CN109517908A - A kind of SNP site for the AB form variation type causing hemolytic blood transfusion reaction - Google Patents
A kind of SNP site for the AB form variation type causing hemolytic blood transfusion reaction Download PDFInfo
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- CN109517908A CN109517908A CN201910062490.0A CN201910062490A CN109517908A CN 109517908 A CN109517908 A CN 109517908A CN 201910062490 A CN201910062490 A CN 201910062490A CN 109517908 A CN109517908 A CN 109517908A
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- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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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 object of the present invention is to provide a kind of for detecting the SNP site for causing acute or delayed hemolytic transfusion reaction AB form variation type.It is abo blood group gene coding region by the 803rd bit base initiation codon, is the mutation of G > C.The present invention provides the new applications of abo blood group AwB type 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 AwB type gene mutation site.
Description
Technical field
The invention belongs to gene diagnosis product technique fields, and in particular to one kind causes acute or Delayed onset for detecting
The SNP site of the AB form variation type (AwB) of hemolytic blood transfusion reaction.
Background technique
Normal abo blood group is divided into A type, Type B, AB type and O-shaped four kinds of blood groups, but morphs in abo blood group gene
In the case where such as SNP, missing, insertion, repetition, cause abo blood group erythrocyte surface antigen to become with the antibody in blood plasma
To change, such as AwB type blood group in this ABO blood group system found in the present invention, Staphylococal Protein A is weaker than normal A type people on red blood cell,
But there are the antibody of anti-A1 cell in blood plasma, once infusion AB type human blood, easily occurs transfusion hemolytic reaction.
Hemolytic blood transfusion reaction (Hemolytic Transfusion Reaction；HTR) it is divided into acute molten with Delayed onset
Hemorrhagic transfusion reaction, the blood transfusion that ABO system is not harmonious is generally acute hemolytic transfusion reaction, very harmful.Generally transfusing blood
It is interior for 24 hours afterwards to occur, occur immediately more than after blood transfusion, be the blood transfusion side reaction of most serious, once occurring to rescue immediately, otherwise causes
Dead rate is very high.
Either at home or external, the mistake infusion of abo blood group is to cause acute hemolytic transfusion reaction the most normal
The factor seen.Anti- A or anti-B antibody may be generated in the serum of AwB type people.If input AwB type blood results in acute
Or Delayed onset immune hemolysis reaction.For such situation, it is necessary to emphasize the importance of prevention.AwB blood group is in neonatal hemolytic
It is also of great significance in disease diagnosis and antenatal exaination, when AwB type pregnant woman has bred non-homotype or non-O-shaped fetus, if serum
In there are IgG anti-A and/or anti-B, will lead to fetus and neonatal hemolytic disease occur, serious person can cause neonatal death.
Liu Jianling etc. reports that twin is B (A) blood group in family, is diagnosed as xeroderma pitmentosum after raw, if
It is related to B (A) blood group to there is no theoretical foundation that seek.Using the method for family line investigation, observes poor antigen and transmits situation in family,
Sometimes the property that can speculate poor antigen has certain help to identification hypotype.
As PCR amplification is in the introducing and development of blood group research field, the scholars from all over the world wish from
Science accurately parsing is obtained on molecular level, and Genotyping and sequencing analysis are carried out to the individual of ABO subgroup phenotype one after another.
Summary of the invention
Cause acute or delayed hemolytic transfusion reaction AB type for detecting the object of the present invention is to provide a kind of
The SNP site of anomaly (AwB), to make up the deficiencies in the prior art.
Family member of the applicant to an AwB type blood group gene in autosome 9q34.1-9q34.2 dominant inheritance
It is sequenced, has found the site for the pathogenic mutation that family member AB form variation type gene exists genetically, the site
Mutation causes the change of Staphylococal Protein A quality and quantity, and generates anti-A1 antibody by immunostimulation, once wrong blood occurs
The infusion (infusion AB type blood) of type, it is more likely that fatal harm occurs.
The present invention provides a kind of for detecting acute or delayed hemolytic transfusion reaction the AB form variation type of initiation
(AwB) SNP site is abo blood group gene coding region by the 803rd bit base initiation codon, is the mutation of G > C；
It is the product for being used to prepare detection AB form variation type the present invention also provides the purposes of above-mentioned SNP site；
The product, preferably PCR amplification detection kit；
Another aspect of the present invention provides a kind of PCR amplification detection kit for detecting ABO blood group system AB form variation type；
The PCR amplification detection kit, which includes at least, the molecular labeling for detecting above-mentioned SNP site；
As the preferred of embodiment, the molecular labeling is sequenced for the primer pair or probe or haplotype of PCR amplification
Primer；
Direct amplification and sequencing primer information wherein for detecting above-mentioned SNP site is as follows:
ABO-E7F:5'-TTCCTGGAGACGGCGGAGAA-3'SEQ ID NO:1
ABO-E7R:5'-GTGGTCGCGGAACTCCATGT-3'SEQ ID NO:2
It is as follows for detecting above-mentioned SNP site haplotype sequencing primer information:
ABO-E6m:5'-CGGGATCCATGTGGGTGGCACCCTGCCA-3'SEQ ID NO:3；
ABO-E7mo:5'-GGGCCTAGGCTTCAGTTACTC-3'SEQ ID NO:4.
The present invention provides the new applications of abo blood group AwB type genetic test, thus provide it is a kind of effectively avoid it is acute
The approach of gene diagnosis, prenatal gene screening and genetic counselling that hemolytic blood transfusion reaction occurs, application effect show the present invention
The SNP site and detection primer of provided gene can be effectively used for clinical patients peripheral blood and carry out abo blood group AwB type base
Because of the quick detection in mutational site.
Detailed description of the invention
Abo blood group AwB form variation type gene propositus's gene mutation site sequencer map.Fig. 1: normal A type reference sequences, ginseng
BGMUT (international blood group gene mutation library) is derived from than sequence；
Fig. 2: propositus's direct Sequencing figure；
Fig. 3: propositus's cloning and sequencing figure, the gene coding region propositus ABO the 803rd bit base by initiation codon are sent out
Mutation (nt803G > C) is given birth to.
Specific embodiment
Applicant has found pathogenic SNP site in an abo blood group AwB type gene family, and confirms the prominent of the gene
Change is to cause AB type blood group to morph to cause red blood cell Staphylococal Protein A weaker than normal A type human red blood cells Staphylococal Protein A, and in blood plasma
Generate anti-A1 antibody of the same race.Once inputting AB type blood, it is likely that cause acute hemolytic transfusion reaction, in order to accomplish in time
Detection and prevention, to facilitate the present invention.
A anomaly is located at chromosome 9q34.1-9q34.2, and (NCBI accession number is the transcribed mRNA at about 1065bp
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: from the prominent of abo blood group AwB form variation type gene propositus's genetic test confirmation AB form variation type (AwB)
Displacement point.
1, peripheral blood genomic DNA is extracted:
Meet national relevant policies regulation, and on the basis of sampling object agreement, extracts abo blood group AB form variation type
Gene family member peripheric venous blood 2-5ml, is put into EDTA-Na2In anticoagulant tube, -80 DEG C freeze it is spare；DNA, which is extracted, to be used
The DNA extraction kit of LIFE company, 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 LTE (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 AwB type 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'-TTCCTGGAGACGGCGGAGAA-3'；ABO-E7R:5'-GTGGTCGGAACTCCATGT-3' is sent out in propositus
For existing ABO gene in addition to GC heterozygosis does not occur for 803 bases of the 7th exon, remaining site meets A102B101 feature.Repeatedly sequencing
The result shows that there is the mutation really in the mutational site, it is not because of (Fig. 2) that amplification or sequencing mistake are introduced.
The verifying in the site embodiment 2SNP
3, haplotype sequencing is carried out to the 6th, 7 exon of ABO gene and the 6th introne, determines propositus's AB anomaly base
The mutational site of cause
PCR amplification target fragment primer: ABO-E6mo:5'-CGGGATCCATGTGGGTGGCACCCTGCCA-3'；ABO-
E7mo:5'-GGGCCTAGGCTTCAGTTACTC-3'.
PCR amplification primer can also select other primer pairs that can expand above-mentioned SNP site.
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'-CGGGATCCATGTGGGTGGCACCCTGCCA-3'；ABO-E7mo:5'-
GGGCCTAGGCTTCAGTTACTC-3' determines the haplotype sequence (Fig. 3) of sample.This sports a newfound mutation.It should
Mutation is not present in three following databases: single nucleotide polymorphism database (ftp: //ftp.ncbi.nih.gov/
Snp/database/), blood group gene mutation library (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/
Xslcgi.fcgi? cmd=bgmut/systems_info&system=abo), show that the mutation is very rare, which leads
The gene coding region A the 803rd bit base by initiation codon has been caused to result in the 268th amino acids by glycine by G > C
Become alanine；The mutation of amino acid occurs missing antigen so as to cause this AwB type individual Staphylococal Protein A epitope and weakens, and leads to this
Kind of individual does not need immunostimulation and can generate that anti-A1 antibody (innate immunity) of the same race can be generated in blood plasma, works as infusion
Acute hemolytic transfusion reaction will occur when the AB type blood of normal person, to jeopardize patient vitals.And nearly more than 400 example just
The Mutation Screening that the site is often carried out in the peripheral blood genomic DNA sample of locality crowd, does not find the mutation.
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, and direct Sequencing testing result shows A, B blood
The presence of type gene；It was found that except 803 bases of the 7th exon GC does not occur for the ABO gene of the Liang Ming family member of the propositus
Outside heterozygosis, remaining site meets A102B101 feature.Continue to carry out ABO gene the 7th to the Liang Wei family member of discovery SNP mutation
The haplotype of exon is sequenced, using said extracted a family member (father propositus) genomic DNA as template
PCR product is cloned into carrier T by the amplification that PCR target fragment is carried out with ABO-E7mo primer, and the multiple bacterium colonies of picking carry out single times
Type sequencing, haplotype sequencing primer use above-mentioned primer, determine the haplotype sequence of sample.Haplotype sequencing discovery, two
For the 803rd bit base of the position gene coding region family member A by G > C, resulting in the 268th amino acids from glycine becomes the third ammonia
Acid, it is completely the same with propositus, and all there is anti-A1 antibody of the same race in this family individual blood plasma.
Pass through above-mentioned analysis, it was demonstrated that the primer of inventor's design can be used to detect whether individual abo blood group gene is sent out
Raw A gene mutation, to there is the danger of acute blood transfusion type hemolytic reaction after potential transfuse blood.
Sequence table
<110>Qingdao downtown blood station
<120>a kind of SNP site for the AB form variation type for causing hemolytic blood transfusion reaction
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ttcctggaga cggcggagaa 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtggtcgcgg aactccatgt 20
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cgggatccat gtgggtggca ccctgcca 28
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gggcctaggc ttcagttact c 21
Claims (7)
1. it is a kind of for detecting the SNP site for causing acute or delayed hemolytic transfusion reaction AB form variation type, it is special
Sign is that it is G > C's that the SNP site, which is abo blood group gene coding region by the 803rd bit base initiation codon,
Mutation.
2. application of the SNP site described in claim 1 in the product of preparation detection AB form variation type.
3. application as claimed in claim 2, which is characterized in that the product is PCR amplification detection kit.
4. a kind of PCR amplification detection kit for detecting ABO blood group system AB form variation type；It is characterized in that, the PCR expands
Increase the molecular labeling that detection kit includes detection SNP site described in claim 1.
5. kit as claimed in claim 4, which is characterized in that the molecular labeling is the primer pair of PCR amplification, visits
Needle or haplotype sequencing primer.
6. kit as claimed in claim 5, which is characterized in that the primer pair of the PCR amplification, sequence SEQ
ID NO:1、SEQ ID NO:2。
7. kit as claimed in claim 5, which is characterized in that the sequence of the haplotype sequencing primer is SEQ ID
NO:3 or SEQ ID NO:4.
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| CN201910062490.0A CN109517908A (en) | 2019-01-23 | 2019-01-23 | A kind of SNP site for the AB form variation type causing hemolytic blood transfusion reaction |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304311A (en) * | 2019-12-24 | 2020-06-19 | 青岛市中心血站 | SNP marker for detecting O gene mutation in ABO blood group system |
| CN112063704A (en) * | 2020-10-15 | 2020-12-11 | 青岛市中心血站 | SNP (single nucleotide polymorphism) site related to A variant in ABO (anaerobic-baffled oxide) blood group system |
| CN113249466A (en) * | 2021-06-01 | 2021-08-13 | 青岛大学附属医院 | SNP site of RHCE blood group gene for triggering hemolytic transfusion reaction and severe neonatal hemolytic disease |
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| CN113249466A (en) * | 2021-06-01 | 2021-08-13 | 青岛大学附属医院 | SNP site of RHCE blood group gene for triggering hemolytic transfusion reaction and severe neonatal hemolytic disease |
| CN113249466B (en) * | 2021-06-01 | 2022-04-15 | 青岛大学附属医院 | SNP site of RHCE blood group gene for triggering hemolytic transfusion reaction and severe neonatal hemolytic disease |
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Application publication date: 20190326 |