CN103937806B - A kind of Rh blood group DEL type RHD838G > A allelotrope and detection method thereof - Google Patents
A kind of Rh blood group DEL type RHD838G > A allelotrope and detection method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of <i>RHD</iGreatT. GreaT.GT838G & gt; A allelotrope and detection method thereof, belong to molecular biological analysis detection technique field.It goes out the primer sequence that can increase to the region comprising the target site with mutant gene sequence by PCR-Auele Specific Primer technical project, special in specific <i>RHD</iGreatT. GreaT.GT838G & gt by the regioselectivity ground of gene amplification method to the target site comprising mutator gene; A allelotrope carries out pcr amplification detection.The present invention adopts molecular biology method to carry out the detection of gene level, and the method substantially increases susceptibility, the specificity of detection, easy, quick.Owing to there is the difference of <i>RHD</iGreatT. GreaT.GT gene between different nationalities; on the basis of correlative study; according to Chinese <i>RHD</iGreatT. GreaT.GT gene molecule background, specially for <i>RHD</iGreatT. GreaT.GT838G & gt; A allelotrope designs.The present invention not only has important clinical practice meaning in the deficiency making up serological technique, but also has research application value widely.
Description
Technical field
The present invention relates to a kind of RHD838G>A allelotrope and detection method thereof, be specifically related to a kind of method of the known mutations gene be doped in wild type gene bunch, belong to molecular biological analysis detection technique field.
Background technology
Rh blood group is system that is the most complicated in human erythrocyte's blood group system, most polymorphism, is also the main erythrocyte blood type causing clinical blood transfusion reaction and serious hemolytic disease of newborn.Found more than 50 kind of Rh blood group antigen at present, wherein RhD antigen has very strong immunogenicity, is to be produced by RHD genes encoding, is the emphasis of blood group research.D antigen whether detected according to Surface of Erythrocytes clinically, Rh blood group antigen are divided into the RhD positive and the negative two large classes of RhD.
Japanese scholars Okubo in 1984 etc. are detected by absorption and elution test further to the RhD negative sample confirmed through direct agglutination test and indirect antihuman globulin test (IAT), find that a part of individual erythrocyte surface still can detect the existence of D antigen, be referred to as D and diffuse type (DEL type).It belongs to the one of weak D type, and antigenicity is extremely weak.Compared with white people, DEL type ratio in Chinese RhD Population with Negative is very high.Shao etc. are to the large sample investigation result display of ShenZhen,GuangDong area, and in the RhD Population with Negative that DEL type is identified by conventional serological in China, shared ratio is about 26%, and the ratio of Taiwan's scholars report this area is 32.6%.Wagner etc. carry out large sample investigation and find that DEL type is rare in the European white race.The not agnate difference because there is genetic background in different areas, also there is larger difference in the genotype of DEL blood group D antigen.In recent years about the research of DEL blood group RHD gene becomes the focus of erythrocyte blood type research.Find have new allelotrope to exist in DEL blood group RHD gene successively, and there is obvious hereditary property.
At present, the common detection methods of DEL blood group D antigen adopts serology salt water law and indirect antihuman globulin test and absorption and elution test to identify.Also there is unstable in the method not only complex operation, result.In addition, due to the impact of disease or other factors, red corpuscle result when serology shaping of some individuality is difficult to judge; Chronic long blood transfusion patients Serological presents the phenomenon in " the mixing visual field " sometimes; When the red corpuscle or red corpuscle sample that cannot obtain sample are not enough, as fetal blood group qualification, forensic identification hangover etc., Serologic detection can not obtain correct result.
Specificity and the working method of the Antibodies Against Rhesus D Antigen of use is depended on the detection of method to DEL blood group of immunoserology, reliability is lower, and being not suitable for clinical large-scale application due to complex steps, reliable method is that the method that applying gene detects detects DEL blood group.Now determining DEL blood group D antigen genotype by detecting DNA, not only there is in the deficiency making up serological technique important clinical practice meaning, but also there is research application value widely.DEL blood group D antigen constantly has new allelotrope to be found in recent years, this research group have also discovered the neomorph that 1 routine DEL blood group RHD genotype RHD838G>A suddenlys change recently, and establishes corresponding detection method for newfound RHD838G>A allelotrope.
Summary of the invention
The object of the present invention is to provide Rh blood group DEL type RHD838G>A mutator gene and detection method thereof, by detecting RHD838G>A transgenation and carry out Rh blood group DEL type gene type assay and as the phenotypic evaluation index of DEL blood group.
Detection method of the present invention is the change in the specific sequence site utilizing mankind DEL blood group different genotype, designs primer sequence targetedly, carries out that polymerase chain reaction come.
According to technical scheme provided by the invention, a kind of Rh blood group DEL type RHD838G>A allelotrope, its wild type gene DNA sequence dna is as shown in SEQIDNO:1, and mutator gene DNA sequence dna is as shown in SEQIDNO:2.
The sequence obtaining nucleotide sequence and RHD wild-type from testing sample compares, and whether check sudden change is 838G → A sudden change.The coded protein of described RHD mutator gene has the change of 280A → T aminoacid sequence.
The allelic detection method of described Rh blood group DEL type RHD838G>A, is optionally increased by the detection zone of gene amplification method to the target site comprising mutator gene, detects the presence or absence of this mutator gene thus; Described detection method is as follows:
(1) DNA in sample to be checked is extracted;
(2) with this DNA for template, near RHD838G>A mutator gene coding region design PCR primer carry out PCR reaction, obtain PCR reaction product;
(3) the nucleotide sequence composition of PCR reaction product is measured;
(4) sequence of nucleotide sequence and RHD wild type gene is compared, determined whether that 838G → A suddenlys change.
By database BLAST comparison function, carry out translating to determine whether there is 280A → T amino acid mutation site according to normal single open reading frame.PCR reaction product can be checked order by sequenator by the nucleotide sequence composition of described PCR reaction product.
The allelic detection method of described Rh blood group DEL type RHD838G>A, concrete steps are as follows:
(1) design of primers: for RHD allelic sequences, designs a pair specific oligonucleotide primer sequence, and wherein forward primer is RHD838A-F, and its sequence is as shown in SEQIDNO:3; Reverse primer is RHD838A-R, and its sequence is as shown in SEQIDNO:4; Amplified production fragment length is 163bp; And introduce 1 pair of internal reference primer sequence as DNA sample amplification positive internal control photograph, forward primer is β-globin-F, and its sequence is as shown in SEQIDNO:5; Reverse primer is β-globin-R, and its sequence is as shown in SEQIDNO:6, and internal reference amplified production fragment length is 102bp;
(2) RHD838G>A allelotrope increases expansion: reaction system cumulative volume 50 μ L; Wherein containing the Taq polysaccharase 1.0 μ L of PCR5 × damping fluid 10 μ L, DNA profiling 200ng, 1U/ μ L, MgCl
2final concentration is 2.0mmol/L, dNTP final concentration is 200nmol/L, and the final concentration of specific forward primer and reverse primer is 200nmol/L; Add sterilization distilled water to reaction system cumulative volume 50 μ L;
Above-mentioned reaction system reacted in PCR instrument, reaction conditions: 95 DEG C of denaturation 5min, then 94 DEG C of sex change 30s successively, then 62 DEG C of annealing 40s, 72 DEG C extend 1min, totally 35 circulations;
(3) RHD838G>A allelotrope detects: carry out electrophoresis with sepharose to thing of the increasing of step (2) gained being expanded production, whether have object fragment to detect it.
Beneficial effect of the present invention: the present invention adopts molecular biology method to carry out the detection of gene level, highly sensitive and high precision test can be doped in the presence or absence of the mutator gene in gene pool.Owing to there is the difference of RHD gene between different nationalities, on the basis of correlative study, according to Chinese RHD gene molecule background, design for RHD838G>A allelotrope newfound in Chinese specially.By detecting RHD838G>A transgenation and carry out Rh blood group DEL type gene type assay and as the phenotypic evaluation index of DEL blood group.The present invention not only has important clinical practice meaning in the deficiency making up serological technique, but also has research application value widely.
Accompanying drawing explanation
Fig. 1 is that in embodiment, RHD838G>A allelotrope detects gel electrophoresis figure.
Fig. 2 is RHD838G>A allelic mutation sequencer map in embodiment.
Embodiment
In the present invention, " wild type gene " refers to and does not undergo mutation, and has the gene containing genetic information of normal function originally; " mutated genes " refers to the gene that there occurs sudden change; " sudden change " refers to the change of the nucleotide sequence of DNA, RNA etc., the base substitution being equivalent to use in genetics, insert in, lack, inversion, repetition, transposition etc.; " gene pool " refers to the genes population be made up of lots of genes.
The preparation of embodiment 1DNA template
Adopt the test kit purchased to extract Whole Blood Genomic DNA, concrete steps are as follows:
(1) get aseptic 2.0mL centrifuge tube one, add 1mL cell pyrolysis liquid.
(2) shake gently so through the whole blood sample of EDTA anti-freezing, until thoroughly mix; Then drawing 500 μ L blood samples adds above-mentioned containing in the centrifuge tube of cell pyrolysis liquid, topples over centrifuge tube 5-6 mixing gently.
(3) incubated at room 10 minutes (period puts upside down centrifuge tube 2-3 mixing).
(4) centrifugal 5 minutes of 12000rpm room temperature.
(5) as far as possible supernatant liquor is moved slowly with pipettor and abandon clean, note not by the white mass sucking-off of two-phase intersection.
(6) turbula shaker (Votex) is used acutely to mix, until white corpuscle resuspended (10-15 second).
(7) in re-suspended cell solution, karyorhexis liquid 300 μ L is added.Inhale with liquid transfer gun head and put solution 5-6 cracking white corpuscle.Now solution should become very thickness.If visible cell agglomerate after mixing, then solution is placed in 37 DEG C and hatches until agglomerate dissipates.If to hatch after 1 hour still visible cell agglomerate, then separately add karyorhexis liquid 100 μ L and repeat to be placed in 37 DEG C and hatch.
(8) in karyorhexis thing, albumen precipitation liquid 100 μ L is added, with turbula shaker concuss 10-20 second.
(9) centrifugal 5 minutes of 12000rpm room temperature.
(10) its supernatant liquor is gone to adding in the 2.0mL centrifuge tube of 300 μ L room temperature Virahols of reference numeral.
(11) put upside down gently to mix solution, until white linear DNA forms precipitation.
(12) centrifugal 1 minute of 12000rpm room temperature.
(13) abandoning supernatant, adding isopyknic room temperature volume concentration with sample size is the ethanol of 70%, puts upside down centrifuge tube gently for several times.
(14) with pipettor as far as possible ethanol is moved slowly abandon clean.Centrifuge tube is placed in 50 DEG C of baking 5-10 minute, allows remaining ethanol volatilization clean as far as possible.
(15) in centrifuge tube, add the DNA lysate of 50-100 μ L, mix gently.
(16) DNA extraction effect is assessed with 1% agarose gel electrophoresis, Nanodrop nucleic acid instrument detection level, quantitatively to 20ng/ μ L ,-20 DEG C of preservations.
Embodiment 2RHD838G>A allele detection technique scheme:
Instrument: Veriti96 type PCR instrument, BIO-RADGelDocXR+ type gel imaging instrument (Bio Rad Laboratories), gel-electrophoretic apparatus (Beijing 61 company).
Reagent: QIAampDNA extracts test kit (German Qiagen company); DNAIsolationKit extracts test kit (Beijing PELFREEZ company); PCR damping fluid, dNTP, Taq enzyme (American AB I company); Primer and probe are synthesized by the Shanghai biological company limited of raw work.
(1) design of primers: the RHD gene (sequence number: BN000065) recorded according to American National Bioinformatics Institute (NCBI) GenBank and the allelic sequence of the various RHD of Chinese, by Oligo6.0 primer software design primer, finally determine 1 couple of specific oligonucleotide primer sequence (RHD838A-F, CGGTGTTGGCAGGAGGCGTGG; RHD838A-R, CTTCAGCCAAAGCAGAGGAGG), amplified production fragment length is 163bp;
And introduce 1 pair of internal reference primer sequence and to increase positive internal control (β-globin-F, GTGCACCTGACTCCTGAGGAGA as DNA sample; β-globin-R, CCTTGATACCAACCTGCCCAG), amplified production fragment length is 102bp.
RHD838G>A allelotrope detect primer sequence and atopic as shown in table 1.
Table 1
Note: * F=forward primer; R=reverse primer.
The position of the exon base sequence involved by # primer nucleotide sequences refers to the whole numbering that puts in order of 10 exons by ATG start code; The position of involved intron base sequence refers to the single numbering that puts in order of each intron.
(2) reaction conditions: reaction cumulative volume: 50 μ L, containing PCR5 × damping fluid 10 μ L, DNA profiling 200ng, Taq polysaccharase (1U/ μ L) 1.0 μ L, MgCl
2final concentration 2.0mmol/L, dNTP final concentration 200nmol/L, and specificity upper and lower primer final concentration 200nmol/L, and add sterilization distilled water to cumulative volume 50 μ L.
Reaction conditions: 95 DEG C of denaturations 5 minutes, then 94 DEG C of sex change 30 seconds successively, then 62 DEG C of annealing 40 seconds, 72 DEG C extend 1 minute, totally 35 circulations.
(3) allelotrope detects: carry out electrophoresis with 1.5% sepharose to thing of the increasing of step (2) gained being expanded production, whether have object fragment to detect it.Taken pictures by gel imaging instrument observations, PCR primer is shown as single band after electrophoresis, without assorted band, then points out PCR primer single, without non-specific amplification.If pillar location is positioned at the position of suitable size, then fragment for the purpose of.Be 163bp according to the object fragment PCR products of above-mentioned primer amplification, reaction result is shown in accompanying drawing 1, visible object band, illustrates that design of primers is reasonable, can amplify object band.
Wherein, detect gained gel electrophoresis figure as shown in Figure 1, by figure M:50bp ladder molecular weight markers thing, 1: blank, 2-3:RHD gene wild type control, 4-7:RHD838G>A saltant type sample.
Detect gained transgenation sequencer map as shown in Figure 2, in figure, arrow is depicted as RHD gene the 6th exon 838 position and has occurred G>A sudden change.
Claims (1)
1. a Rh blood group DEL type
rHD838G>A allelotrope detection primer pair, is characterized in that:
rHD838A-F, CGGTGTTGGCAGGAGGCGTGA;
rHD838A-R, CTTCAGCCAAAGCAGAGGAGG.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304211A (en) * | 2020-03-10 | 2020-06-19 | 无锡市第五人民医院 | RHD-T268A mutant and detection thereof |
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| CN111154850A (en) * | 2020-01-16 | 2020-05-15 | 安徽省第二人民医院(安徽医学高等专科学校附属医院、安徽省职业病防治院) | RHD939G & gtA allele and detection method thereof |
| CN111154766B (en) * | 2020-01-16 | 2023-04-25 | 安徽省第二人民医院(安徽医学高等专科学校附属医院、安徽省职业病防治院) | RHD-S68R mutant and detection method thereof |
| CN111172297B (en) * | 2020-03-10 | 2021-01-12 | 无锡市第五人民医院 | RhD blood type gene RHD993C & gtT allele and application thereof |
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| CN1552918A (en) * | 2003-12-15 | 2004-12-08 | 深圳市血液中心 | Primer, reagent box and sizing method for Chinese the Han nationality crowd differential RHD gene sizing |
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| CN1552918A (en) * | 2003-12-15 | 2004-12-08 | 深圳市血液中心 | Primer, reagent box and sizing method for Chinese the Han nationality crowd differential RHD gene sizing |
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| Systematic RH genotyping and variant identification in French donors of African origin.;Kappler-Gratias S et al.;《blood transfus》;20130617;第12卷(第suppl 1期);第s264－272页 * |
| 中国人群RhD阴性个体中D基因多态性的研究;苏宇清等;《临床输血与检验》;20030531;第5卷(第2期);第91-94页 * |
| 在中国人群中首次发现1例Rh血型弱D12型;孙国栋等;《中国输血杂志》;20060228;第19卷(第1期);摘要，第16页第1栏第1段 * |
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| CN111304211A (en) * | 2020-03-10 | 2020-06-19 | 无锡市第五人民医院 | RHD-T268A mutant and detection thereof |
| CN111304211B (en) * | 2020-03-10 | 2020-12-01 | 无锡市第五人民医院 | RHD-T268A mutant and detection thereof |
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