CN111808937A - Fut 1508 dupT allele of Bombay blood group and detection method and application thereof - Google Patents
Fut 1508 dupT allele of Bombay blood group and detection method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology analysis and detection, and relates to a Monsantha speciosaFUT1508dupT allele and detection method and application thereof, namely Bombay blood typeFUT1508dupT allele, a variant of the Hh blood group systemFUT1The coding region of the gene is repeated one T base from the 508 th position of the initiation codon. The Bombay blood typeFUT1Use of the 508dupT allele for the preparation of a preparation of red blood cells of the Bombay blood group. The detection of the Bombay-like blood groupFUT1508dupT allele is obtained by performing PCR amplification on DNA of a blood sample to be detected by adopting the primer to obtain a specific amplification band patternFUT1Comparing the wild amplified banding patterns to determine whether the sample to be tested has a Bombay-like blood groupFUT1The 508dupT allele. The detection method can be used forFor specifying individualsFUT1Gene identification and Bombay blood type identification, and can be used for screening a large number of samples due to low screening cost and simple operation, so as to establish a Bombay blood type donor database and provide corresponding blood for Bombay blood type patients.
Description
Technical Field
The invention belongs to the technical field of molecular biology analysis and detection, relates to a kind of Mumbay FUT 1508 dupT allele and a detection method and application thereof, and particularly relates to a known mutant gene detection method doped in a wild-type gene cluster.
Background
The blood group system refers to the typing of blood based on differences in antigens on the red blood cell membrane. The human red blood cell blood group system has been found to be 39. The Hh blood group system, also known as the punch blood group system, is the human blood group system that types blood based on the presence of H antigen on the surface of red blood cells. The H antigen is the precursor of each ABO blood group antigen. The FUT1 gene determining the H antigen is more than 5000 base pairs long on chromosome 19 and contains 4 exons. The FUT1 gene has two alleles H and H, the H allele encodes a fucosyltransferase, which links fucose to galactose at the end of a sugar chain to form an H antigen, and the H allele does not encode an active fucosyltransferase. hh homozygote individuals are very rare in humans and include predominantly the montmorillonoid form (non-secretory with complete loss of the H antigen) and the montmorillonoid form (partial loss of the H antigen).
The Bombay blood group is extremely rare in China, and because individuals of the Bombay blood group cannot synthesize the antigen A and the antigen B and the antigen H which are precursors of the antigen A and the antigen B, and simultaneously because of natural immunity, anti-A, anti-B and anti-H antibodies are generated in vivo, the individuals cannot receive blood of any ABO blood group. In the routine detection of ABO blood type, a blood type of Bombay is often mistaken for O type, and if O type blood is mistakenly input without further detailed examination, anti-H immunoglobulin activates the complement cascade reaction, so that red blood cells are dissolved and acute hemolytic transfusion reaction is initiated. In order to ensure the safety of blood transfusion of patients, the detection of the Bombay blood type has very important clinical value.
Even if a patient is clearly assigned to Bombay type(s), it is extremely difficult to find a donor to whom rare Bombay type(s) match. It is therefore necessary to build a library of (generic) punch blood group donors. Because the blood type is extremely rare and a large number of blood donors need to be screened, a first blood donor and a second blood donor can be found, the method which is simple to operate, accurate in result, low in technical requirement, low in screening cost and the like and is suitable for detecting a large number of samples is urgently needed to be developed.
Currently, the conventional detection method for blood type of Bombay is a serological method, which includes: ABO positive typing, ABO reverse typing, H blood type positive typing, H blood type reverse typing, an anti-H titer test, an absorption and diffusion test and the like. The method is not only complicated to operate, but also has instability and uncertainty of results, and is not suitable for large-scale clinical application. In addition, the anti-H agents are expensive and not easy to store for a long time. In contrast, the gene detection can be used for batch detection, and the detection result of the Bombay blood type is reliable and accurate. The similar Monsanto phenotype is determined by detecting the FUT1 genotype, 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 application value. At present, the identification of FUT1 gene mutation is carried out by a sequencing method, but the method has some defects which are difficult to overcome: the operation process is complex, the technical requirement is high, the reagent is expensive, the detection cost is high, the popularity of the sequencer is low, and the sequencer cannot be developed in a common laboratory, and the defects cause that the sequencer is greatly limited in clinical application. Therefore, a method for detecting the Bombay blood type of a large number of samples, which has the advantages of simple operation, accurate result, low technical requirement and low screening cost, is an urgent problem to be solved.
Disclosure of Invention
In view of the problems of the prior art, the present invention aims to provide the FUT 1508 dupT allele and the detection method and application thereof. The invention is designed according to a new allele 508dupT found on the patient FUT1 gene, and establishes a new method for detecting the FUT1 gene mutation based on a PCR method. The difference between the mutant gene and the wild gene in the base sequence of the mutant point is used to design a sequence specific primer, and the individual carrying the mutant gene is identified by a gene amplification method. The detection method of the FUT1 gene 508dupT mutant gene of the Bombay blood group can be used for the identification of the FUT1 gene of a specific individual and the identification of the Bombay blood group(s), and can also be used for the screening of a large number of samples due to low screening cost and simple operation so as to establish a Bombay blood group donor database and provide combined blood for Bombay blood group(s) patients.
The invention carries out the FUT1 gene analysis of the Bombay-like blood group by detecting the 508dupT gene mutation and uses the gene analysis as an index of the blood grouping of the Bombay-like blood group. The detection method of the 508dupT allele of the FUT1 gene of the Bombay-like blood group is completed by utilizing the specific sequence change of the mutant site of the FUT1 gene of the Bombay-like blood group, designing a primer aiming at the changed base sequence and carrying out polymerase chain reaction.
In order to achieve the purpose, the invention adopts the following technical scheme.
A kind of the Monsanto blood group FUT 1508 dupT allele, which is the coding region of the Hh blood group system variant FUT1 gene, is repeated by one T base from the 508 th bit of the initiation codon.
Further, the use of the FUT 1508 dupT allele of the punch-like blood group for the preparation of a preparation of red blood cells of the punch-like blood group.
A product for detecting the Bombay blood group of red blood cells is used for detecting whether a sample to be detected has the FUT 1508 dupT allele of the Bombay blood group.
Furthermore, the primer for detecting the blood type FUT 1508 dupT allele of Bombay is used for carrying out PCR amplification on the blood sample DNA to be detected to obtain a specific amplification band pattern, and the specific amplification band pattern is compared with the amplification band pattern of the wild type FUT1 to determine whether the blood type FUT 1508 dupT allele of Bombay exists in the sample to be detected.
Further, the sequences of the primer pair are SEQ ID NO:1-SEQ ID NO:3, and the primer sequences of the 508dupT allele of the FUT1 gene of the Bombay-like blood group are as follows:
508dupT-F：5’-tctctggcttcccctgctT-3’(SEQ ID NO:1)，
10077-R：5’-ggtgaagttggccaggtaga-3’(SEQ ID NO:2)；
the primer sequences for the wild-type FUT1 gene are as follows:
508wild-F：5’-tctctggcttcccctgctC-3’(SEQ ID NO:3)，
10077-R：5’-ggtgaagttggccaggtaga-3’(SEQ ID NO:2)；
the reverse primer sequence is shared by the mutant type and the wild type, and has no mutation identification specificity.
The primer pair is used for detecting the FUT 1508 dupT allele of the Bombay-like blood group.
The product is a detection kit or a reagent.
The preparation comprises the primer pair.
The detection method of the 508dupT allele of the FUT1 gene of the Bombay type comprises the following steps of selectively amplifying a target fragment containing a mutation site by a gene amplification method, and detecting the existence of the mutation gene.
(1) Extracting the genome DNA in the sample to be tested.
(2) The DNA is used as a template, specific PCR primers are designed aiming at the 508dupT mutation site, and PCR reaction is carried out to obtain a PCR reaction product.
(3) Detecting the banding pattern of the PCR reaction product by using a gel electrophoresis method.
(4) Judging whether the detected sample carries the 508dupT allele or not according to the pattern of the amplified product bands, and identifying the homozygous type or the heterozygous type.
Further, the detection method of the 508dupT allele of the FUT1 gene of the Bombay-like blood group comprises the following specific steps.
(1) Designing a primer: specific oligonucleotide primer sequences are designed aiming at FUT 1508 dupT allele sequences, wherein a forward primer is 508dupT-F (with 508dupT sequence specificity), a reverse primer is 10077-R (without mutation identification specificity), and the length of an amplification product fragment is 498 bp; specific oligonucleotide primer sequences are designed aiming at FUT1 wild-type gene sequences, wherein a forward primer is 508wild-F (with 508 wild-type sequence specificity), a reverse primer is 10077-R (without mutation identification specificity), and the length of an amplification product fragment is 498 bp; simultaneously introducing 1 pair of housekeeping gene beta-actin primers as a DNA sample amplification positive internal reference, wherein the forward primer is beta-actin-F, and the sequence is as follows: 5'-ctccatcctggcctcgctgt-3' (SEQ ID NO: 4); the reverse primer is beta-actin-R, and the sequence thereof is as follows: 5'-gctgtcaccttcaccgttcc-3', the length of the internal reference amplification product fragment is 268 bp.
(2)508dupT allele/wild type gene amplification: the total volume of the amplification reaction system is 10 mu L, the amplification reaction system comprises 50ng of DNA template, the final concentration of forward and reverse primers is 0.4 mu M respectively, and 2 XTaq PCR MasterMix 5ul (TIANGEN). PCR amplification conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 66 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; finally, the extension is carried out for 10min at 72 ℃ and the temperature is cooled to 4 ℃.
(3) FUT 1508 dupT allele/wild type gene assay: and (3) carrying out electrophoresis on the amplification product obtained in the step (2) by using agarose gel to obtain the band pattern of the amplification product, thereby judging the genotype of the amplification product, namely whether the amplification product carries the 508dupT allele and is homozygous or heterozygous.
The beneficial effects of the invention are as follows.
The FUT1 gene 508dupT mutant gene of blood group similar to Bombay and the detection method thereof are designed according to FUT1 new allele 508dupT identified in a laboratory, and the detection of the gene level is carried out by adopting a molecular biological method with higher sensitivity and precision, so that the existence of the mutant gene can be detected, and the homozygote or heterozygote can also be judged. If the blood type is the homozygous type, the fact that the detected person is the Bombay blood type can be accurately judged, and the method is suitable for screening the Bombay blood type individuals in one step.
The detection method of the FUT1 gene 508dupT mutant gene of Bombay blood group provided by the invention can be used for the identification of the FUT1 gene of a specific individual and the identification of the Bombay blood group (group), and can also be used for the screening of a large number of samples due to the advantages of low screening cost, simple operation, low technical requirement, reliable result and the like, thereby providing an efficient and accurate method for establishing a Bombay blood group donor database (group). The aim of establishing a database of Bombay blood type donors is to provide compatible blood for Bombay blood type patients and solve the problem of difficult blood transfusion.
Drawings
FIG. 1 is the gel electrophoresis image of the FUT 1508 dupT allele detection in the example. Wherein M is a DNA Marker which is respectively 100bp, 200bp, 300bp, 400bp, 500bp, 700bp and 1000bp from bottom to top. DNA sample: 1 and 3: 508dupT/wild heterozygous mutant individuals; 2 and 4: a wild-type individual. An amplification primer: 1 and 2: 508dupT-F and 10077-R; 3 and 4: 508 weld-F and 10077-R. Internal control gene: beta-actin, product 268 bp. A is the amplification result under the reaction condition (1), and B is the amplification result under the reaction condition (2).
FIG. 2 is a diagram of the mutant sequencing of the FUT 1508 dupT allele in the examples.
Detailed Description
In the present invention, the "wild-type gene" refers to a gene containing genetic information, which has no mutation and has an original normal function; "mutant gene" refers to a gene in which a mutation has occurred; "mutation" refers to a change in the nucleic acid sequence of DNA, RNA, or the like, and corresponds to a base substitution, insertion, deletion, inversion, duplication, translocation, or the like used in genetics.
Example 1 the class Bombay blood group FUT 1508 dupT allele detection method.
Firstly, preparing a DNA template.
The method adopts a purchased kit to extract the whole blood genome DNA and comprises the following specific steps.
(1) And (3) adding 20 mu L of protease K solution into 200 mu L of EDTA anticoagulated whole blood sample, and uniformly mixing.
(2) Adding 200 μ L buffer GB, mixing thoroughly, standing at 56 deg.C for 10min, mixing several times, and making the solution become clear.
(3) Add 200. mu.L of absolute ethanol and mix well by inversion, at which point a flocculent precipitate may appear.
(4) The solution and flocculent precipitate obtained in the previous step are transferred to an adsorption column CB 3. Centrifuging at 12000rpm for 30sec, pouring out waste liquid in the collecting tube, and placing adsorption column CB3 in the collecting tube.
(5) To adsorption column CB3, 500. mu.L of buffer GD was added, centrifuged at 12000rpm for 30sec, the waste liquid in the collection tube was discarded, and adsorption column CB3 was placed in the collection tube.
(6) 600. mu.L of the rinsing solution PW was added to the adsorption column CB3, and centrifuged at 12000rpm for 30sec to discard the waste liquid in the collection tube, and the adsorption column CB3 was put in the collection tube.
(7) And (6) repeating the step.
(8) Centrifuging at 12000rpm for 2min, and discarding waste liquid. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
(9) Transferring the adsorption column CB3 into a 1.5ml centrifuge tube, suspending 50 muL of DNasefree deionized water at the middle position of the adsorption film, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.
(10) The effect of DNA extraction was evaluated by 1% agarose gel electrophoresis, and DNA concentration and purity were determined by a Merinton SMA4000 ultra-micro UV-Vis nucleic acid protein analyzer.
Second, 508dupT allele detection method.
1. An apparatus.
GeneAmp PCR Sytem 9700 PCR instrument (ABI, USA), ENDUROTM GEL DOCUMENT TOUCH User Manual GEL imager (Labnet, USA), and GEL electrophoresis instrument (Hexay, Beijing).
2. And (3) a reagent.
A blood genome DNA extraction kit (Beijing Tiangen Biochemical technology Co., Ltd.) and 2 XTaq PCRmastermix (Beijing Tiangen Biochemical technology Co., Ltd.) were synthesized by Shanghai Ying Weiji trading Co., Ltd.
3. And (3) designing a primer.
2 pairs of specific oligonucleotide primer sequences (508dupT-F, 5'-tctctggcttcccctgctT-3', 10077-R, 5'-ggtgaagttggccaggtaga-3' amplified fragment length 498 bp; 508wild-F, 5'-tctctggcttcccctgctC-3', 10077-R, 5'-ggtgaagttggccaggtaga-3' amplified fragment length 498bp) were finally determined by designing primers through Oligo 6.0 primer software according to FUT1 (SEQ ID NO: NG-007510.1) recorded by the National Center for Biological Information (NCBI) GenBank.
1 pair of primer of housekeeping gene beta-actin is introduced as positive control for DNA sample amplification (the sequence is as described in [0017 ]), and the length of the amplified product fragment is 268 bp.
The 508dupT allele detection primer sequences and reaction specificities are shown in Table 1.
Table 1.508 dupT allele detection primer sequences and reaction specificity.
Note: f ═ forward primer; r is a reverse primer.
The position of the nucleotide sequence of the # primer is referred to the FUT1 standard sequence (SEQ ID NO: NG 007510.1).
4. And (3) reaction conditions.
(1) Total volume of reaction: 10 μ L, including 50ng of DNA template, 0.4 μ M each of forward and reverse primers, 0.25u (TaKaRa) of Ex Taq DNApolymerase, 25mM TAPS buffer (pH9.3), 50mM KCl, 3mM MgSO4, 1 μ g of gluclease-free BSA, 0.2mM each of dNTP mix. Pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 66 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; finally, the extension is carried out for 10min at 72 ℃ and the temperature is cooled to 4 ℃.
(2) Total volume of reaction: 10 μ L, including 50ng of DNA template, final forward and reverse primer concentrations of 0.4 μ M each, 2 × TaqPCR MasterMix 5ul (TIANGEN). Pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 66 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; finally, the extension is carried out for 10min at 72 ℃ and the temperature is cooled to 4 ℃.
5. And (4) detecting the allele.
Electrophoresis was performed on a 1.5% agarose gel at 150V for 15min, and the amplification effect was visualized by UV gel imaging as shown in FIG. 1. The 508dupT allele carrier sample showed a clear and bright 508dupT specific amplification product band, while the non-carrier sample did not have the 508dupT specific amplification product band; the detected sample has both a 508dupT specific amplification product band and a 508wild specific amplification product band, and the detected sample is proved to be a carrier of the heterozygous 508dupT allele; only the 508dupT specific amplification product band and not the 508wild specific amplification product band proved to be homozygous 508dupT allele carriers.
The electrophoretogram of the gel obtained by detection is shown in FIG. 1. The detected sample is a carrier of the heterozygous 508dupT allele, and the identification method is proved to be effective, feasible and reliable.
The sequencing chart of the gene mutation obtained by detection is shown in FIG. 2.
Claims (10)
1. Bombay blood typeFUT1508dupT allele, whichCharacterized in that the Monsanto-like blood typeFUT1508dupT allele is a variant of the Hh blood group systemFUT1The coding region of the gene is repeated one T base from the 508 th position of the initiation codon.
2. Bombay blood group of the class of claim 1FUT1Use of the 508dupT allele for the preparation of a preparation of red blood cells of the Bombay blood group.
3. A product for detecting the Bombay blood type of red blood cells is characterized in that the product is used for detecting whether a sample to be detected stores the Bombay blood typeFUT1The 508dupT allele.
4. The article for detecting the Bombay blood group of erythrocytes according to claim 3, wherein said article for detecting the Bombay blood group of erythrocytes is characterized in that it comprises at least one of the following componentsFUT1508dupT allele is a primer for PCR amplification of blood sample DNA to be detected to obtain specific amplified band patternFUT1Comparing the wild amplified banding patterns to determine whether the sample to be tested has a Bombay-like blood groupFUT1The 508dupT allele.
5. The article for detecting Bombay blood group in erythrocytes of claim 4, wherein the sequence of the primer pair is SEQ ID NO. 1-SEQ ID NO. 3, and the Bombay blood group isFUT1The primer sequences for the gene 508dupT allele are as follows:
508dupT-F：5’-tctctggcttcccctgctT -3’（SEQ ID NO:1），
10077-R：5’-ggtgaagttggccaggtaga -3’ （SEQ ID NO:2）；
against the wild typeFUT1The primer sequences of the genes are as follows:
508 wild-F：5’-tctctggcttcccctgctC -3’ （SEQ ID NO:3），
10077-R：5’-ggtgaagttggccaggtaga -3’ （SEQ ID NO:2）；
the reverse primer sequence is shared by the mutant type and the wild type, and has no mutation identification specificity.
6. Primer pair with sequence shown as SEQ ID NO. 1-SEQ ID NO. 3 for detecting Bombay-like blood groupFUT1The 508dupT allele.
7. The article for detecting the Bombay blood group of erythrocytes according to claim 3, wherein said article is a detection kit or a reagent.
8. The article of claim 7, wherein said article comprises primer pairs of SEQ ID No. 1 to SEQ ID No. 3.
9. Bombay blood typeFUT1A method for detecting a 508dupT allele, which comprises selectively amplifying a target fragment containing a mutation site by a gene amplification method to thereby detect the presence or absence of the mutant gene, the detection method comprising the steps of:
(1) extracting genome DNA in a sample to be detected;
(2) designing a specific PCR primer aiming at the 508dupT mutation site by taking the DNA as a template and carrying out PCR reaction to obtain a PCR reaction product;
(3) detecting the strip pattern of the PCR reaction product by using a gel electrophoresis method;
(4) judging whether the detected sample carries the 508dupT allele or not according to the pattern of the amplified product bands, and identifying the homozygous type or the heterozygous type.
10. The Bombay-like blood group of claim 1FUT1The detection method of the gene 508dupT allele is characterized by comprising the following specific steps:
(1) designing a primer: to is directed atFUT1508dupT allelic gene sequence, designing specific oligonucleotide primer sequence, wherein the forward primer is 508dupT-F, the reverse primer is 10077-R, and the length of the amplified product fragment is 498 bp; to is directed atFUT1Designing specific oligonucleotide primer sequence with forward primer 508wild-F and reverse primer 10077-R for wild gene sequence, and amplifyingThe length of the product fragment is 498 bp; simultaneously introducing 1 pair of housekeeping gene beta-actin primers as a DNA sample amplification positive internal reference, wherein the forward primer is beta-actin-F, and the sequence is as follows: 5'-ctccatcctggcctcgctgt-3' (SEQ ID NO: 4); the reverse primer is beta-actin-R, and the sequence thereof is as follows: 5'-gctgtcaccttcaccgttcc-3' (SEQ ID NO: 5), the length of the internal reference amplification product fragment is 268 bp;
(2)508dupT allele/wild type gene amplification: the total volume of the amplification reaction system is 10 mu L, the amplification reaction system comprises 50ng of DNA template, the final concentration of forward and reverse primers is 0.4 mu M respectively, and 2 XTaq PCR MasterMix is 5 ul; PCR amplification conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 66 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; finally, extending for 10min at 72 ℃, and cooling to 4 ℃;
（3）FUT1508dupT allele/wild type gene assay: and (3) carrying out electrophoresis on the amplification product obtained in the step (2) by using agarose gel to obtain the band pattern of the amplification product, thereby judging the genotype of the amplification product, namely whether the amplification product carries the 508dupT allele and is homozygous or heterozygous.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113667673B (en) * | 2021-07-15 | 2024-02-06 | 武汉血液中心 | Montellike FUT1236delG allele and detection method and application thereof |
| CN116287321A (en) * | 2023-04-28 | 2023-06-23 | 浙江省血液中心 | SNP site of H blood group system antigen deletion related to immune hemolytic transfusion reaction, application and reagent |
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