CN111304211A - RHD-T268A mutant and detection thereof - Google Patents
RHD-T268A mutant and detection thereof Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a RhD blood type antigen RHD-T268A mutant RHD802A > G allele, wherein a wild type RHD gene is shown as a sequence of SEQ ID NO. 1, and a mutant type RHD gene is shown as a sequence of SEQ ID NO. 2; a specific primer for detecting RhD blood group antigen mutant is shown as an upstream primer in SEQ ID NO. 3 and a downstream primer in SEQ ID NO. 4. The RHD-T268A mutant and the detection thereof can detect the existence of the mutant gene doped in a gene library with high sensitivity and high precision.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an RHD-T268A mutant and detection thereof.
Background
The Rh blood group is the most complex and polymorphic system of the human erythrocyte blood group system and is also the main erythrocyte blood group causing clinical transfusion reactions and severe neonatal hemolytic disease. More than 50 Rh blood group antigens are found, wherein RhD antigens have strong immunogenicity, are coded by RHD genes and are the key points of blood group research. Clinically, Rh blood group antigens are classified into two main types, namely RhD positive and RhD negative, according to whether D antigen is detected on the surface of an erythrocyte membrane.
Currently, the conventional method for detecting Rh blood group D antigen is to identify by adopting a serological saline method, an indirect anti-human globulin test and an absorption and diffusion test. Serological techniques, however, have certain limitations. The results of some individuals when serotyped are difficult to determine due to disease or other factors; serological results of chronic long-term transfusion patients sometimes exhibit a phenomenon of "mixed visual field"; serological tests also fail to obtain correct results when samples cannot be obtained or when there are insufficient red blood cells or red blood cell samples, such as fetal blood grouping, forensic remains, etc.
The detection of RhD blood type by means of immunoserology depends mainly on the specificity of the anti-D antibodies and the amount of antigen expressed. Currently, with the development of molecular biotechnology, RhD mutants increase year by year, and the antigen expression amount and the gene mutation site thereof are different. The detection of the gene mutants mainly determines the Rh blood group D antigen genotype by a molecular biological method, has important clinical practical significance on making up the defects of a serology technology, and also has wide scientific research and application values.
Disclosure of Invention
The invention aims to provide a RhD blood group antigen RHD-T268A mutant RHD802A > G allele and a detection method thereof, and Rh blood group genotype analysis is carried out by detecting the RHD802A > G allele.
The technical scheme is as follows: a RhD blood group antigen RHD-T268A mutant RHD802A > G allele, wherein a wild type RHD gene is shown as a SEQ ID NO:1 sequence, and a mutant type RHD gene is shown as a SEQ ID NO:2 sequence is shown in the specification; compared with a wild RHD gene, the mutant RHD gene locus has g.25303322A > G mutation, namely the 257 th base A of the mutant RHD gene sequence is mutated into G; the amino acid sequence derived from the coding sequence of the wild type RHD gene is shown as SEQ ID NO: 5, the amino acid sequence obtained from the mutant RHD gene coding sequence is shown as SEQ ID NO: 6, amino acid sequence SEQ ID NO: 6 compared to the amino acid sequence of SEQ ID NO: in contrast, position 268 was converted from threonine T to alanine A.
Further, a specific primer for detecting mutant RHD802A > G allele, wherein the upstream primer sequence of the specific primer is shown as SEQ ID NO. 3, and the downstream primer sequence is shown as SEQ ID NO. 4.
The beneficial effects of the invention are as follows: the RHD-T268A mutant and the detection thereof of the invention adopt a molecular biology method to detect the gene level, and can detect the existence of the mutant gene doped in a gene library with high sensitivity and high precision. Because the difference of RHD genes exists among different nationalities, the gene is designed specially aiming at the newly discovered RHD802A G allele in Chinese on the basis of related research according to the molecular background of the RHD gene of Chinese. The invention not only has important clinical practical significance in making up the defects of the serology technology, but also has wide scientific research application value.
Drawings
FIG. 1 is a gel electrophoresis chart of the RHD802A > G allele detection in the example;
FIG. 2 is a sequence chart of the RHD802A > G allele mutation in the examples.
Detailed Description
The invention will be further described with reference to the accompanying figures 1 to 2.
A RhD blood group antigen RHD-T268A mutant means that the mutation occurs at the 25303322 position of chromosome 1, the 257 th base A is mutated into G, the wild type RHD gene is shown as the sequence of SEQ ID NO:1, and the mutant type RHD gene is shown as the sequence of SEQ ID NO:2 sequence is shown in the specification; the number of the gene in NCBl reference database GRCh38.p13 is NC-000001.11 (25272393 and 25330445). The wild amino acid sequence of the RHD gene coding sequence is shown as SEQ ID NO: 5, wherein the amino acid change is as set forth in SEQ ID NO: the 268 position of the 6 sequence is converted from threonine T to alanine A.
SEQ ID NO:1
GACTTCCCAGCTCATTCCCTAAATGCTGCACAATCAGGGTAACTGTGCCCTGAGCCTAAGAGGCAGTAGTGAGCTGGCCCATCATGTCCACTGATGAAGGACACGTAGCCCCAACACAGGGGAGAAGTGGTTTCAGGATCAGCAAAGCAGGGAGGATGTTACAGGGTTGCCTTGTTCCCAGCGTGCTGGTCACTTGCAGCAAGATGGTGTTCTCTCTCTACCTTGCTTCCTTTACCCACACGCTATTTCTTTGCAGACTTATGTGCACAGTGCGGTGTTGGCAGGAGGCGTGGCTGTGGGTACCTCGTGTCACCTGATCCCTTCTCCGT
SEQ ID NO:1
GACTTCCCAGCTCATTCCCTAAATGCTGCACAATCAGGGTAACTGTGCCCTGAGCCTAAGAGGCAGTAGTGAGCTGGCCCATCATGTCCACTGATGAAGGACACGTAGCCCCAACACAGGGGAGAAGTGGTTTCAGGATCAGCAAAGCAGGGAGGATGTTACAGGGTTGCCTTGTTCCCAGCGTGCTGGTCACTTGCAGCAAGATGGTGTTCTCTCTCTACCTTGCTTCCTTTACCCACACGCTATTTCTTTGCAGACTTATGTGCACAGTGCGGTGTTGGCAGGAGGCGTGGCTGTGGGTACCTCGTGTCACCTGATCCCTTCTCCGT
SEQ ID NO:2
GACTTCCCAGCTCATTCCCTAAATGCTGCACAATCAGGGTAACTGTGCCCTGAGCCTAAGAGGCAGTAGTGAGCTGGCCCATCATGTCCACTGATGAAGGACACGTAGCCCCAACACAGGGGAGAAGTGGTTTCAGGATCAGCAAAGCAGGGAGGATGTTACAGGGTTGCCTTGTTCCCAGCGTGCTGGTCACTTGCAGCAAGATGGTGTTCTCTCTCTACCTTGCTTCCTTTACCCACACGCTATTTCTTTGCAGGCTTATGTGCACAGTGCGGTGTTGGCAGGAGGCGTGGCTGTGGGTACCTCGTGTCACCTGATCCCTTCTCCGT
SEQ ID NO:2
GACTTCCCAGCTCATTCCCTAAATGCTGCACAATCAGGGTAACTGTGCCCTGAGCCTAAGAGGCAGTAGTGAGCTGGCCCATCATGTCCACTGATGAAGGACACGTAGCCCCAACACAGGGGAGAAGTGGTTTCAGGATCAGCAAAGCAGGGAGGATGTTACAGGGTTGCCTTGTTCCCAGCGTGCTGGTCACTTGCAGCAAGATGGTGTTCTCTCTCTACCTTGCTTCCTTTACCCACACGCTATTTCTTTGCAGGCTTATGTGCACAGTGCGGTGTTGGCAGGAGGCGTGGCTGTGGGTACCTCGTGTCACCTGATCCCTTCTCCGT
SEQ ID NO:5
MSSKYPRSVRRCLPLWALTLEAALILLFYFFTHYDASLEDQKGLVASYQVGQDLTVMAAIGLGFLTSSFRRHSWSSVAFNLFMLALGVQWAILLDGFLSQFPSGKVVITLFSIRLATMSALSVLISVDAVLGKVNLAQLVVMVLVEVTALGNLRMVISNIFNTDYHMNMMHIYVFAAYFGLSVAWCLPKPLPEGTEDKDQTATIPSLSAMLGALFLWMFWPSFNSALLRSPIERKNAVFNTYYAVAVSVVTAISGSSLAHPQGKISKTYVHSAVL
SEQ ID NO:6
MSSKYPRSVRRCLPLWALTLEAALILLFYFFTHYDASLEDQKGLVASYQVGQDLTVMAAIGLGFLTSSFRRHSWSSVAFNLFMLALGVQWAILLDGFLSQFPSGKVVITLFSIRLATMSALSVLISVDAVLGKVNLAQLVVMVLVEVTALGNLRMVISNIFNTDYHMNMMHIYVFAAYFGLSVAWCLPKPLPEGTEDKDQTATIPSLSAMLGALFLWMFWPSFNSALLRSPIERKNAVFNTYYAVAVSVVTAISGSSLAHPQGKISKAYVHSAVL
The detection of the RhD blood group antigen RHD-T268A mutant RHD802A > G allele detects the existence of the mutant gene by selectively amplifying a detection area of a target part containing the mutant gene by a gene amplification method. The detection method comprises the following steps:
(1) extracting DNA in a sample to be detected;
(2) carrying out PCR reaction by using the DNA as a template and a PCR primer designed aiming at a coding region near the RHD802A G mutant gene to obtain a PCR reaction product;
(3) measuring the nucleotide sequence composition of the PCR reaction product;
(4) the nucleotide sequence was compared to the sequence of the RHD wild-type gene to determine whether there was an 802A → G mutation. The nucleotide sequence composition of the PCR reaction product can be used for sequencing the PCR reaction product through a sequencer.
The detection of the RhD blood group antigen RHD-T268A mutant RHD802A > G allele comprises the following specific steps:
(1) designing a primer: according to the sequence number of the RHD gene recorded in the GenBank of the National Center for Biological Information (NCBI): NC-000001.11, designing primers through Oligo 6.0 primer software, and finally determining 1 pair of specific oligonucleotide primer sequences, wherein the sequence of the upstream primer is shown as SEQID NO. 3 in Table 1; the sequence of the downstream primer is shown as SEQID NO. 4, and the length of the amplified product fragment is 198 bp.
TABLE 1 RHD802A > G allele detection primer sequences and reaction specificity
Note: the position of the exon base sequence referred to by the oligonucleotide primer sequence refers to the entire arrangement of 10 exons starting from the ATG start codon; the position of the base sequence of the intron in question refers to the single order of arrangement of each intron.
(2)RHD802A>Amplification of the G allele: the total volume of the reaction system is 50 mu L; wherein the PCR reaction solution contains 10 μ L of PCR 5 × buffer solution, 5.0 μ L of DNA template, 1.0 μ L of Taq polymerase and 1.0 μ L MgCl2The final concentration is 2.0mmol/L, the final concentration of dNTP is 200nmol/L, and the final concentrations of the specificity forward primer and the specificity reverse primer are both 200 nmol/L; adding sterilized double distilled water to the total volume of the reaction system to be 50 mu L; and (2) reacting the reaction system in a PCR instrument under the following reaction conditions: pre-denaturation at 95 ℃ for 5min, then denaturation at 94 ℃ for 30s in sequence, annealing at 63 ℃ for 40s, and extension at 72 ℃ for 1min for 32 cycles;
(3) RHD802A > G allele detection: and (3) carrying out electrophoresis on the amplified product obtained in the step (2) by using an agarose gel to detect whether the amplified product contains the target fragment.
Preparation of DNA template
Whole blood genomic DNA was extracted using a commercially available kit. The method comprises the following specific steps:
(1) taking one sterile 2.0mL centrifuge tube, and adding 1mL cell lysate;
(2) gently shaking the whole blood sample anticoagulated by EDTA until the whole blood sample is thoroughly mixed; then, adding 500 mu L of blood sample into the centrifuge tube containing the cell lysate, slightly pouring the centrifuge tube for 5-6 times, and uniformly mixing;
(3) incubate for 10 minutes at room temperature (during which the tube is inverted for 2-3 rounds of mixing);
(4) centrifuging at 12000rpm for 5 minutes at room temperature;
(5) the supernatant is removed as far as possible slowly by a liquid shifter, and the white substance at the interface of the two phases is not sucked out;
(6) mix vigorously using a vortex shaker (Votex) until the leukocytes are resuspended (10-15 seconds);
(7) to the resuspended cell solution was added 300. mu.L of the lysis solution. The solution was pipetted 5-6 times to lyse the leukocytes. At which point the solution should become very viscous. If a clump of cells is visible after mixing, the solution is incubated at 37 ℃ until the clump dissipates. If cell clumps remain visible after 1 hour of incubation, an additional 100. mu.L of lysis buffer was added and incubation at 37 ℃ repeated;
(8) adding 100 mu L of protein precipitation solution into the nuclear lysate, and violently shaking for 10-20 seconds by using a vortex oscillator;
(9) centrifuging at 12000rpm for 5 minutes at room temperature;
(10) transferring the supernatant to a corresponding number of 2.0mL centrifuge tube added with 300 μ L of room temperature isopropanol;
(11) gently invert to mix the solution until white linear DNA forms a precipitate;
(12) centrifuging at 12000rpm for 1min at room temperature;
(13) discarding the supernatant, adding room-temperature 70% ethanol with the volume equal to that of the sample, and slightly inverting the centrifuge tube for several times;
(14) the ethanol solution was removed as slowly as possible by pipette. Baking the centrifugal tube at 50 ℃ for 5-10 minutes to completely volatilize residual ethanol liquid as much as possible;
(15) adding 50-100 mu L of DNA solution into a centrifuge tube, and gently mixing uniformly;
(16) the DNA extraction effect was evaluated by 1% agarose gel electrophoresis, and the content was quantified by Nanodrop nucleic acid analyzer and stored at-20 ℃.
RHD802A > G allele detection:
the instrument comprises the following steps: veriti 96 type PCR instrument, BIO-RAD Gel Doc XR + type Gel imager (Berle, USA), Gel electrophoresis instrument (Hex, Beijing).
Reagent: QIAamp DNA extraction kit (Qiagen, Germany); DNA Isolation Kit extraction Kit (Beijing PELFREEZ company); PCR buffer, dNTP, Taq enzyme (ABI, USA); the primers were synthesized by Shanghai Biometrics, Inc.
(1)RHD802A>Amplification of the G allele: total volume of reaction: 50 μ L, 10 μ L of PCR-containing 5 Xbuffer, 5.0 μ L of DNA template, 1.0 μ L of Taq polymerase (1U/. mu.L), MgCl2Final concentration 2.0mmol/L, dNTP final concentration 200nmol/L, and specificity upper and lower primer final concentration 200nmol/L, and adding sterilized double distilled water to total volume of 50 μ L. Reaction conditions are as follows: pre-denatured at 95 ℃ for 5 minutes, thenDenaturation at 94 ℃ for 30 seconds followed by annealing at 63 ℃ for 40 seconds and extension at 72 ℃ for 1 minute for 32 cycles;
(2) RHD802A > G allele detection: the amplified product obtained in step (1) was subjected to electrophoresis using 1.5% agarose gel to detect the presence or absence of the desired fragment. And observing and photographing the result by a gel imager, and displaying the PCR product as a single band after electrophoresis without a miscellaneous band, thus prompting that the PCR product is single and has no non-specific amplification. If the position of the stripe is in a position with proper size, the target segment is obtained. As shown in fig. 1, M: 50bp gradient molecular weight markers, 1: blank control, 2: wild-type control, 3: RHD802A > G mutant samples;
(3) and (3) purifying an amplification product: the PCR product after Agarose Gel electrophoresis is purified and recovered by adopting an Agarose Gel DNA purification kit of Takara company to prepare sequencing;
(4) sanger sequencing and result judgment: the purified PCR product was sequenced on an ABI3730 type fully automatic DNA sequencer. The sequencing results were aligned with the RHD wild-type Reference Sequence (NCBI Reference Sequence: NC-000001.11) and the results were reported as a function of the actual mutation. The gene mutation map obtained by sequencing is shown in figure 2, and the arrow in the figure shows the RHD gene locus g.25303322A > G mutation.
Sequence listing
<110> fifth people hospital in Wuxi city
<120> RHD-T268A mutant and detection thereof
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>329
<212>DNA
<213>Homo sapiens
<400>1
gacttcccag ctcattccct aaatgctgca caatcagggt aactgtgccc tgagcctaag 60
aggcagtagt gagctggccc atcatgtcca ctgatgaagg acacgtagcc ccaacacagg 120
ggagaagtgg tttcaggatc agcaaagcag ggaggatgtt acagggttgc cttgttccca 180
gcgtgctggt cacttgcagc aagatggtgt tctctctcta ccttgcttcc tttacccaca 240
cgctatttct ttgcagactt atgtgcacag tgcggtgttg gcaggaggcg tggctgtggg 300
tacctcgtgt cacctgatcc cttctccgt 329
<210>2
<211>329
<212>DNA
<213>Homo sapiens
<400>2
gacttcccag ctcattccct aaatgctgca caatcagggt aactgtgccc tgagcctaag 60
aggcagtagt gagctggccc atcatgtcca ctgatgaagg acacgtagcc ccaacacagg 120
ggagaagtgg tttcaggatc agcaaagcag ggaggatgtt acagggttgc cttgttccca 180
gcgtgctggt cacttgcagc aagatggtgt tctctctcta ccttgcttcc tttacccaca 240
cgctatttct ttgcaggctt atgtgcacag tgcggtgttg gcaggaggcg tggctgtggg 300
tacctcgtgt cacctgatcc cttctccgt 329
<210>3
<211>28
<212>DNA
<213>Homo sapiens
<400>3
ctttacccac acgctatttc tttgcagg 28
<210>4
<211>26
<212>DNA
<213>Homo sapiens
<400>4
aaagcagagg aggttagttg tctagt 26
<210>5
<211>275
<212>PRT
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<400>5
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20 25 30
His TyrAsp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr
35 40 45
Gln Val Gly Gln Asp Leu Thr Val Met Ala Ala Ile Gly Leu Gly Phe
50 55 60
Leu Thr Ser Ser Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn
65 70 75 80
Leu Phe Met Leu Ala Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly
85 90 95
Phe Leu Ser Gln Phe Pro Ser Gly Lys Val Val Ile Thr Leu Phe Ser
100 105 110
Ile Arg Leu Ala Thr Met Ser Ala Leu Ser Val Leu Ile Ser Val Asp
115 120 125
Ala Val Leu Gly Lys Val Asn Leu Ala Gln Leu Val Val Met Val Leu
130 135 140
Val Glu Val Thr Ala Leu Gly Asn Leu Arg Met Val Ile Ser Asn Ile
145 150 155 160
Phe Asn Thr Asp Tyr His Met Asn Met Met His Ile Tyr Val Phe Ala
165 170 175
Ala Tyr Phe Gly Leu Ser Val Ala Trp Cys Leu Pro Lys Pro Leu Pro
180 185 190
Glu Gly Thr Glu AspLys Asp Gln Thr Ala Thr Ile Pro Ser Leu Ser
195 200 205
Ala Met Leu Gly Ala Leu Phe Leu Trp Met Phe Trp Pro Ser Phe Asn
210 215 220
Ser Ala Leu Leu Arg Ser Pro Ile Glu Arg Lys Asn Ala Val Phe Asn
225 230 235 240
Thr Tyr Tyr Ala Val Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255
Ser Leu Ala His Pro Gln Gly Lys Ile Ser Lys Thr Tyr Val His Ser
260 265 270
Ala Val Leu
275
<210>6
<211>275
<212>PRT
<213>Homo sapiens
<400>6
Met Ser Ser Lys Tyr Pro Arg Ser Val Arg Arg Cys Leu Pro Leu Trp
1 5 10 15
Ala Leu Thr Leu Glu Ala Ala Leu Ile Leu Leu Phe Tyr Phe Phe Thr
20 25 30
His Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr
35 40 45
Gln Val Gly Gln Asp Leu Thr Val MetAla Ala Ile Gly Leu Gly Phe
50 55 60
Leu Thr Ser Ser Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn
65 70 75 80
Leu Phe Met Leu Ala Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly
85 90 95
Phe Leu Ser Gln Phe Pro Ser Gly Lys Val Val Ile Thr Leu Phe Ser
100 105 110
Ile Arg Leu Ala Thr Met Ser Ala Leu Ser Val Leu Ile Ser Val Asp
115 120 125
Ala Val Leu Gly Lys Val Asn Leu Ala Gln Leu Val Val Met Val Leu
130 135 140
Val Glu Val Thr Ala Leu Gly Asn Leu Arg Met Val Ile Ser Asn Ile
145 150 155 160
Phe Asn Thr Asp Tyr His Met Asn Met Met His Ile Tyr Val Phe Ala
165 170 175
Ala Tyr Phe Gly Leu Ser Val Ala Trp Cys Leu Pro Lys Pro Leu Pro
180 185 190
Glu Gly Thr Glu Asp Lys Asp Gln Thr Ala Thr Ile Pro Ser Leu Ser
195 200 205
Ala Met Leu Gly Ala Leu Phe Leu Trp Met Phe TrpPro Ser Phe Asn
210 215 220
Ser Ala Leu Leu Arg Ser Pro Ile Glu Arg Lys Asn Ala Val Phe Asn
225 230 235 240
Thr Tyr Tyr Ala Val Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255
Ser Leu Ala His Pro Gln Gly Lys Ile Ser Lys Ala Tyr Val His Ser
260 265 270
Ala Val Leu
275
Claims (2)
1. A RhD blood group antigen RHD-T268A mutant RHD802A > G allele characterized by: the wild type RHD gene is shown as a SEQ ID NO:1 sequence, and the mutant type RHD gene is shown as a SEQ ID NO:2 sequence is shown in the specification; compared with a wild RHD gene, the mutant RHD gene locus has g.25303322A > G mutation, namely the 257 th base A of the mutant RHD gene sequence is mutated into G; the amino acid sequence derived from the coding sequence of the wild type RHD gene is shown as SEQ ID NO: 5, the amino acid sequence obtained from the mutant RHD gene coding sequence is shown as SEQ ID NO: and 6.
2. A specific primer for the detection of a mutant RHD802A > G allele, characterized in that: the upstream primer sequence of the specific primer is shown as SEQ ID NO. 3, and the downstream primer sequence is shown as SEQ ID NO. 4.
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| CN112940099A (en) * | 2021-03-24 | 2021-06-11 | 无锡市第五人民医院 | RhD-T163P mutant and detection thereof |
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| CN112940099A (en) * | 2021-03-24 | 2021-06-11 | 无锡市第五人民医院 | RhD-T163P mutant and detection thereof |
| CN112940099B (en) * | 2021-03-24 | 2022-07-05 | 无锡市第五人民医院 | RhD-T163P mutant and detection thereof |
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