CN113667673A - Fut1236delG allele of Bombay-like blood group and detection method and application thereof - Google Patents
Fut1236delG allele of Bombay-like blood group and detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a Bombay blood group FUT1236delG allele and a detection method and application thereof, wherein the Bombay blood group FUT1236delG allele is obtained by deleting 1G from the 236 th base of an initiation codon in an encoding region of an Hh blood group system variant FUT1 gene. The detection product and the detection method of the FUT1 gene mutation are also provided, a sequence specific primer is designed by utilizing the difference of the mutant gene and the wild gene at the base sequence of the mutation point, and an individual carrying the mutant gene is identified by a gene amplification method; the detection product and the detection method can detect the existence of the mutant gene doped in the gene library with high sensitivity and high precision. The method can be used for the identification of the FUT1 gene and the Bombay blood group (group) of a specific individual, and can also be used for the screening of a large number of samples to provide the combined blood for Bombay blood group (group) patients due to low screening cost and simple operation.
Description
Technical Field
The invention relates to the technical field of molecular biology analysis and detection, and relates to a Monsanto FUT1236delG allele and a detection method and application thereof.
Background
The blood group system refers to the typing of blood based on differences in antigens on the red blood cell membrane. By 2 months 2021, the international blood transfusion society (ISBT) confirmed a total of 43 in the human red blood cell blood group system. 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, anti-H agents are expensive and not easily stored for long periods. 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 blood type detection method of Bombay which has simple operation, accurate result, low technical requirement and low screening cost and is suitable for a large number of samples is an urgent problem to be solved.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a Bombay FUT1236delG allele and a detection method and application thereof, which can be used for not only the identification of the FUT1 gene of a specific individual and the identification of Bombay blood group(s), but also 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 adopts the following technical scheme:
in a first aspect of the embodiments of the present invention, there is provided a Bombay blood group FUT1236delG allele in which 1G is deleted from the 236 th base of the start codon in the coding region of the Hh blood group system variant FUT1 gene.
In a second aspect of the embodiments of the present invention, there is provided the use of said pythagorean-like FUT1236delG allele for the preparation of a product for the detection of pythagorean-like blood groups.
In a third aspect of the embodiments of the present invention, there is provided a primer pair for detecting a Bombay blood type of red blood cells, wherein the sequences of the primer pair are shown as SEQ ID NO:1-SEQ ID NO:3, and the primer sequences of the 236delG alleles of the FUT1 gene of the Bombay blood type are as follows:
236 delG-F: as shown in SEQ ID NO. 1,
440 Wild-R: as shown in SEQ ID NO. 2;
the primer sequences for the wild-type FUT1 gene are as follows:
236 Wild-F: as shown in SEQ ID NO. 3,
reverse primer: as shown in SEQ ID NO. 2; the reverse primer sequence is shared by the mutant type and the wild type, and has no mutation identification specificity.
In a fourth aspect of an embodiment of the present invention, there is provided a product for the detection of the blood type of red blood cells, said product being used for detecting the presence of the FUT1236delG allele of the blood type of monta according to claim 1 in a sample to be tested.
Further, the product comprises a PCR amplification sequencing detection kit, and the PCR amplification sequencing detection kit comprises the primer pair.
Further, the PCR amplification sequencing detection kit also comprises a 2 xTaq PCR MasterMix and an internal reference primer pair.
In a fifth aspect of the embodiments of the present invention, there is provided a method for detecting 236delG allele of FUT1 gene belonging to the Bombay class, the method comprising: selectively amplifying a target fragment containing a mutation site by a gene amplification method, thereby detecting the presence or absence of the mutant gene, the detection method comprising the steps of:
extracting genome DNA in a sample to be detected;
carrying out PCR reaction by using the genome DNA as a template and adopting a primer pair for detecting the red blood cell Bombay blood type to obtain a PCR reaction product;
detecting the band pattern of the PCR reaction product by using a gel electrophoresis method;
and judging whether the detected sample carries 236delG alleles according to the banding pattern, and identifying homozygote or heterozygote.
Further, the judging whether the detected sample carries 236delG alleles according to the banding pattern and identifying homozygote or heterozygote specifically comprises:
if the strip pattern of the sample to be detected is: the absence of a 236 delG-specific amplification product band indicates that the sample to be detected is a non-carrier;
if the strip pattern of the sample to be detected is: a 236delG specific amplification product band shows that the sample to be detected is a 236delG allele carrier; the 236delG allele carrier is further judged to be homozygous or heterozygous, including:
if the strip pattern of the sample to be detected is: the band of the 236delG specific amplification product and the band of the 440wild specific amplification product show that the sample to be detected is a carrier of the heterozygote 236delG allele;
if the strip pattern of the sample to be detected is: only the 236 delG-specific amplification product band but not the 440 wild-specific amplification product band indicates that the sample to be tested is a homozygous 236delG allele carrier.
Further, the primer pair for detecting the red blood cell Bombay type is shown in claim 3, and the length of the 236 delG-specific amplification product band and the length of the 440 wild-specific amplification product band are both 250 bp;
simultaneously, 1 pair of housekeeping gene beta-actin primers are introduced to be used as a positive internal reference for DNA sample amplification, the sequences of the internal reference gene primer pairs are shown as SEQ ID NO. 4-NO. 5, and the length of the internal reference amplification product fragment is 200 bp.
Further, the total volume of the reaction system for amplifying the 236delG allele/wild type gene is 10 muL, the reaction system comprises 50ng of DNA template, the final concentration of forward and reverse primers is 0.4 muM respectively, and 2 XTaq PCR MasterMix is 5 ul;
the conditions of the PCR amplification are as follows: 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 ℃.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the embodiment of the invention provides a Bombay FUT1236delG allele and a detection method and application thereof, wherein the Bombay FUT1236delG allele is a G base which is deleted from the 236 th position of an initiation codon in an encoding region of an Hh blood group system variant FUT1 gene. The detection method and the application of the FuT1236delG allele of the blood group similar to Bombay can detect the existence of the FUT1236delG mutant gene doped in a gene bank with high sensitivity and high precision. The detection of the analogous Bombay blood group FUT1236delG allele is to perform PCR amplification on DNA of a blood sample to be detected by adopting the primers to obtain a specific amplification band pattern to be compared with the amplification band pattern of the wild type FUT1 so as to determine whether the analogous Bombay blood group FUT1236delG allele exists in a sample to be detected. Due to the difference of FUT1 gene among different nationalities, on the basis of related research, the gene was specially designed for the newly discovered FUT1236delG allele in Chinese according to the molecular background of the FUT1 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. The detection method can be used for the identification of the FUT1 gene and the identification of the Bombay blood group(s) of a specific individual, 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 the corresponding blood for Bombay blood group(s) patients.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a gel electrophoresis image of the detection of FUT1236delG allele according to the present invention; wherein M is DNAmarker which is respectively 150bp, 200bp, 250bp, 300bp, 350bp and 400bp from bottom to top; lane 1 shows DNA as wild type and primers as wild type primers; lane 2 shows the DNA as wild type and the primers as mutant primers; lane 3 shows DNA as mutant and primers as wild-type; lane 4 shows that the DNA is mutant and the primers are mutant primers; lane 5 shows DNA as heterozygous, wild-type primer; lane 6 shows that the DNA is heterozygous and the primer is a mutant primer; internal control gene: beta-actin, product 200 bp;
FIG. 2 is a chart of sequencing of FUT1236delG allele mutations in examples of the invention; FIG. 2A shows the result of the heterozygous mutant type of the sample 3; FIG. 2B shows the wild-type result of the sample 1,
FIG. 3 is a 3D mimetic diagram of an enzyme structure, wherein FIG. 3A is a 3D mimetic diagram of a wild-type enzyme structure; FIG. 3B is a 3D mimetic diagram of mutant translation products.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
the present application found that the new allele 236delG existing in the FUT1 gene, namely the FUT1236delG allele of Bombay-like blood group, is 1G deleted from the 236 th base of the initiation codon in the coding region of the FUT1 gene of the Hh blood group system variant.
Then, a new PCR-based detection product and detection method for FUT1 gene mutation are established according to the new allele 236delG found on the FUT1 gene. Specifically, a sequence specific primer is designed by utilizing the difference of the mutant gene and the wild type gene in the base sequence of the mutation point, and an individual carrying the mutant gene is identified by a gene amplification method. The detection product and the detection method can detect the existence of the mutant gene doped in the gene library with high sensitivity and high precision. The upstream primer sequence of the specific primer is shown as SEQ ID NO. 1, and the downstream primer sequence is shown as SEQ ID NO. 2, and the specific primer is used for detecting FUT1236delG allele. The primer sequences aiming at the wild type FUT1 gene are shown as SEQ ID NO. 3 and 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 detection method of the Bombay blood group FUT1 gene 236delG mutant gene can be used for the identification of the FUT1 gene and the Bombay blood group of a specific individual, and can also be used for screening 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 patients.
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.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
The FuT1236delG allele, a species of Mumbay, and methods and uses thereof, of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 Bombay blood group FUT1236delG allele
The embodiment of the invention provides an allele mutant gene FUT1236delG, wherein a wild type FUT1 gene and a mutant type FUT1 gene are respectively shown in figure 2; compared with the wild-type FUT1 gene, the mutant FUT1 gene has 1G deletion at the 236 th base of the gene sequence.
As shown in FIG. 3, the mutation type resulted in premature termination of translation due to deletion at 236delG bases. The translation product is an alpha helix structure with no actual function.
Example 2 detection of FUT1236delG alleles of Bombay blood group and method of detection
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 of LProteinase K solution into 200 mu 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 mu LDNasefree deionized water at the middle position of an adsorption film, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.
(10) The DNA extraction effect was evaluated by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined by a NanoDrop ND-1000 ultra-micro UV-Vis nucleic acid protein analyzer.
Second, 236delG allele detection method.
1. Instrument for measuring the position of a moving object
GeneAmpPCRSytem model 9700 PCR instrument (ABI, USA), synGENE-GCXX9 gel imager (synGENE, USA), and gel electrophoresis instrument (Hexay, Beijing).
2. Reagent
The blood genome DNA extraction kit (Beijing Tiangen Biochemical technology Co., Ltd.) and 2 XTaqPCRmastermix 5ul (Beijing Tiangen Biochemical technology Co., Ltd.) were synthesized by Jiangsu Weihe Biotechnology Co., Ltd.
3. Primer design
The primers were designed by Oligo6.0 primer software according to FUT1 (SEQ ID NO: NG-007510.1) recorded in the GenBank of the National Center for Biotechnology Information (NCBI) of America, to finally determine 2 pairs of specific oligonucleotide primer sequences:
236delG-F5’-CAGCACCCTGCTTCCCTCTCCGC-3’
the amplification size of 440Wild-R5 '-GTACTCCTCCGACATCCAGTCG-3' is 250 bp;
236Wild-F5’-CAGCACCCTGCTTCCCTCTCCGG-3’，
440Wild-R5 '-GTACTCCTCCGACATCCAGTCG-3' amplified size is 250 bp.
Meanwhile, 1 pair of primers of housekeeping gene beta-actin is introduced as a positive control for DNA sample amplification, the sequence of the primer pair of beta-actin is shown as SEQ ID NO. 4-NO. 5, and the specific steps are as follows:
and (3) primer application: BactinF: 5'-CATTAAGGAGAAGCTGTGCTAC-3' (SEQ ID NO: 4): bactin R: 5'-GAAGGTAGTTTCGTGGATG-3' (SEQ ID NO:5)
The length of the amplified product fragment is 200 bp.
The allele detection primer sequences and reaction specificities are shown in Table 1.
Table 1.236delG 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 of ExTaqDNApolymerase0.25u (TaKaRa), 25mM MTAPS buffer (pH9.3), 50mM KCl, 3mM MgSO4, 1 μ g of gluclease-freeBSA, 0.2mM each of dNTPmix. 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, with final forward and reverse primer concentrations of 0.4 μ M each, 2 × TaqPCRMastermix5ul (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 judgment principle is as follows:
236delG allele carrier samples present a brightly clear 236 delG-specific amplification product band, whereas non-carrier samples do not have a 236 delG-specific amplification product band;
the detected sample has both 236delG specific amplification product band and 440wild specific amplification product band, and the detected sample is proved to be a carrier of the heterozygous 236delG allele; only the 236 delG-specific amplification product band, but not the 440 wild-specific amplification product band, proved to be carriers of the homozygous 236delG allele.
As can be seen from the figure 1, it is,
respectively detecting the sample 1 to be detected by using a wild type primer and a mutant type primer, and respectively detecting the results in a lane 1 and a lane 2, which show that the sample 1 to be detected has a 440wild specific amplification product strip but does not have a 236delG specific amplification product strip, and judging that the sample 1 to be detected is a wild type;
the sample 2 to be detected is detected by respectively adopting a wild type primer and a mutant type primer, and the results are respectively a Lane 3 and a Lane 4, which indicate that the sample 2 to be detected does not have a 440wild specific amplification product strip and has a 236delG specific amplification product strip, and the sample 2 to be detected is judged to be a mutant type and a homozygous 236delG allele carrier;
and (3) respectively detecting the sample to be detected 3 by using a wild type primer and a mutant type primer, and respectively detecting the result in a lane 5 and a lane 6, which shows that the sample to be detected 3 has a 440wild specific amplification product band and a 236delG specific amplification product band, and the sample to be detected 3 is judged to be a mutant type and a heterozygous 236delG allele carrier.
Through sequencing verification, fig. 2A shows the result of the heterozygous mutant type of the sample 3 to be detected; FIG. 2B shows the wild type result of the sample 1 to be tested, and the sequencing result is consistent with the judgment result of the invention, which shows that the invention has correct judgment and the method is reliable.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<120> one kind of Bombay blood group FUT1236delG allele and detection method and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cagcaccctg cttccctctc cgc 23
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtactcctcc gacatccagt cg 22
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cagcaccctg cttccctctc cgg 23
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cattaaggag aagctgtgct ac 22
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaaggtagtt tcgtggatg 19
Claims (10)
1. A Bombay blood group FUT1236delG allele, wherein the Bombay blood group FUT1236delG allele is a variant of the Hh blood group system FUT1 gene coding region in which 1G is deleted from the 236 th base of the initiation codon.
2. Use of the FUT1236delG alleles of Bombay blood groups according to claim 1 for the preparation of a preparation for the detection of Bombay blood groups.
3. A primer pair for detecting erythrocyte Bombay type is characterized in that the sequence of the primer pair is shown as SEQ ID NO 1-SEQ ID NO 3, wherein the primer sequences aiming at 236delG allele of the FUT1 gene of the Bombay type are shown as SEQ ID NO 1 and SEQ ID NO 2, the primer sequence aiming at the wild FUT1 gene is shown as SEQ ID NO 3 and SEQ ID NO 2,
4. a product for the detection of the blood type of the red blood cells montmoriy, characterized in that it is used to detect the presence of the blood type FUT1236delG allele of claim 1 in a sample to be tested.
5. The article of claim 4, wherein the article comprises a PCR amplification sequencing assay kit, and the PCR amplification sequencing assay kit comprises the primer pair of claim 3.
6. The article for detecting red blood cell Bombay type according to claim 5, wherein the PCR amplification sequencing detection kit further comprises 2 XTaq PCR MasterMix and an internal reference primer pair.
7. A method for detecting FUT1236delG alleles of the Bombay blood group, comprising: selectively amplifying a target fragment containing a mutation site by a gene amplification method, thereby detecting the presence or absence of the mutant gene, the detection method comprising the steps of:
extracting genome DNA in a sample to be detected;
carrying out PCR reaction by using the genome DNA as a template and adopting a primer pair for detecting the red blood cell Bombay blood type to obtain a PCR reaction product;
detecting the band pattern of the PCR reaction product by using a gel electrophoresis method;
and judging whether the detected sample carries 236delG alleles according to the banding pattern, and identifying homozygote or heterozygote.
8. The method for detecting FUT1236delG alleles of Bombay blood group according to claim 7, wherein said determining whether the sample carries 236delG alleles according to said banding pattern and identifying homozygotes or heterozygotes comprises:
if the strip pattern of the sample to be detected is: the absence of a 236 delG-specific amplification product band indicates that the sample to be detected is a non-carrier;
if the strip pattern of the sample to be detected is: a 236delG specific amplification product band shows that the sample to be detected is a 236delG allele carrier; the 236delG allele carrier is further judged to be homozygous or heterozygous, including:
if the strip pattern of the sample to be detected is: the band of the 236delG specific amplification product and the band of the 440wild specific amplification product show that the sample to be detected is a carrier of the heterozygote 236delG allele;
if the strip pattern of the sample to be detected is: only the 236 delG-specific amplification product band but not the 440 wild-specific amplification product band indicates that the sample to be tested is a homozygous 236delG allele carrier.
9. The method for detecting FUT1236delG alleles of Bombay blood group of claim 7, wherein the primer pair for detecting erythrocyte Bombay blood group is shown in claim 3, and the length of the 236 delG-specific amplification product band and the length of the 440 wild-specific amplification product band are both 250 bp;
simultaneously, 1 pair of housekeeping gene beta-actin primers are introduced to be used as a positive internal reference for DNA sample amplification, the sequences of the internal reference gene primer pairs are shown as SEQ ID NO. 4-NO. 5, and the length of the internal reference amplification product fragment is 200 bp.
10. The method for detecting FUT1236delG alleles of Bombay type according to claim 7, wherein the 236delG alleles/wild type gene amplification reaction system has a total volume of 10 μ L, including 50ng DNA template, final forward and reverse primer concentrations of 0.4 μ M each, and 2 XTaq PCR MasterMix5 ul;
the conditions of the PCR amplification are as follows: 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 ℃.
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