CN111197091A - Rh blood type DEL type RHD1073T & gtA allele and application thereof - Google Patents
Rh blood type DEL type RHD1073T & gtA allele and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a DEL type RHD1073T A allele of Rh blood type and application thereof, wherein a wild type RHD gene is shown as SEQ ID NO. 1, and a mutant type RHD gene is shown as SEQ ID NO. 2; a specific primer for detecting mutant genes of the mutant genes is disclosed, wherein the sequence of an upstream primer of the specific primer is shown as SEQ ID NO. 3, and the sequence of a downstream primer is shown as SEQ ID NO. 4, and the specific primer is used for detecting RHD1073T > A alleles. The Rh blood type DEL type RHD1073T > A allele and the application thereof can detect the existence of mutant genes 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 Rh blood type DEL type RHD1073T > A allele and application 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.
In 1984, Okubo et al further examined RhD-negative samples confirmed by direct agglutination test and indirect anti-human globulin test (IAT) by absorptiometry, and found that the presence of D antigen was detectable on the surface of some individual erythrocytes, which was called D-diffused type (DEL type). It is one of weak D-forms, with very weak antigenicity. DEL type is at a much higher rate in RhD negative populations in China compared to caucasian. According to the related studies, DEL type accounts for about 26% of the RhD-negative population identified by routine serology in our country, 32.6% in Taiwan, and DEL type is rare in European white race. Different ethnic groups in different regions have different genetic backgrounds, and the gene types of the DEL blood type D antigens also have larger differences. In recent years, research on DEL blood group RHD genes has become a hot spot in research on erythrocyte blood groups. The new allele existing in the RHD gene of DEL blood type is discovered in succession, and the gene has obvious genetic characteristics.
Currently, the conventional method for detecting DEL blood group D antigen is to identify it by serological saline method and indirect anti-human globulin test as well as absorption and diffusion test. The method is not only complicated to operate, but also has unstable results. In addition, the results of some individuals in serotyping are difficult to judge 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 the DEL blood type by the method of immune serology depends on the specificity and the operation method of the used anti-D antibody, the reliability is low, and the method is not suitable for large-scale clinical application due to complicated steps, and the reliable method is to detect the DEL blood type by the method of gene detection. At the moment, the DEL blood type D antigen genotype is determined by detecting DNA, so that the method not only has important clinical practical significance in making up for the defects of the serology technology, but also has wide scientific research and application values.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides the Rh blood type DEL type RHD1073T > A mutant gene and the detection primer thereof, which can detect the existence of the mutant gene doped in a gene library with high sensitivity and high precision.
The technical scheme is as follows: in order to realize the purpose, the Rh blood type DEL type RHD1073T > A mutant gene of the invention has the wild type RHD gene shown as SEQ ID NO. 1 and the mutant type RHD gene shown as SEQ ID NO. 2; compared with the wild RHD gene, the mutant RHD gene has mutation from base T to base A at position 158 of the gene sequence.
A specific primer for detecting Rh blood type DEL type RHD1073T > A mutant genes is disclosed, wherein the sequence of an upstream primer of the specific primer is shown as SEQ ID NO. 3, and the sequence of a downstream primer is shown as SEQ ID NO. 4, and is used for detecting RHD1073T > A alleles.
The beneficial effects of the invention are as follows: an RHD-S68R mutant and detection thereof, provides a mutant RHD1073T > A gene, can detect the existence of the mutant gene doped in a gene library with high sensitivity and high precision; due to the difference of RHD genes among different nationalities, the gene is specially aimed at the newly discovered RHD1073T > A allele in Chinese 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 image of the RHD1073T > A allele detection in this example;
FIG. 2 is a sequence chart of the RHD1073T > A allelic variant in this example.
Detailed Description
The invention will be further described with reference to the accompanying figures 1 to 2.
An Rh blood group DEL type RHD1073T > A allelic mutant gene, the mutation occurs at the position 25306729 of the chromosome 1, the number of the gene is NC-000001.11 (25272393-25330445) in NCBl reference database GRCh38.p13, the wild type RHD gene is shown as SEQ ID NO:1, and the mutant type RHD gene is shown as SEQ ID NO: 2; compared with the wild RHD gene, the mutant RHD gene has mutation from base T to base A at position 158 of the gene sequence.
SEQ ID NO:1
GCTTATAATAACACTTGTCCACAGGGGTGTTGTAACCGAGTGCTGGGGATTC CCCACAGCTCCATCATGGGCTACAACTTCAGCTTGCTGGGTCTGCTTGGAGAGAT CATCTACATTGTGCTGCTGGTGCTTGATACCGTCGGAGCCGGCAATGGCATGTGG GTCACTGGGCTTACCCCCCATCCCCTTAACACTCCCCTCCAACTCAGGAAGAAATGTGTGCAGAGTCCTTAGCTGGGGCGTGTGCACTCGGGGCCAGGTGCTCAGTAGGC TTCGGTGAATATTTGTTGGCTGATTTATTCAGAAATTCTGTCCAGCCCCTACCTTG GATGGATTTATCACCTCTCCAGGCCACCTCTTCTTTCCA
SEQ ID NO:2
GCTTATAATAACACTTGTCCACAGGGGTGTTGTAACCGAGTGCTGGGGATTC CCCACAGCTCCATCATGGGCTACAACTTCAGCTTGCTGGGTCTGCTTGGAGAGAT CATCTACATTGTGCTGCTGGTGCTTGATACCGTCGGAGCCGGCAATGGCAAGTGG GTCACTGGGCTTACCCCCCATCCCCTTAACACTCCCCTCCAACTCAGGAAGAAATGTGTGCAGAGTCCTTAGCTGGGGCGTGTGCACTCGGGGCCAGGTGCTCAGTAGGC TTCGGTGAATATTTGTTGGCTGATTTATTCAGAAATTCTGTCCAGCCCCTACCTTG GATGGATTTATCACCTCTCCAGGCCACCTCTTCTTTCCA
The detection method of the Rh blood type DEL type RHD1073T > A allele detects the existence of the mutant gene by selectively amplifying a detection area of a target position 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) using the DNA as a template, and carrying out PCR reaction on a PCR primer designed aiming at a coding region near the RHD1073T & gtA 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 a 1073T → a mutation. The nucleotide sequence composition of the PCR reaction product can be used for sequencing the PCR reaction product through a sequencer.
The detection method of the Rh blood type DEL type RHD1073T > A allele comprises the following specific steps:
(1) designing a primer: designing primers through Oligo 6.0 primer software according to RHD gene (sequence number: NC-000001.11) recorded by the GenBank of National Center for Biotechnology Information (NCBI), 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 199 bp;
TABLE 1 RHD1073T > A 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)RHD1073T>Amplification of a 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 61 ℃ for 40s, and extension at 72 ℃ for 1min for 32 cycles;
(3) RHD1073T > A 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
The method adopts a purchased kit to extract the whole blood genome DNA, and 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 cell clumps are visible after mixing, incubating the solution at 37 ℃ until the clumps dissipate, and if cell clumps are still visible after incubating for 1 hour, adding 100 mu L of lysis buffer and repeatedly incubating at 37 ℃;
(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 ℃.
RHD1073T > A allele detection technical scheme:
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); DNAIsolation Kit extraction Kit (PELFREEZ corporation, beijing); PCR buffer, dNTP, Taq enzyme (ABI, USA); primers and probes were synthesized by Shanghai Biotechnology Ltd.
(1)RHD1073T>Amplification of a 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-denaturation at 95 ℃ for 5min followed by denaturation at 94 ℃ for 30 sec followed by annealing at 61 ℃ for 40 sec and extension at 72 ℃ for 1min for 32 cycles.
(2) RHD1073T > A 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: RHD1073T > A mutant samples.
(3) And (3) purifying an amplification product: in the research, PCR products after Agarose Gel electrophoresis are purified and recovered by adopting an Agarose Gel DNAPurification Kit of Takara company, and sequencing is prepared.
(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 pattern of the detected gene mutation is shown in FIG. 2, in which the RHD gene shows the g.25306729T > A mutation as shown by the arrow.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Sequence listing
<110> second people hospital of Anhui province (subsidiary hospital of higher specialty school of Anhui medical science, prevention and treatment hospital of occupational diseases of Anhui province)
<120> Rh blood type DEL type RHD1073T > A allele and application thereof
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ctgctggtgc ttgataccgt cggagccggc aatggcaagt gggtcactgg gcttaccccc 180
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cgtgtgcact cggggccagg tgctcagtag gcttcggtga atatttgttg gctgatttat 300
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ccatccaagg taggggctgg acag 24
Claims (2)
1. An Rh blood type DEL type RHD1073T > A mutant gene, which is characterized in that: the wild type RHD gene is shown as SEQ ID NO. 1, and the mutant type RHD gene is shown as SEQ ID NO. 2; compared with the wild RHD gene, the mutant RHD gene has mutation from base T to base A at position 158 of the gene sequence.
2. A specific primer for detecting the mutant gene of Rh blood type DEL type RHD1073T > a, characterized in that: the sequence of the upstream primer of the specific primer is shown as SEQ ID NO. 3, and the sequence of the downstream primer is shown as SEQ ID NO. 4, and the specific primer is used for detecting RHD1073T > A allele.
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| US20110070590A1 (en) * | 2009-09-22 | 2011-03-24 | Jan Rohozinski | Primers and Methods for Determining RhD Zygosity |
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2020
- 2020-01-16 CN CN202010046597.9A patent/CN111197091A/en active Pending
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|---|---|---|---|---|
| US20110070590A1 (en) * | 2009-09-22 | 2011-03-24 | Jan Rohozinski | Primers and Methods for Determining RhD Zygosity |
| CN106834287A (en) * | 2017-04-11 | 2017-06-13 | 青岛市中心血站 | A kind of SNP marker for detecting RhD negative phenotypes |
| CN106967808A (en) * | 2017-04-11 | 2017-07-21 | 青岛市中心血站（青岛市公民义务献血办公室青岛市输血医学研究所） | A kind of primer sets and its application for being used to detect RhD negative blood groups |
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| 孙国栋等: ""在中国人群中首次发现1例 Rh 血型弱 D12型"", 《中国输血杂志》 * |
| 谢俊杰等: ""Rh血型D放散型(Del)"", 《中国输血杂志》 * |
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