CN108410972A - A kind of gene parting detecting reagent for mankind Rh 23 gene locis of blood group - Google Patents
A kind of gene parting detecting reagent for mankind Rh 23 gene locis of blood group Download PDFInfo
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Abstract
The invention belongs to people's nucleic acid in vitro detection technique fields, and in particular to the gene parting detecting reagent of the mankind No. 5, No. 19, No. 21 and 23 gene locis of X/Y chromosomes.The present invention devises the composite PCR amplification system that 23 gene locis being distributed on the mankind No. 5, No. 19, No. 21 and X/Y chromosomes are carried out at the same time with Genotyping first, and wherein PCR amplification primer sets are：SEQ ID No.1~SEQ ID No.53；The gene parting detecting reagent of the present invention includes Primer composition container, PCR reaction mother liquor containers；Primer composition container includes that primer SEQ ID No.1~SEQ ID No.53 compositions store liquid.The present invention realizes the Single tube amplification to 23 gene locis, and the gene loci amplification in the interior and different fluorescence channels of each fluorescence channel is balanced, and parting collection of illustrative plates is good；And cost, manpower and time can be saved significantly on, improve working efficiency.
Description
Technical field
The invention belongs to people's nucleic acid in vitro detection technique fields, and in particular to having polymorphism in human gene group DNA
The detection of the genotype of gene loci, and in particular to one using multiplex polymerase chain re-action to the mankind No. 5, No. 19, No. 21
And 23 gene locis carry out the kit of Rh blood group gene partings detection on X/Y chromosomes.
Background technology
Rh feminine genders and the positive two kinds of blood groups of Rh can be distinguished according to the presence or absence of Rh factors, this blood group system is known as Rh blood groups
System.Rh is two letters of head of rhesus macaque (Rhesus Macacus) foreign language title.The scientists such as blue moral stainer are 1940
When year does zoopery, it is found that there are the antigenic substances of Rh blood groups on the red blood cell in rhesus macaque and majority's body, so name
's.There are Rh agglutinogen persons on every blood of human body red blood cell, is Rh positive.Otherwise it is feminine gender.
Rh blood group systems are the most blood group systems of polymorphism in human erythrocyte's blood group system, it has the immune of height
Source property is the important blood group system for being only second to abo blood group, has important clinical meaning.The Rh system antigens that can be identified at present
There are more than 40 to plant, but during clinical common antigen has 5, according to its antigenicity from being followed successively by D to weak by force>C>E>c>E, wherein D antigens
Presence or absence is often used to represent Rh positive or negatives, and writing Rh (D)+or Rh (D)-, alphabetical D add round bracket to indicate antigen (no
Italic should be write by adding when round bracket indicates corresponding gene).Draw in the clinical blood transfusion treatment of Rh (D) Population with Negative and Maternal-fetal immunity
Have great importance in the neonatal hemolytic risen.Rh (D) negative patient ratio in China's Chinese Han Population is about 0.3-0.5%,
But Rh (D) negative patients ratio can be up to 10% in Some Minority Races crowd.Foreign literature is reported in the crowd of Caucasia
Rh (D) negative patient ratios can be up to 10-18%.
In recent years, as RhD the and RhCE genes of coding Rh albumen are cloned, are sequenced, in Rh blood group system molecular genetics
The research of mechanism and the parting context of detection of Rh blood groups achieve great progress.By Southern hybridization analysis, really
Determine Rh " locus " to be made of two homologous structure genes：One coding Rh (D) polypeptide, another coding (CcEe) polypeptide.
After research finds that the cDNA of 1 single RhCE goes to K562 cells, this overall length peptide chain can both express E/e and C/c antigens.
There is RhD and RhCE genes high homology, the nucleotide sequence of the two to only have about 7% difference, and scholar thinks that this may be
Repeat function originating from common ancestral gene.
The genetic polymorphism of RhD is complicated and enriches, including the complete missing of RhD genes, the excalation of RhD genes, base
The point mutation of cause and RhD Ψ pseudogenes etc.；Not only genetic mechanism is various for Rh (D) antigen, and there is also a variety of variations for Rh (D) antigens
Body, including weak D types, part D types and the weak phenotypes of other D at least define the part D of 42 kinds of weak D types and 6 classifications at present.
Domestic and international several scholars have found that weak D types donor blood, which is accidentally defeated by the negative receptors of Rh (D), with blood group serology method generates primary
The case of anti-D also has more because of the immune report 15-20 for generating anti-D of part D.The research of Caucasia crowd is found
The overwhelming majority is accounted in Rh (D) Population with Negative caused by RhD gene delections, the Caucasia crowd of only 0.2-1% shows as weak D
Type, wherein about 95% is weak DI-IV types.And it is domestic because lacking ripe detection method, domestic people Rh blood groups molecular background with
Crowds Distribute still lacks system research, but has research to point out that the Rh blood group inheritance mechanism of China is various and complicated, and China is differently
Find that China Rh (D) Population with Negative only has about 50% and causes for RhD gene delections in the research in area, it is to put to have about 20-30%
Type is dissipated, wherein being 1227G more than 95%>The mutation of A, residue is weak D phenotypes there are about 10-20%, wherein 60% or more is weak D
In weak D15 types, 30% or more is the DVI Type3 types in the D of part.
In view of in Rh blood groups, part D, weak D are in clinical blood transfusion and Rh blood groups fetus, new life caused by female tire blood group incompatibility
The uncertainty of importance and these Rh (D) antigenic variants in conventional serological detection in youngster's hemolytic disease,
College of American Pathologists (College of American Pathologists, CAP) in 2015, U.S.'s blood transfusion medical resource
Dispatch claims jointly for the committee (Transfusion Medicine Resource Committee, TMRC), should reinforce to Rh blood
The application of type, the genotyping technique of especially weak D types in clinic.
Invention content
The purpose of the present invention aiming in Rh blood groupings in the demand and clinic of the Genotypings such as part D, weak D
Demand to the detection of Rh blood group Common genes partings, providing one kind can be simultaneously on the mankind No. 5, No. 19, No. 21 and X/Y chromosome
23 gene locis carry out the kit of Rh blood group gene partings detection.
First aspect present invention provide it is a kind of to 23 gene locis of the mankind No. 5, No. 19, No. 21 and X/Y chromosomes simultaneously
The composite PCR amplification system of Genotyping is carried out, 23 gene locis are：AMEL、SID1、SID2、SID3、SID4、
Ref19、Box、pEx01、IN01、IN02、Ex03、IN03、IN07、IN08、wD1_gT、wD2_Gc、wD3_Cg、wD4_Cg、
wD15_aG、1227Ga、Ex10、Dc、Ee.According to each gene loci amplicon length distribution range, by 23 gene positions
Point is divided into two groups, and first group is 12 gene locis：Box、IN07、pEx01、IN02、IN08、Ref19、SID1、SID2、
Ex03, IN01, IN03, SID3, second group is 11 gene locis：AMEL、1227Ga、wD2_Gc、wD15_aG、wD1_gT、
Dc、wD3_Cg、wD4_Cg、Ex10、Ee、SID4.The composite PCR amplification system includes the PCR described in first group and second group
Amplimer, wherein first group includes the corresponding PCR amplification primer in the following group site：Site Box, corresponding primer sequence such as SEQ ID
Shown in No.1, SEQ ID No.2；Site IN07, corresponding primer sequence is as shown in SEQ ID No.3, SEQ ID No.4；Site
PEx01, corresponding primer sequence is as shown in SEQ ID No.5, SEQ ID No.6；Site IN02, corresponding primer sequence such as SEQ ID
Shown in No.7, SEQ ID No.8；Site IN08, corresponding primer sequence is as shown in SEQ ID No.9, SEQ ID No.10；Site
Ref19, corresponding primer sequence is as shown in SEQ ID No.11, SEQ ID No.12；Site SID1, corresponding primer sequence such as SEQ
Shown in ID No.13, SEQ ID No.14；Site SID2, corresponding primer sequence such as SEQ IDNo.15, SEQ ID No.16 institutes
Show；Site Ex03, corresponding primer sequence is as shown in SEQ ID No.17, SEQ ID No.18；Site IN01, corresponding primer sequence
As shown in SEQ ID No.19, SEQ ID No.20；Site IN03, corresponding primer sequence such as SEQ ID No.21, SEQ ID
Shown in No.22；Site SID3, corresponding primer sequence is as shown in SEQ IDNo.23, SEQ ID No.24；
There is a primer glimmering using FAM in wherein first group of gene loci in the corresponding PCR amplification primer in each site
Light element is marked；Draw the wherein corresponding upstreams gene loci Box, pEx01, IN02, IN08, Ref19, SID1, SID2, SID3
Object is all made of FAM fluoresceins and is marked；The corresponding downstream primer of gene loci IN07, Ex03, IN01, IN03 is all made of FAM
Fluorescein is marked.
Second group includes the corresponding PCR amplification primer in the following group site：Site AMEL, corresponding primer sequence such as SEQ
Shown in IDNo.25, SEQ ID No.26；Site 12 27Ga, corresponding primer sequence such as SEQ ID No.27, SEQ ID No.28,
Shown in SEQ ID No.29；Site wD2_Gc, corresponding primer sequence such as SEQ ID No.30, SEQ ID No.31, SEQID
Shown in No.32；Site wD15_aG, corresponding primer sequence is as shown in SEQ ID No.33, SEQ ID No.34, ID No.35；Position
Point wD1_gT, corresponding primer sequence is as shown in SEQ ID No.36, SEQ ID No.37, SEQ ID No.38；Site Dc, accordingly
Primer sequence is as shown in SEQ ID No.39, SEQ ID No.40；Site wD3_Cg, corresponding primer sequence such as SEQ ID
No.41, SEQ ID No.42, shown in SEQ ID No.43；Site wD4_Cg, corresponding primer sequence such as SEQ ID No.44, SEQ
Shown in ID No.45, SEQ ID No.46；Site Ex10, corresponding primer sequence such as SEQ ID No.47, SEQ ID No.48 institutes
Show；Site Ee, corresponding primer sequence is as shown in SEQ ID No.49, SEQID No.50, SEQ ID No.51；Site SID4, phase
Answer primer sequence as shown in SEQ ID No.52, SEQ ID No.53；
At least a primer uses in the corresponding PCR amplification primer in each site in wherein second group of gene loci
TAMRA fluoresceins are marked；Wherein gene loci AMEL, 1227Ga, wD2_Gc, wD1_gT, wD3_Cg, Ex10, SID4 couple
The downstream primer answered is marked using TAMRA；The corresponding sense primer of gene loci wD15_aG, Dc, wD4_Cg, Ee uses TAMRA
Label.
Preferably, the corresponding composite PCR amplimer of 23 gene locis and fluorescein decorative features such as table 1 and table
Shown in 2：
Table 1
Table 2
Each group of gene loci pcr amplification product length does not overlap each other.It is dense to adjust each gene loci PCR primer work
Degree, makes in same group between homozygote or segment peak height differs within 40% between heterozygote.First group and second group of gene loci
The working concentration of corresponding PCR amplification primer is 80~900nmol/L.
In PCR amplification system of the present invention, primer oligonucleotides shown in SEQ ID No.1 to SEQ ID No.53
The dry powder of the PCR amplification primer of sequence or solution composition.
23 chromogene sites provided by the present invention composite amplification system, RHD is detected including 18 respectively
Common 4 positions in mutational site genetic marker Quality Control site on gene extron and RHD genes, 1 gender Quality Control site, 1
Quality Control site on other chromosomes.
(1) 18 detection site is respectively to Rh blood group common mutations types, including RHD genes lack entirely, common RhD genes
Excalation, 1227G>A, weak D 1-4 and weak D15 types are detected.
(2) sites Ref19：The site expands No. 19 chromosome segments using one couple of PCR primers, is with No. 19 chromosomes
PCR system Quality Control.
(3) 4 short tandem repeats (Short tandem repeat, STR) polymorphic markers be located at No. 5,
On No. 20 and No. 21 chromosomes, str locus seat is 3 nucleotide or 4 nucleotide core recurring units, is shown in Chinese Han Population
Go out high polymorphism, can be used for sample confirmation and linkage inheritance is traced to the source, while being also experiment Quality Control site.
Quality Control of (4) 1 gender sites for determining detection sample gender and experiment.
It is a plurality of specific that 23 gene locis of multiplexed PCR amplification system detectio provided by the present invention are respectively distributed to the mankind
Chromosome on, formed 6 function modules：Respectively sample molecules label, PCR inner quality controls, RhD genes lack detection work(entirely
Can area, type detection function area and C/c, E/e detection function area and are diffused at weak D by excalation detection function area.Wherein, RhD
Gene lacks detection function entirely and excalation detection function area is located at FAM fluorescence channels, and genetic marker has half-quantitative detection
Function, each genetic marker all have with reference to peak, and to calculate the copy number of RhD specific amplification segments with reference to peak peak height.Weak D
Type, diffuse type, the inspection of C/c, E/e are located at TAMRA fluorescence channels, be only used as qualitative detection, carry out corresponding site saltant type
While qualitative detection, wild-type allele as to lack entirely or the supplement of excalation inspection result verify.
Second aspect of the present invention provides PCR amplification system described in first aspect present invention and is preparing to the mankind No. 5,19
Number, 23 gene locis carry out the purposes in Rh blood group gene parting detecting reagents on No. 21 and X/Y chromosomes.
Third aspect present invention provides a kind of to 23 gene locis on the mankind No. 5, No. 19, No. 21 and X/Y chromosomes
Rh blood group gene parting detecting reagents are carried out, it includes the primer sets containing PCR amplification system described in first aspect present invention
Close object container and PCR reaction mother liquor containers, wherein
The Primer composition container contains the storage liquid for the primer that sequence is SEQ ID No.1 to SEQ ID No.53,
Store a concentration of corresponding primer working solution concentration of each primer in liquid 4 times；
PCR reaction mother liquors containing 2 times of reaction densities for carrying out PCR reactions in the PCR reaction mother liquors container, PCR are anti-
It includes the archaeal dna polymerase that enzyme activity is 0.5~5U, the magnesium ion of a concentration of 1~5mmol/L, a concentration of 40~400 μ to answer mother liquor
The Tris-HCl buffer solutions of the dNTP of mol/L and a concentration of 20~100mmol/L.
Fourth aspect present invention provides a kind of application method of detection kit described in third aspect present invention, specifically such as
Under：
(1) human gene group DNA is extracted, ultraviolet spectrometry quantifies a concentration of 2~10ng/ μ L of genomic DNA；
(2) multiplexed PCR amplification：Overall reaction system is 20 μ L, and reaction system composition is as follows：
(3) PCR reactions (such as ABI9700, ABI9600, Bio-Rad C1000) on regular-PCR instrument carry out, and PCR is anti-
Answer condition as follows：95 DEG C 15 minutes start after, 95 DEG C 30 seconds, 60 DEG C 40 seconds, 72 DEG C 60 seconds, totally 28 cycle, then 72 DEG C guarantor
Temperature 20 minutes, then 4 DEG C of heat preservations；
(4) PCR product takes 1 μ L, routinely operates and carries out Capillary Electrophoresis on genetic analyzer.
(5) data after electrophoresis are analyzed using exclusive data analysis software (such as GeneMapper), can be obtained every
The parting collection of illustrative plates and data of one gene loci.
Wherein, the DNA profiling refers to human genome DNA.Various conventional methods can be used (such as in human genome DNA
Human gene group DNA's extracts kit etc. of Chelex-100 methods, paramagnetic particle method, phenol chloroform method and extensive stock) from the following group
Knit or sample in extract and obtain：Human blood, blood cake, sperm, seminal stain, saliva, salivary stain, amniotic fluid, hair, musculature, bone
Deng.Preferably, in the PCR amplification system of 20 μ L, DNA profiling amount can obtain preferably expanding in the range of 2ng to 5ng with
Genotyping result.
Fifth aspect present invention provides detection kit described in third aspect present invention in nondiagnostic progress Rh blood groups
Purposes in Genotyping detection.
Genetic analysis can be used in the pcr amplification product of 23 gene locis composite PCR amplification system provided by the present invention
Instrument (such as AB3130XL, AB3500Genetic Analyzer) carries out capillary electrophoresis analysis, and the data after electrophoresis can be
It is analyzed in the Data Analysis Software such as GeneMapper, obtains the parting collection of illustrative plates and data of each gene loci.
Beneficial effects of the present invention：
Due in establishing multiplexed PCR amplification systematic procedure, with the increase of detected gene loci number, each pair of primer
Between interfere with each other and can also increase.The present invention realizes the Single tube amplification to 23 gene locis by the design of optimization, each glimmering
Gene loci amplification in optical channel and in different fluorescence channels is balanced, and parting collection of illustrative plates is good.
Using the gene parting detecting reagent of 23 gene locis of mankind Rh blood groups provided by the present invention, one can be passed through
Secondary response can effectively solve the confirmation work that common Rh blood groupings and sample are traced to the source, either PCR amplification link also
It is genetic analyzer detection, can all saves significantly on cost, manpower and time, to improves working efficiency.
Description of the drawings
23 bases that the full missing carrier DNA's sample of RhD genes list copy is obtained using kit of the present invention as an example of Fig. 1
Because of site parting collection of illustrative plates.
23 genes that RhD-RhCE fusions carrier DNA's sample is obtained using kit of the present invention as an example of Fig. 2
Site parting collection of illustrative plates.
RhD genes lack 23 bases that Rh blood group negative patient DNA samples are obtained using kit of the present invention entirely as an example of Fig. 3
Because of site parting collection of illustrative plates.
RhD genes do not lack as an example of Fig. 4, and wD15 carriers of mutation DNA samples use 23 that kit of the present invention obtains
A gene loci parting collection of illustrative plates.
Specific implementation mode
Content for a better understanding of the present invention is detected as specific reality with 23 gene loci partings in human blood sample sheet below
Example is applied to be described further.It should be understood that following specific examples is merely to illustrate the present invention, rather than limiting the invention.
The nucleotides sequence included by Primer composition in PCR reaction systems reagent of the present invention is classified as SEQ ID No.1 extremely
The primer of SEQ ID No.53 is inventor by design of primers, is closed according to general conventional in this field by DNA Synesis Company
At method synthesis, it is then formulated as 80~900nmol/L of working concentration.
Amplified reaction carries out on 9700 thermal cyclers of ABI in the present embodiment, and electrophoresis and detection are in 3500 heredity of ABI
It is carried out on analyzer, data analysis uses GeneMapper ID v3.2 softwares.Use other reagents and material such as internal standard,
POP7, capillary electrophoresis buffer, Hi-Di, allelic ladder (ladder) are that those skilled in the art are commonly conventional
Material.
Embodiment
1：It is prepared by this genomic DNA of human blood sample
Test sample is contributed under informed consent by volunteer.Peripheric venous blood 1mL, EDTA anti-freezing is adopted by medical routine.
Genomic DNA is extracted using the human peripheral genome extraction agent box of QIAGEN companies, elution volume is 100 microlitres, is used
Ultraviolet spectrometry quantitative instrument is quantitative, dilution gene group DNA concentration to 2.5ng/ μ L.
2：PCR system is prepared
PCR reaction systems are prepared by following system (overall reaction system is 20 μ L)：
Primer composition container, PCR reaction mother liquor containers are taken out in kit.It is multiplied by above-mentioned reactant by overall reaction number
Requirement is individually reacted in system calculates separately out required PCR reaction mother liquors amount, Primer composition amount, PCR reaction auxiliary liquid
Amount and ddH2O amounts, mentioned reagent is mixed in a 1.5mL EP pipe it is even, dispensed by 19 μ L of each PCR reaction tubes,
Number, is then separately added into 1 μ L of sample DNA template, and mixing again by sample number.
3：PCR reacts
PCR reactions are carried out using ABI 9700PCR instrument.
PCR conditions are as follows：95 DEG C 15 minutes start after, 95 DEG C 30 seconds, 60 DEG C 40 seconds, 72 DEG C 60 seconds, totally 28 cycle, so
Keep the temperature 20 minutes for 72 DEG C afterwards, then 4 DEG C of heat preservations.
4：Capillary Electrophoresis parting detects
(1) (+10 μ L Hi-Di of 1 μ L internal standards) × (sample number+1) is taken to be made into mixed liquor, by often 10 μ L packing of pipe after mixing
In 96 hole board pores.1 μ L allelic ladders (Ladder) are added in a wherein hole.
(2) 1 μ L PCR products is taken to be added in the board pore of corresponding 96 hole by sample number.
(3) 95 DEG C of sample is denaturalized 5 minutes, then rapid cooled on ice 4 minutes.
(4) sample is put into the sample tray of Genetic Analyser, routinely parameter carries out Capillary Electrophoresis (referring to heredity
Analyzer manufacturer operational manual).
(5) Capillary Electrophoresis terminates, and parting collection of illustrative plates is obtained with GeneMapper softwares analysis experimental data, referring to Fig. 1, figure
2, shown in Fig. 3 and Fig. 4.
Mono- copy missing carrier (the parting ccDdEe of RhD as shown in Figure 1:RhD*D-) genomic DNA parting collection of illustrative plates,
FAM label 12 gene locis (be followed successively by from left to right Box, IN07, pEx01, IN02, Ref19, IN08, SID1, SID2,
Ex03, IN01, IN03, SID3), 11 gene locis of TAMRA labels (be followed successively by from left to right AMEL, 1227Ga, wD15_aG,
WD2_Gc, wD3_Cg, wD4_Cg, wD1_gT, Dc, Ex10, Ee, SID4), uniform understands.
Analysis：
(1) PCR quality control of procedure：There is corresponding amplified production peak in SID1, SID2, SID3, SID4, Ref19 and C1401L,
The sites AMEL are in XY.PCR processes are normal.
(2) Box approximations 1:2 peak types, prompt the full missing of RhD gene lists copy, pEx01, IN01, Ex03, IN03, IN07,
IN08 is consistent with the sites Box, prompts for single copy and lacks.IN02 is approximation 2:1 peak (being cc types in RhCE), supports RhD mono-
Copy missing.
(3) channels TAMRA：Each site 1227Ga, wD15_aG, wD2_Gc, wD3_Cg, wD4_Cg, wD1_gT is wild
Homozygous, Ex09 exists with Ex10 amplified peaks, and the sites Dc RhD specific peaks exist with c specific peaks.The sites Ee are in heterozygosis
Type.The sites C are without amplified production peak.
RhD-RhCE fusions (parting CcDdEe as shown in Figure 2:RhD*D-CE (2-9) D2) carrier's parting figure
Spectrum.12 gene locis of FAM labels, 11 gene locis of TAMRA labels, uniform understand.
Analysis：
(1) PCR quality control of procedure：There is corresponding amplified production peak in SID1, SID2, SID3, SID4, Ref19 and C1401L,
The sites AMEL are in XY.PCR processes are normal.
(2) Box approximations 1:1 peak type prompts RhD genes to lack entirely；PEx01 is approximation 1:1 peak type；IN01 is approximation 1:1
Peak type；IN02 is approximation 3:1 peak (being Cc types in RhCE), shows that RhD intrones 2 have single copy missing；Ex03、IN03、IN07、
IN08 is approximation 2:1 peak type prompts corresponding region to there is single copy and lacks.
(3) channels TAMRA：Each site 1227Ga, wD15_aG, wD2_Gc, wD3_Cg, wD4_Cg, wD1_gT is wild
Homozygous, Ex09 exists with Ex10 amplified peaks, and the sites Dc RhD specific peaks exist with c specific peaks.The sites Ee are in heterozygosis
Type.The sites C amplified production peak exists.
RhD genes as shown in Figure 3 lack (parting ccddEe entirely:RhD*D-/RhD*D-) Rh negative blood groups person parting
Collection of illustrative plates.12 gene locis of FAM labels, 11 gene locis of TAMRA labels, uniform understand.
Analysis：
(1) PCR quality control of procedure：There is corresponding amplified production peak in SID1, SID2, SID3, SID4, Ref19 and C1401L,
The sites AMEL are in XY.PCR processes are normal.
(2) sites Box be in downstream box single peak type, prompt RhD genes lack entirely it is homozygous, pEx01, IN01, IN02,
Ex03, IN03, IN07, IN08 are in RhCE specific fragment single peak types, consistent with the sites Box, and it is pure to support that RhD genes lack entirely
Mould assembly.
(3) channels TAMRA：Each site 1227Ga, wD15_aG, wD2_Gc, wD3_Cg, wD4_Cg, wD1_gT, Ex10 is equal
Without amplified production peak, the sites Dc RhD specific peaks disappear, and it is homozygous to support that Box site RhD are lacked entirely.The c of the sites Dc RhCE is special
Anisotropic peak exists.The sites Ee are in heterozygous.The sites C are without amplified production peak.
WD15 as shown in Figure 4 is mutated (parting CcDdEE:RhD*845A) carrier's parting collection of illustrative plates.The 12 of FAM labels
A gene loci, 11 gene locis of TAMRA labels, uniform understand.
Analysis：
(1) PCR quality control of procedure：There is corresponding amplified production peak in SID1, SID2, SID3, SID4, Ref19 and C1401L,
The sites AMEL are in XX.PCR processes are normal.
(2) Box approximations 1:1 peak type prompts RhD genes without full missing, pEx01, IN01, Ex03, IN03, IN07, IN08
It is approximation 1:1 peak, it is consistent with the sites Box.IN02 is approximation 3:1 peak (RhCE is Cc types), it is consistent with the sites Box.
(3) channels TAMRA：The sites wD15_aG are in heterozygous, and 1227Ga, wD2_Gc, wD3_Cg, wD4_Cg, wD1_gT are each
Site is wild homozygous, and Ex09 exists with Ex10 amplified peaks, and the sites Dc RhD specific peaks exist with c specific peaks.Ee
Site short-movie section E is homozygous.The sites C amplified production peak exists.
A kind of gene parting detecting reagent of 23 gene locis of mankind Rh blood groups proposed by the invention can with
PCR instrument and the laboratory steady implementation of genetic analysis, detection time are about 3-4 hours.Meanwhile reagent provided by the present invention
It can easily be produced and in biomedical testing agency in biotech company for detecting, have industrialization and popularization
The condition of application.
The present invention is described with reference to its specific embodiment.To those skilled in the art, according to front
Description, may be the present invention various modifications or transformation, including (but being not limited only to)：Change different groups of other fluorescein labels,
Change fluorescein-labeled primer (label downstream primer is such as changed by label sense primer), according to each gene loci equipotential base
Because range change genome grouping arrangement, according to others PCR reaction mother liquors to PCR amplification condition, primer reaction density into
Row optimization, and change recommended reaction system etc..It will be apparent that above-mentioned modification or transformation are to those skilled in the art
All it is possible, but these modifications and transformation are without departing from the spirit and scope of the present invention.
Sequence table
<110>Shanghai Wuseshi Medical Research Co., Ltd.
<120>A kind of gene parting detecting reagent for mankind Rh 23 gene locis of blood group
<130> Z001039180031CN
<160> 53
<170> PatentIn version 3.5
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agaagaggtt ctgggtgttg gt 22
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acatttctgc attcctctag tgaca 25
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gccactggcc actgagtgta 20
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ttacaagggc attggcactt aatag 25
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ataagtgggt gttcctagtg atgg 24
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attaggtgcc caacagtgtt tg 22
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caggtaattg gtatgtgaaa aagtgt 26
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gaatggaggg aaagtgggaa g 21
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ccctgggctc tgtaaagaat agtg 24
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atcagagctt aaactgggaa gctg 24
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actcataaac agcaagtcaa catatattct 30
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ggagaaaaag gatttctgtt gacat 25
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attccatatt ttaagattta ggagcagac 29
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attcaggagg cgtggctgta gg 22
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caggaggcgt ggctgttga 19
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gttgtctagt ttcttaccgg cacgt 25
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attgcagtag tgagctggca catca 25
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attgccaaca ccgcactgta ca 22
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gccaacaccg cactgttcc 19
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tgataactta gcaaatggct attgga 26
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attcgagacc aacctgacgc ac 22
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gagacggaca caggatgagg tc 22
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attgagacgg acacaggatg acctg 25
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tgctatttgc tcctgtgacc agtt 24
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aatccaagga ctatcagggc gtg 23
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gacaaactgg gtatcgttgg tg 22
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attcagacaa actgggtatc gttgttc 27
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attgtaatga gacatttagg ctgtttcaa 29
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aatggtgaga ttctcctcaa agagt 25
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attgattgga cttctcagca cagc 24
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gcttagagct tcctttgcca tct 23
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tgtcgttgag gctacccagt c 21
Claims (8)
1. a kind of composite PCR that 23 gene locis of the mankind No. 5, No. 19, No. 21 and X/Y chromosomes are carried out at the same time with Genotyping
Amplification system, 23 gene locis are divided into two groups, and first group is 12 gene locis：Box、IN07、pEx01、IN02、
IN08、Ref19、SID1、SID2、Ex03、IN01、IN03、SID3；Second group is 11 gene locis：AMEL、1227Ga、
wD2_Gc、wD15_aG、wD1_gT、Dc、wD3_Cg、wD4_Cg、Ex10、Ee、SID4；The composite PCR amplification system includes the
PCR amplification primer described in one group and second group, wherein first group includes the corresponding PCR amplification primer in the following group site：
Site Box, corresponding primer sequence is as shown in SEQ ID No.1, SEQ ID No.2；
Site IN07, corresponding primer sequence is as shown in SEQ ID No.3, SEQ ID No.4；
Site pEx01, corresponding primer sequence is as shown in SEQ ID No.5, SEQ ID No.6；
Site IN02, corresponding primer sequence is as shown in SEQ ID No.7, SEQ ID No.8；
Site IN08, corresponding primer sequence is as shown in SEQ ID No.9, SEQ ID No.10；
Site Ref19, corresponding primer sequence is as shown in SEQ ID No.11, SEQ ID No.12；
Site SID1, corresponding primer sequence is as shown in SEQ ID No.13, SEQ ID No.14；
Site SID2, corresponding primer sequence is as shown in SEQ ID No.15, SEQ ID No.16；
Site Ex03, corresponding primer sequence is as shown in SEQ ID No.17, SEQ ID No.18；
Site IN01, corresponding primer sequence is as shown in SEQ ID No.19, SEQ ID No.20；
Site IN03, corresponding primer sequence is as shown in SEQ ID No.21, SEQ ID No.22；
Site SID3, corresponding primer sequence is as shown in SEQ ID No.23, SEQ ID No.24；
The wherein corresponding sense primers of gene loci Box, pEx01, IN02, IN08, Ref19, SID1, SID2, SID3 are all made of
FAM fluoresceins are marked；The corresponding downstream primer of gene loci IN07, Ex03, IN01, IN03 be all made of FAM fluoresceins into
Line flag；
Second group includes the corresponding PCR amplification primer in the following group site：
Site AMEL, corresponding primer sequence is as shown in SEQ ID No.25, SEQ ID No.26；
Site 12 27Ga, corresponding primer sequence is as shown in SEQ ID No.27, SEQ ID No.28, SEQ ID No.29；
Site wD2_Gc, corresponding primer sequence is as shown in SEQ ID No.30, SEQ ID No.31, SEQ ID No.32；
Site wD15_aG, corresponding primer sequence is as shown in SEQ ID No.33, SEQ ID No.34, ID No.35；
Site wD1_gT, corresponding primer sequence is as shown in SEQ ID No.36, SEQ ID No.37, SEQ ID No.38；
Site Dc, corresponding primer sequence is as shown in SEQ ID No.39, SEQ ID No.40；
Site wD3_Cg, corresponding primer sequence is as shown in SEQ ID No.41, SEQ ID No.42, SEQ ID No.43；
Site wD4_Cg, corresponding primer sequence is as shown in SEQ ID No.44, SEQ ID No.45, SEQ ID No.46；
Site Ex10, corresponding primer sequence is as shown in SEQ ID No.47, SEQ ID No.48；
Site Ee, corresponding primer sequence is as shown in SEQ ID No.49, SEQ ID No.50, SEQ ID No.51；
Site SID4, corresponding primer sequence is as shown in SEQ ID No.52, SEQ ID No.53；
The corresponding downstream primers of wherein gene loci AMEL, 1227Ga, wD2_Gc, wD1_gT, wD3_Cg, Ex10, SID4 use
TAMRA is marked；The corresponding sense primer of gene loci wD15_aG, Dc, wD4_Cg, Ee is marked using TAMRA.
2. composite PCR amplification system as described in claim 1, which is characterized in that first group corresponding with second group of gene loci
PCR amplification primer working concentration be 80~900nmol/L.
3. composite PCR amplification system as described in claim 1, which is characterized in that primer is by SEQ ID No.1 to SEQ ID
The dry powder of the PCR amplification primer of oligonucleotide sequence shown in No.53 or solution composition.
4. a kind of carrying out the detection of Rh blood group gene partings to 23 gene locis on the mankind No. 5, No. 19, No. 21 and X/Y chromosomes
Kit, which is characterized in that the kit includes that Primer composition containing PCR amplification system described in claim 1 is held
Device and PCR reaction mother liquor containers, wherein
The Primer composition container contains the storage liquid for the primer that sequence is SEQ ID No.1 to SEQ ID No.53, storage
4 times of a concentration of corresponding primer working solution concentration of each primer in liquid；
PCR reaction mother liquors containing 2 times of reaction densities for carrying out PCR reactions in the PCR reaction mother liquors container, PCR reactions are female
Liquid include enzyme activity be 0.5~5U archaeal dna polymerase, the magnesium ion of a concentration of 1~5mmol/L, a concentration of 40~400 μm of ol/L's
The Tris-HCl buffer solutions of dNTP and a concentration of 20~100mmol/L.
5. purposes of the kit described in claim 4 in nondiagnostic progress Rh blood group gene partings detection.
6. a kind of application method of kit as claimed in claim 4, which is characterized in that including step：
(1) human gene group DNA is extracted, ultraviolet spectrometry quantifies a concentration of 2.5~10ng/ μ L of genomic DNA；
(2) multiplexed PCR amplification：Overall reaction system is 20 μ L, and reaction system composition is as follows：
(3) PCR reactions are carried out in regular-PCR instrument, and PCR reaction conditions are as follows：95 DEG C 15 minutes start after, 95 DEG C 30 seconds, 60 DEG C
40 seconds, 72 DEG C 60 seconds, totally 28 cycle, then 72 DEG C keep the temperature 20 minutes, then 4 DEG C heat preservation；
(4) PCR product takes 1 μ L, routinely operates and carries out Capillary Electrophoresis on genetic analyzer；
(5) to the data after electrophoresis utilize exclusive data analysis software, obtain each gene loci parting collection of illustrative plates and
Data.
7. application method as claimed in claim 6, which is characterized in that the DNA profiling refers to human genome DNA, described
Human genome DNA is in human blood, blood cake, sperm, seminal stain, saliva, salivary stain, amniotic fluid, hair, musculature, bone
It is one or more.
8. PCR amplification system described in claim 1 is being prepared to 23 genes on the mankind No. 5, No. 19, No. 21 and X/Y chromosomes
Site carries out the purposes in the kit of Rh blood group gene partings detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114507724A (en) * | 2022-03-29 | 2022-05-17 | 河南兰德施坦纳基因科技有限公司 | Primer group and kit for detecting human erythrocyte Rh blood group genotyping and application |
-
2018
- 2018-05-17 CN CN201810471969.5A patent/CN108410972B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| CAROLINA TRUCCO BOGGIONE等: "Genotyping approach for non-invasive foetal RHD detection in an admixed population", 《BLOOD TRANSFUS》 * |
| CATHERINE A HYLAND等: "Non-invasive fetal RHD genotyping for RhD negative women stratified into RHD gene deletion or variant groups: comparative accuracy using two blood collection tube types", 《PATHOLOGY》 * |
| 孙学兰等: "新乡地区48例ＲhD阴性汉人ＲHD基因多态性研究", 《现代预防医学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114507724A (en) * | 2022-03-29 | 2022-05-17 | 河南兰德施坦纳基因科技有限公司 | Primer group and kit for detecting human erythrocyte Rh blood group genotyping and application |
| CN114507724B (en) * | 2022-03-29 | 2023-08-22 | 河南兰德施坦纳基因科技有限公司 | Primer group and kit for detecting Rh blood group genotyping of human red blood cells and application of primer group and kit |
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