CN109182511A - It is a kind of for detecting the SNP site of abo blood group anomaly - Google Patents
It is a kind of for detecting the SNP site of abo blood group anomaly Download PDFInfo
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- CN109182511A CN109182511A CN201811341632.9A CN201811341632A CN109182511A CN 109182511 A CN109182511 A CN 109182511A CN 201811341632 A CN201811341632 A CN 201811341632A CN 109182511 A CN109182511 A CN 109182511A
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- snp site
- blood group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention provides a kind of SNP site for causing abo blood group to morph, and is abo blood group gene coding region by initiation codon the 526th, 703,796 or the 803rd bit base;Wherein the 526th bit base is CC homozygosis, the 703rd bit base is GG homozygosis, the 796th bit base is CA heterozygosis, the 803rd bit base is GC heterozygosis.The present invention provides the new applications of abo blood group anomaly genetic test, to provide the approach that one kind effectively avoids the gene diagnosis of acute hemolytic transfusion reaction generation, prenatal gene screening and genetic counselling, application effect shows that the SNP site of gene provided by the present invention and detection primer can be effectively used for the quick detection that clinical patients peripheral blood carries out abo blood group anomaly gene mutation site.
Description
Technical field
The invention belongs to gene diagnosis product technique fields, and in particular to a kind of for detecting the SNP of abo blood group anomaly
Site, i.e. one kind cause abo blood group gene to morph, and cause the SNP site of acute hemolytic transfusion reaction.
Background of invention
Acute hemolytic transfusion reaction (Acute Hemolytic Transfusion Reaction;AHTR) generally defeated
It is interior for 24 hours after blood to occur, occur immediately more than after blood transfusion, is the blood transfusion side reaction of most serious, once occur to rescue immediately, otherwise
Lethality is very high.As long as input 5~10ml ABO incompatible blood may occur in which manifest symptom;Transfusion volume is more than 200ml, it will
Cause serious consequence.Acute hemolytic transfusion reaction clinically often shows as intravascular hemolysis, and patient has fever, shiver with cold, evil
, then there is hemoglobinuria, haemoglobinaemia in the heart, backache, and hypotensive shock should all be thought of as acute intravascular hemolysis
Transfusion reaction should stop rapidly transfusing blood and carry out every inspection.This process can cause complement activation simultaneously, start blood coagulation system
System discharges bradykinin and catecholamine, induces DIC;Circulation and renal failure even occurs;Therefore usually low blood pressure is stopped
Gram, DIC, acute renal failure be known as acute intravascular hemolysis triad.
Abo blood group anomaly patient is because the immune sexual factor stimulation such as blood transfusion or gestation generates anti-A1 or anti-B antibody, when again
It will cause acute or delayed hemolytic transfusion reaction when the secondary blood inputted containing corresponding antigens.In gene level, A type and B
The most common type is A101 and B101 respectively.B101 8 compared with A101 at base mutate, key position nt526G > C,
Nt703A > G, nt796A > C and nt803C > G, cause replacement R176G, G235S, L266M and G268A of following amino acid.
Tetra- amino acid of 176R, 235G, 266L and 268G determine glycosyl transferase A (GTA, N- acetyl-D- galactolipin aminopherase)
The haplotype of specificity, (526C+703G+796C+803G) 4 nucleotide combinations is known as AAAA type;176G, 235S, 266M and
Tetra- amino acid of 268A determine glycosyl transferase B (GTB, D- galactosyl transferase) specificity, (526G+703A+796A+803C)
The haplotype of 4 nucleotide combinations is known as BBBB type.The enzyme that universal A allele encodes is set to AAAA type by people, generally
The enzyme of B allele coding be set to BBBB type, A and B here represents A type glycosyl transferase specificity or the transfer of Type B glycosyl
The amino acid of enzyme spcificity, AAAA type are followed successively by 176 arginine, 235 glycine, 266 leucines, 268 glycine;
BBBB type is followed successively by 176 glycine, 235 serines, 266 methionines, 268 alanine.In addition to most common
AAAA type and BBBB type, it has now been found that and the blood group gene anomaly being proved there are also AAAB type, AABA type, BBAB type,
BBBA type and BABB type.
Summary of the invention
The object of the present invention is to provide a kind of for detecting the SNP site of abo blood group anomaly, which can be used for
Abo blood group anomaly gene is detected, to make up the deficiencies in the prior art.
Family of the applicant to an abo blood group anomaly gene in autosome 9q34.1-9q34.2 dominant inheritance
Member is sequenced, and has found family member's gene in the presence of pathogenic mutation SNP site genetically, the mutation meeting in the site
The change of antigen levels and quality is caused, and anti-A1 antibody can be generated by immunostimulation, causes blood transfusion to cause acute molten
Hemorrhagic transfusion reaction.
The present invention provides a kind of SNP site for causing abo blood group to morph, and is abo blood group gene coding region by
Beginning codon the 526th, 703,796 or the 803rd bit base;Wherein the 526th bit base is CC homozygosis, the 703rd bit base is GG pure
It closes, the 796th bit base is CA heterozygosis, the 803rd bit base is GC heterozygosis.
Another aspect of the present invention provides a kind of method of detection variation abo blood group, is by whether there are SNP above-mentioned
It puts to realize.
It is a kind of for detecting the PCR amplification primer of above-mentioned SNP site, sequence information is as follows:
ABO-E6F:5'-CATGTGGGTGGCACCCTG-3'(SEQ ID NO:1)
ABO-E6R:5'-TCGCCACTGCCTGGGTCTC-3'(SEQ ID NO:2);
The sequence information of another pair primer is as follows:
ABO-E7F:5'-CGTCCGCCTGCCTTGCAG-3'(SEQ ID NO:3)
ABO-E7R:5'-GGACGGACAAAGGAAACAGAGTT-3'(SEQ ID NO:4)
It is as follows for detecting above-mentioned SNP site haplotype amplification and sequencing primer information:
ABO-E6mo:5'-CATGTGGGTGGCACCCTG-3'(SEQ ID NO:5)
ABO-E7mo:5'-GGACGGACAAAGGAAACAGAGTT-3'(SEQ ID NO:6).
The present invention provides the new applications of abo blood group anomaly genetic test, so that providing one kind effectively avoids urgency
Property hemolytic blood transfusion reaction occur gene diagnosis, prenatal gene screening and genetic counselling approach, application effect shows this hair
The SNP site and detection primer of gene provided by bright can be effectively used for clinical patients peripheral blood and carry out abo blood group variation
The quick detection of type gene mutation site.
Detailed description of the invention
Fig. 1: the normal A type reference sequences figure of abo blood group;
Fig. 2: the normal Type B reference sequences figure of abo blood group;
Fig. 3: four critical sites direct Sequencing figures of propositus;
Fig. 4: four critical sites cloning and sequencing figures of propositus;Wherein the gene coding region propositus ABO is by initiation codon
It is (526C+703G+796A+803C) 4 nucleotide combinations, list that son, which plays the 526th, the 703rd, the 796th and the 803rd,
Times type is AABB type.
Specific embodiment
Applicant has found the SNP site of mutation in an abo blood group anomaly gene family, and confirms the SNP of mutation
Site is to cause to generate anti-A1 antibody of the same race to cause the Disease-causing gene of acute hemolytic transfusion reaction, to facilitate this
Invention.
Related anomaly is located at chromosome 9q34.1-9q34.2, and (NCBI is stepped on the transcribed mRNA at about 1065bp
Record number is NM_020469.2), directly translation forms the protein that 355 amino acid forms.
The present invention is described in detail below with reference to embodiment.
Embodiment 1: by abo blood group anomaly propositus's genetic test, confirm SNP site.
1, peripheral blood genomic DNA is extracted:
Meet national relevant policies regulation, and on the basis of sampling object agreement, extracts abo blood group anomaly gene
Family member peripheric venous blood 2-5ml, is put into EDTA-Na2In anticoagulant tube, -80 DEG C freeze it is spare;DNA extracts public using LIFE
The DNA extraction kit of department, concrete operations process are as follows:
The EDTA anticoagulation frozen takes 500ul to be put in centrifuge tube, isometric erythrocyte splitting is added after room temperature thawing
Liquid (pH8.0) is vortexed and mixes 5 minutes, and 12000rpm is centrifuged 5 minutes, abandons supernatant.It repeats that the whirlpool erythrocyte cracked liquid 500ul is added
Rotation mixes 5 minutes, and 12000rpm is centrifuged 5 minutes, abandons supernatant.
Sediment, which is vortexed, to be mixed 5 minutes, and karyorhexis liquid 50ul is added, is vortexed and mixes 5 minutes, be placed in 56 DEG C of metal baths 15 and divide
Clock.Sample is taken out from water-bath, and albumen is added and removes liquid 135ul.Vortex mixes well, as 4 DEG C 30 minutes.
12000rpm is centrifuged 5 minutes at room temperature, and Aspirate supernatant (about 200ul) is into a new centrifuge tube.Ice ethyl alcohol is added
500ul shakes centrifuge tube repeatedly, until white DNA pellet object is precipitated.Room temperature 12000rpm is centrifuged 2 minutes, abandons supernatant.To DNA
75% ethyl alcohol is added in precipitating, and rinsing is primary, and room temperature 12000rpm is centrifuged 3 minutes, abandons supernatant, is placed in room temperature remaining second of volatilizing
Alcohol is eventually adding 50 μ L TE (pH8.0), 4 DEG C of overnight dissolving DNAs.
To the DNA row agarose gel electrophoresis of extraction, and application ultraviolet specrophotometer is in 260nm and 280nm colorimetric, detection
DNA purity and concentration.
2, direct sequencing finds the mutational site of propositus's anomaly gene
PCR amplification target fragment: reaction condition and reaction system:
(1) PCR reaction condition: 95 DEG C initial denaturation 10 minutes;94 DEG C 60 seconds, 63 DEG C 90 seconds, 72 DEG C 60 seconds, 10 circulation;
94 DEG C 60 seconds, 61 DEG C 90 seconds, 72 DEG C 60 seconds, 25 circulation;72 DEG C of extension 10min, 10 DEG C of amplified production heat preservations.
(2) reaction system: (TAKARA Ex-Taq polymerase)
The expansion of the genomic DNA template and above-mentioned ABO-E7F, ABO-E7R primer of propositus is carried out using the reaction system
Increase reaction.
PCR product sequencing: being sequenced above-mentioned PCR product using routine Sanger PCR sequencing PCR, wherein applying primer pair
ABO-E7F:5'-CGTCCGCCTGCCTTGCAG-3';ABO-E7R:5'-GGACGGACAAAGGAAACAGAGTT-3' is formerly demonstrate,proved
Person has found that abo blood group gene coding region the 526th bit base by initiation codon is CC homozygosis, the 703rd bit base is GG pure
It closes, the 796th bit base is CA heterozygosis, the 803rd bit base is GC heterozygosis.Multiple sequencing result show the mutational site be not because
For (Fig. 3) for expanding or being sequenced mistake introduction.
3, haplotype sequencing is carried out to the 6th, 7 exon of ABO gene and the 6th introne, determines propositus's anomaly gene
Mutational site
PCR amplification target fragment primer: ABO-E6mo:5'-CATGTGGGTGGCACCCTG-3';ABO-E7mo:5'-
GGACGGACAAAGGAAACAGAGTT-3'。
The reaction condition and reaction system of PCR amplification target fragment:
(1) PCR reaction condition: 95 DEG C initial denaturation 5 minutes;95 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 50 seconds, 40 circulation;72
DEG C extend 5min, 10 DEG C of amplified production heat preservation.
(2) reaction system: (TAKARA LA-Taq polymerase)
The clone of the genomic DNA template and inventor's design that carry out every family member respectively using the reaction system surveys
The primer of sequence carries out amplification reaction.PCR product is cloned into carrier T, the multiple bacterium colonies of picking carry out haplotype sequencing, Dan Bei
Type sequencing primer uses ABO-E6mo:5'-CATGTGGGTGGCACCCTG-3';ABO-E7mo:5'-
GGACGGACAAAGGAAACAGAGTT-3' determines the haplotype sequence (Fig. 4) of sample.The SNP site form is not present in following
Three databases in: single nucleotide polymorphism database (ftp: //ftp.ncbi.nih.gov/snp/database/), blood
Type gene mutation library (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi? cmd=
Bgmut/systems_info&system=abo), show that the SNP site is very rare, and result in encoding glycophorin
The nucleotide of composition changes, which shows by initiation codon the 526th, the 703rd, the 796th and
803 are (526C+703G+796A+803C) 4 nucleotide combinations, and haplotype is AABB type, and corresponding amino acid is followed successively by
176 arginine, 235 glycine, 266 methionines, 268 alanine.The change of amino acid causes individual Staphylococal Protein A
Epitope moiety missing, anti-A1 antibody (innate immunity) of the same race can be generated by causing this individual not need immunostimulation, and
Easily missing inspection is Type B, acute hemolytic transfusion reaction can occur when being transfused normal Type B blood, to jeopardize patient vitals.And
The Mutation Screening that the site is carried out in the peripheral blood genomic DNA sample of nearly 300 normal local crowds, does not find that this is prominent
Become.
The propositus family member collected by the blood transfusion medical research of Qingdao downtown blood station, extracts their peripheral blood base
Because of a group DNA, the amplification of PCR target fragment is carried out as template and ABO-E7F, ABO-E7R primer using above-mentioned DNA, it is conventional
Sanger PCR sequencing PCR carries out direct Sequencing to primer pair PCR product using above-mentioned this, finds the Liang Ming family member of the propositus
Abo blood group gene deposit the SNP site form found in the present invention, i.e. coding region the 526th alkali by initiation codon
Base is CC homozygosis, the 703rd bit base is GG homozygosis, the 796th bit base is CA heterozygosis, the 803rd bit base is GC heterozygosis.Continue into
The sequencing of the haplotype of the 6th, 7 exon of row ABO gene and the 6th introne, using the genomic DNA of said extracted as template and
ABO-E6mo, ABO-E7mo primer carry out the amplification of PCR target fragment, and PCR product is cloned into carrier T, the multiple bacterium colonies of picking
Haplotype sequencing is carried out, haplotype sequencing primer uses above-mentioned primer, determines the haplotype sequence of sample.Haplotype sequencing
It was found that coding region is (526C+703G+796A+ for the 526th, the 703rd, the 796th and the 803rd by initiation codon
803C) 4 nucleotide combinations, haplotype are AABB type (Fig. 4).This result and propositus are completely the same, and this two famous expert
It is all there is anti-A1 antibody of the same race in individual blood plasma.
Pass through above-mentioned analysis, it was demonstrated that the primer of SNP site and detection that the present invention is screened can be used to detect a
Whether body abo blood group gene occurs gene mutation, so that it is determined that being that acute blood transfusion type haemolysis is anti-after there is potential transfuse blood
The danger answered.
Sequence table
<110>Qingdao downtown blood station
<120>a kind of for detecting the SNP site of abo blood group anomaly
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
catgtgggtg gcaccctg 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tcgccactgc ctgggtctc 19
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cgtccgcctg ccttgcag 18
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggacggacaa aggaaacaga gtt 23
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
catgtgggtg gcaccctg 18
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ggacggacaa aggaaacaga gtt 23
Claims (8)
1. a kind of SNP site for causing abo blood group to morph, which is characterized in that the SNP site is abo blood group gene
Coding region is by initiation codon the 526th, 703,796 or the 803rd bit base;Wherein the 526th bit base is CC homozygosis, the 703rd
Bit base is GG homozygosis, the 796th bit base is CA heterozygosis, the 803rd bit base is GC heterozygosis.
2. application of the SNP site described in claim 1 in detection abo blood group variation.
3. the application in product of the SNP site preparation described in claim 1 for detecting abo blood group variation.
4. a kind of method of detection variation abo blood group, which is characterized in that the method is to detect whether that there are claims 1
The SNP site.
5. a kind of method for detecting SNP site described in claim 1, which is characterized in that the method is using PCR amplification
Primer or sequencing primer detect SNP site described in claim 1.
6. a kind of PCR amplification primer for detecting SNP site described in claim 1, which is characterized in that the primer, thereon
Downstream primer sequence is respectively SEQ ID NO:1, SEQ ID NO:2.
7. a kind of PCR amplification primer for detecting SNP site described in claim 1, which is characterized in that the primer, thereon
Downstream primer sequence is respectively SEQ ID NO:3, SEQ ID NO:4.
8. a kind of sequencing primer for detecting SNP site described in claim 1, which is characterized in that the sequence of the primer is
SEQ ID NO:5 or SEQ ID NO:6.
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Cited By (3)
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CN109517908A (en) * | 2019-01-23 | 2019-03-26 | 青岛市中心血站 | A kind of SNP site for the AB form variation type causing hemolytic blood transfusion reaction |
CN110042165A (en) * | 2019-03-21 | 2019-07-23 | 苏州西山生物技术有限公司 | Macaque abo blood group methods of genotyping |
CN111593120A (en) * | 2020-07-02 | 2020-08-28 | 青岛市中心血站 | SNP site of AB blood type variant for triggering hemolytic blood transfusion reaction |
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CN101921834A (en) * | 2010-05-17 | 2010-12-22 | 浙江省血液中心 | Polymerase chain reaction-sequence based typing (PCR-SBT) method for ABO blood type genotyping and reagent |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109517908A (en) * | 2019-01-23 | 2019-03-26 | 青岛市中心血站 | A kind of SNP site for the AB form variation type causing hemolytic blood transfusion reaction |
CN110042165A (en) * | 2019-03-21 | 2019-07-23 | 苏州西山生物技术有限公司 | Macaque abo blood group methods of genotyping |
CN110042165B (en) * | 2019-03-21 | 2022-08-12 | 苏州西山生物技术有限公司 | Macaque ABO blood type genotyping method |
CN111593120A (en) * | 2020-07-02 | 2020-08-28 | 青岛市中心血站 | SNP site of AB blood type variant for triggering hemolytic blood transfusion reaction |
CN111593120B (en) * | 2020-07-02 | 2021-04-13 | 青岛市中心血站 | SNP site of AB blood type variant for triggering hemolytic blood transfusion reaction |
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