/L), ddH 20.The primer 1 of PCR reaction is forward primer, and primer 2 is reverse primer, and forward and reverse primer concentration is 10pmol/L.
PCR reaction system is for 25 uL systems: primer 1 and each 0.5mL of primer 2,10 × Pfu Buffer 2.5mL, dNTP Mixture(2.5 mmol/L) 2.5mL, Pfu DNA Polymerase enzyme (2.5U/ul) 0.25mL, ddH 20 18.25mL, remaining 0.5mL volume is template DNA.Namely for each component proportions in the reagent mixed liquor of first time PCR reaction be, primer 1: primer 2: 10 × Pfu Buffer: dNTP Mixture: Pfu DNA Polymerase enzyme: ddH 20=2: 2: 10: 10: 1: 73.
Add Related Component in proportion according to the total amount of system during experiment and mix, adding template DNA and carry out first time PCR and react.
(2) second time PCR(inner primer) test kit:
Test kit comprises outer primer (primer 3 and primer 4), and the primer 3 of PCR reaction is forward primer, and primer 4 is reverse primer, and forward and reverse primer concentration is 10pmol/L.Except primer, other Related Components are identical with first time PCR kit with the ratio of each composition.I.e. primer 3: primer 4: 10 × Pfu Buffer: dNTP Mixture: Pfu DNA Polymerase enzyme: ddH20=2: 2: 10: 10: 1: 73.
Add Related Component in proportion according to the total amount of system during experiment and mix, to add template DNA carrying out second time PCR reaction.
the nest-type PRC reaction kit that embodiment 2 utilizes embodiment 1 to prepare detects milk cow citrullinemia
1, the full extraction organizing DNA in bovine blood
The blood sample 5mL/ head of jugular vein blood collection method random acquisition Jiangsu isolation camp 243 holstein cows, and be placed in vacuum heparin sodium anticoagulant tube and shake up rear centrifuge tube packing often pipe 2ml ,-20 DEG C are frozen for subsequent use.
Adopt Promega Maxwell 16 nucleic acid automatic extracting instrument to carry out the extraction of poba gene group DNA, use matched reagent box (AS1010 of Promega company).The AS1010 test kit of Promega comprises Maxwell 16 Blood DNA Cartridges, Purification Plungers, Elution Tubes, Elution buffer.Operation is undertaken by test kit specification sheets, and after this instrument starts, approximately every 28min can complete the extraction of 16 sample DNAs.After DNA extraction, it be transferred to from wash-out pipe in centrifuge tube (Ep pipe), it is stand-by to be placed in-20 DEG C of preservations.
Through measuring, the concentration that the bovine blood of extraction organizes DNA sample is entirely 83ng/ μ L.
2, first time PCR(outer primer) reaction
The DNA obtained with above-mentioned extracting is template, increases by the first time PCR kit in embodiment 1.System is 25 μ L: template DNA 0.5mL(41.5 ng), first time PCR reagent mixture 24.5mL(is comprising primer 1 and each 0.5mL of primer 2,10 × Pfu Buffer 2.5mL, dNTP Mixture(2.5 mmol/L) 2.5mL, Pfu DNA Polymerase enzyme (2.5U/ul) 0.25mL, ddH 20 18.25mL).Amplification condition is: 94 DEG C of 3min, 94 DEG C of 30s, 62.2 DEG C of 30s, 70 DEG C of 15s, 30cycles, 70 DEG C of 1min.
Be that template is as negative control with sterilized water.
3, second time PCR(inner primer) reaction
Get 5mL first time PCR primer and dilute 100 times as the template of second time PCR reaction, increase by the second time PCR kit in embodiment 1.System is 50 μ L: template DNA 1mL, second time PCR reagent mixture 49mL.Amplification condition is: 94 DEG C of 3min, 94 DEG C of 30s, 61.2 DEG C of 30s, 70.4 DEG C of 15s, 30 cycles, 70.4 DEG C of 1min.
4, check order (check order completed) by the prosperous bio tech ltd of Beijing AudioCodes to first time PCR and second time PCR primer respectively, the sequencing result DNAMAN software analysis of amplified production, in accompanying drawing, arrow institute mark point is the SNP site causing CN.Check whether ox citrullinemia deleterious gene carrier mutational site changes according to sequencer map, if in sequencer map this site be unimodal C(as shown in Figure 1, arrow place is mutational site), be then non-citrullinemia deleterious gene carrier (normal type); If this site is bimodal, as shown in Figure 2 containing C and T(, arrow place is mutational site simultaneously), be then citrullinemia deleterious gene carrier (heterozygote); If this site is unimodal T, be then citrullinemia patient (saltant type does not find this type of in examination).
Statistical result showed, in 243 increment product, CN normal type 238 parts, CN heterozygote 5 parts, CN saltant type 0 part.
comparative example 1 utilizes HRM detection method to detect milk cow citrullinemia
1, material
(1) test sample: with embodiment 2, the blood sample 5ml/ head of jugular vein blood collection method random acquisition Jiangsu isolation camp 243 holstein cows, and be placed in vacuum heparin sodium anticoagulant tube and shake up rear centrifuge tube packing often pipe 2ml ,-20 DEG C are frozen for subsequent use.
DNA extraction method is with embodiment 2.
(2) reagent and test kit
General T aq enzyme reagent kit, purchased from water source biotechnology (Shanghai) Co., Ltd.;
Gel extraction kit, purchased from JaRa bio tech ltd, Shanghai;
100 bp DNA Ladder Marker, purchased from water source biotechnology (Shanghai) Co., Ltd.;
Hot start Taq enzyme reagent kit, purchased from water source biotechnology (Shanghai) Co., Ltd.;
EverGreen Q-PCR Mix, purchased from water source biotechnology (Shanghai) Co., Ltd.;
T-carrier, purchased from precious biological (Dalian) biotechnology company limited;
SYBR GREEN I, purchased from Pu Boxin bio tech ltd, Beijing.
(3) preparation of main agents
Electrophoretic buffer (50 × TAE stock solution): take Tris 242g, glacial acetic acid 57.1mL, adds 100mL 0.5mol/L EDTA solution (pH8.0), is settled to 1L, with front with distilled water diluting for 1 × working fluid.
SYBR Green I dyestuff (electrophoresis level) working fluid: by the proportional arrangement of 1:3, gets TAE electrophoresis working buffer liquid and SYBR Green I dyestuff.
2, the structure of standard plasmid
Adopt the method for full genome synthesis (being synthesized by JaRa bio tech ltd, Shanghai), build CN standard substance, mutated-genotype plasmid (T/T type), wild-type genotype plasmid (G/G type), then make heterozygous genes masterplate (G/T type) with the mixing of the two equal proportion.
3, the Design and synthesis of HRM primer
According to the CN sequence that Genbank has delivered, the primer of application DNAstar software design specific PCR.Primer is synthesized by JaRa bio tech ltd, Shanghai.Concrete primer is as follows:
F1：5’- AAGGAGTTTGTGGAGGAGTTCATC-3’；
R1: 5’-GAGAGGTGCCCAGGAGGTATC-3’。
The product size of primer is 82bp, and annealing temperature is 60 DEG C.
4, the preliminary foundation of HRM detection method
The genotypic defining method of the blood sample DNA of (1) 243 holstein cow is, the tracing pattern of reference standard plasmid is determined.
(2) homozygosity and the mixing plasmid DNA of getting the wild of each standard plasmid of 1 μ L and sudden change do template respectively, carry out pcr amplification according to following system.PCR system is 30 μ L, comprises the ddH of 2 × Mix buffer of 15 μ L, the dNTP of 5 μ L, each 0.5 μ L of upstream and downstream primer, 1 μ L template DNA, 13 μ L 2o.
Reaction conditions is 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, and 60 DEG C of annealing 30s, 72 DEG C extend 30s, and 40 circulations, react and carry out on Roche480 quantitative real time PCR Instrument, carry out HRM analysis after reaction terminates.HRM analyzes and adopts Roche480 quantitative real time PCR Instrument, and testing conditions is 95 DEG C of sex change 1min, 40 DEG C of renaturation 1min, 50 DEG C keep 10s, from 50 DEG C, gather melting curve data with 0.01 DEG C/s heat-up rate, collection 40 secondary data per second, terminates fluorescent collecting to 95 DEG C.
5, data analysis
The HRM analysis software adopting Roche 480 to carry is analyzed.First homogenization (normalization) is carried out to original melting curve, software can divide into groups to curve type automatically, the sample with identical or close tracing pattern will be classified as one group, then be showed the result of grouping by the differential display function of software.With the same sample organized of known standard plasmid, namely there is the genotype of this plasmid gene type.
Result as shown in accompanying drawing 3 and accompanying drawing 4, the two obvious difference of the normal type (T/T) of CN gene and saltant type (C/C).
Statistical result showed, in 243 increment product, CN normal type 238 parts, CN heterozygote 5 parts, CN saltant type 0 part.
The result that result and nested PCR method of the present invention combine the detection method gained checked order is consistent.
In sum, it is simple, easy to operate that the present invention utilizes nested PCR method to combine the detrimental method of order-checking detection ox citrullinemia, and accuracy is high, is applicable to the needs of quick, accurate, high-throughout detection technique.
SEQUENCE LISTING
 
<110> Agricultural University Of South China
 
The Primer composition that a <120> ox citrullinemia deleterious gene detects and test kit thereof and application
 
<130>
 
<160> 6
 
<170> PatentIn version 3.3
 
<210> 1
<211> 23
<212> DNA
<213> first time PCR upstream primer sequence
 
<400> 1
actgcatttt caagaccacc ctg 23
 
 
<210> 2
<211> 24
<212> DNA
<213> first time PCR downstream primer sequence
 
<400> 2
tctgggcggg aaccaacgat tgtc 24
 
 
<210> 3
<211> 21
<212> DNA
<213> second time PCR upstream primer sequence
 
<400> 3
attgaggaca tcagcaagga g 21
 
 
<210> 4
<211> 20
<212> DNA
<213> second time PCR downstream primer sequence
 
<400> 4
cacatacttg gctccttctc 20
 
 
<210> 5
<211> 420
<212> DNA
<213> first time PCR primer sequence
 
<400> 5
actgcatttt caagaccacc ctgttagctg cgtctgcctt agacacaggc catcggagac 60
 
tctgtcttct gacggtgccc tgtccttgcc aaggctccct cccatgggct gctctcccac 120
 
aggtgttcat tgaggacatc agcaaggagt ttgtggagga gttcatctgg ccggccatcc 180
 
agtccagcgc actgtacgag gaccgatacc tcctgggcac ctctctcgcc aggccctgca 240
 
tcgcccgcaa gcaggtggag atcgcccagc gagaaggagc caagtatgtg tctcacggcg 300
 
ccacaggaaa ggtgaggcca gcatgtgtgg ctggagaggg tgaggagggg ggctgccagg 360
 
gggaagtggg cagcggggcc tgatgggcgg tcctcggaca atcgttggtt cccgcccaga 420
 
 
<210> 6
<211> 162
<212> DNA
<213> second time PCR primer sequence
 
<400> 6
attgaggaca tcagcaagga gtttgtggag gagttcatct ggccggccat ccagtccagc 60
 
gcactgtacg aggaccgata cctcctgggc acctctctcg ccaggccctg catcgcccgc 120
 
aagcaggtgg agatcgccca gcgagaagga gccaagtatg tg 162
 
 

Claims (4)

1. a Primer composition for ox citrullinemia deleterious gene detection, is characterized in that, be made up of primer sets A and primer sets B; Described primer sets A is made up of primer 1 and primer 2; Described primer sets B is made up of primer 3 and primer 4; The nucleotide sequence of primer 1 ~ 4 is respectively as shown in SEQ ID NO.1 ~ 4.
2. the Primer composition of ox citrullinemia deleterious gene detection according to claim 1, it is characterized in that, described ox citrullinemia deleterious gene is the gene of the 86th amino acid codes generation nonsense mutation of argininosuccinate synthase gene.
3. the Primer composition of ox citrullinemia deleterious gene detection according to claim 1, it is characterized in that, the mol ratio of primer 1 and primer 2 is 1: 1, and the mol ratio of primer 3 and primer 4 is 1: 1.
4. a test kit for the Primer composition containing the arbitrary described ox citrullinemia deleterious gene detection of claims 1 to 3, is characterized in that, also include following component: archaeal dna polymerase, PCR reaction buffer, dNTP and aqua sterilisa.
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CN101265504A (en) * 2008-03-17 2008-09-17 中国农业大学 Kit for screening cattle citrullinemia carrier
CN102181529A (en) * 2011-03-04 2011-09-14 中国农业大学 Method for detecting deleterious genes of bovine citrullinemia and special primers thereof

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CN101265504A (en) * 2008-03-17 2008-09-17 中国农业大学 Kit for screening cattle citrullinemia carrier
CN102181529A (en) * 2011-03-04 2011-09-14 中国农业大学 Method for detecting deleterious genes of bovine citrullinemia and special primers thereof

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