Human RHCE blood type genotyping primer group and kit based on Sanger sequencing

Abstract

The invention relates to a human RHCE blood type genotyping primer group based on Sanger sequencing and a kit, and belongs to the field of biomedicine clinical molecular detection. The primer group disclosed by the invention comprises a specific PCR (Polymerase Chain Reaction) amplification primer group, wherein the specific PCR amplification primer group comprises 9 pairs of primers designed according to a RHCE gene specific sequence; the 9 pairs of primers are respectively used for amplifying RHCE exons 1 to 10. And the primer group also comprises 10 RHCE (Recombinant Heterogeneous Cell Element) specific sequencing primers corresponding to the amplification primers. Compared with the prior art, the invention has the following technical effects: the specificity of the specific amplification primer designed by combining ARMS with a homologous sequence specific base method is high, the gene sequence of a sample to be detected is detected by matching with the RHCE specific sequencing primer, the RHCE genotype is interpreted by a Sanger sequencing method, and a new mutation point can be found. According to the kit disclosed by the invention, the identification result of the RHCE blood group gene exons 1-10 can be obtained, so that the RHCE high-resolution genetic typing of an experimental sample can be accurately judged.

Description

Technical Field

[0001] The present invention relates to a primer set and a kit for human RHCE blood group gene typing based on Sanger sequencing, belonging to the field of biomedical clinical molecular detection. Background Art

[0002] The RHCE gene and the RHD gene are located on chromosome 1 and have highly similar sequence structures. The RHCE gene mainly consists of 10 exons, and the protein encoded by it is part of a protein complex on the red blood cell membrane, responsible for transporting and maintaining ion balance. Variations in the RHCE protein can lead to different expressions of antigens such as C / c and E / e. The RHCE gene has high polymorphism, which is also one of the important reasons for the complexity of the Rh blood group system. Common polymorphism forms include SNPs (single nucleotide polymorphisms) and gene rearrangements. The allele frequencies of the RHCE gene vary significantly among different populations, resulting in different expression patterns of antigens such as C / c and E / e.

[0003] In recent years, there has been more research on the RHCE gene structure in China, but little research on the RHCE gene typing characteristics. The RHCE gene encodes Rh blood group antigens on the surface of red blood cells, including antigens such as C, c, E, and e. These antigens are of great significance in clinical medicine because they are closely related to hemolytic transfusion reactions and hemolytic disease of the newborn (HDN). In patients with sickle cell disease, accurate identification of the RhCE genotype helps prevent alloimmune reactions. Some rare RHCE variations may lead to specific clinical manifestations. For example, when Rh-negative individuals carry certain specific alleles, they may show the absence or weak expression of certain Rh antigens, which requires special attention in clinical transfusion. In RHCE detection, the C / c and E / e genotypes are the key focuses. This is because the C, c, E, and e antigens have strong antigenicity in the Rh blood group system and are prone to immune reactions in situations such as clinical transfusion and maternal-fetal blood group incompatibility. For example, for Rh-immunized patients with anti-C or anti-e antibodies, gene typing can help predict potential antibody reactions. The C antigen and the c antigen are products encoded by a pair of alleles. If the recipient is of the cc genotype (without C antigen on the red blood cell surface) and receives blood containing the C antigen (such as blood of the Ce genotype), anti-C antibodies may be produced. Similarly, for the E and e antigens, for individuals with the ee genotype, receiving blood containing the E antigen may trigger the production of anti-E antibodies.

[0004] RHCE gene typing can be achieved through various molecular biology techniques, including the PCR-SSO method, PCR-SSP method, PCR-fluorescent probe method, and next-generation sequencing (NGS) technology, etc. PCR-SSO is an efficient and accurate gene typing technique, suitable for gene detection scenarios that require high resolution and high specificity. The whole technique is cumbersome and time-consuming. Due to the complex operation, operation errors are likely to occur during the process, thus affecting the accuracy of the results; for the PCR-SSP method, its principle is to use primers that can specifically recognize specific alleles to detect sequence polymorphisms by PCR amplification, generate corresponding specific amplification product bands, and detect the PCR products by agarose gel electrophoresis, and then perform allele typing according to the presence or absence of the PCR products, also known as allele-specific primer PCR method. This method has a low cost, but the operation is complex, the results cannot be automatically obtained, and the accuracy also needs to be improved; the emergence of the Taqman probe method has well solved the problem of non-specificity of the fluorescent PCR dye method, but the Taqman probe method also has defects. Although the operation is simple and fast, high-resolution typing results cannot be achieved; among the above methods, only the sequencing method can obtain high-resolution results, but first, specific amplification primers need to be designed for PCR amplification; NGS technology has the characteristics of high throughput and high sensitivity, and its powerful analysis ability helps to identify gene mutations, but its high cost and technical threshold also limit the wide application of it.

[0005] Patent CN 114507724A - The invention title is "A primer set, kit and application for detecting human red blood cell Rh blood group gene typing". The primer set includes amplification primers for exons 1-10 and intron 2 of the RHCE gene. The invention is based on gene chip technology, which is a high-throughput and high-sensitivity detection technology. However, due to the huge amount of data generated by gene chips, professional data analysis tools and knowledge are required for processing and interpretation, and the result analysis is difficult; moreover, the costs of gene chip technology equipment and reagents remain high, restricting the popularization and application of this technology. Therefore, in practical applications, it is necessary to combine other detection methods and technical means to make up for its deficiencies and give full play to its advantages.

[0006] In summary, in view of the limitations of the existing technology, there is an urgent need for a faster, more accurate and more convenient detection method for human red blood cell RHCE gene typing to meet clinical needs, improve accuracy, simplify the operation process, reduce costs, and be able to adapt to the needs of different fields. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the existing technology and provide a primer set and kit for human RHCE blood group gene typing based on Sanger sequencing to solve the deficiencies in the existing technology.

[0008] The principle of the present invention is as follows: According to the RHCE gene sequences published in the GENBANK database, specific amplification primers for each subtype of RHCE are designed by using the Amplification refractory mutation system (ARMS) analysis method in combination with the homologous sequence-specific base method. When the primer sequence can completely match the target sequence to be detected, a Polymerase Chain Reaction (PCR) reaction is carried out. During the reaction process, the target nucleic acid fragment will be replicated and amplified, indicating that there is a gene sequence in the sample that is exactly the same as the specific primer, otherwise not. The agarose gel electrophoresis method is used to detect and analyze the results of the PCR reaction. When the electrophoresis gel is stained and analyzed by a gel imaging system, the nucleic acid fragments will be distinguished according to their sizes. The reaction amplification products preliminarily identified by electrophoresis will be purified for the next sequencing analysis to identify the sequences of each allele, so as to achieve high-resolution genotyping of RHCE.

[0009] The Rh blood group system is one of the most important blood group systems in clinical practice and is also the most complex and polymorphic system in the human red blood cell blood group system. RHD and RHCE are two highly homologous genes located on the short arm of human chromosome 1, encoding the RHD and RHCE antigens in the Rh blood group system respectively. These two genes are very similar in structure, having the same number and sequence of exons, but they are different in function, encoding different blood group antigens respectively. The high homology between RHD and RHCE makes it extremely difficult to accurately identify the RHCE subtypes. Ordinary primer design methods have certain limitations in distinguishing RHCE gene subtypes, with low accuracy, or require the design of specific probes, resulting in high costs.

[0010] Due to the high polymorphism of the RHCE gene and its high similarity to the RHD sequence, the sequences of various RHCE gene variants are highly similar, and most of them only have a difference of one base. In actual detection, false positives are likely to occur. In order to perform more accurate detection and avoid the occurrence of false positives, the inventor based on the ARMS primer design method and made improvements on the traditional ARMS. Specific primers for specific positions of the RHD gene are designed by using ARMS in combination with the homologous sequence-specific base method, improving the accuracy of detection. First, find out the specific positions where RHD differs from RHCE, and design forward and reverse primers so that this specific position is located at the ends of the forward and reverse primers; according to the detection results, mismatch bases are added at different positions of the above primers. This design method has high specificity and sensitivity, and is low in cost, and can quickly and accurately detect specific single nucleotide polymorphisms or DNA mutations such as insertions and deletions.

[0011] The technical solution for the present invention to solve the technical problems is specifically as follows:

[0012] In the first aspect of the present invention, a primer set for human RHCE blood group gene typing based on Sanger sequencing is provided. The primer set includes a specific PCR amplification primer set, and the specific PCR amplification primer set contains 9 pairs of primers designed according to the specific sequences of the RHCE gene; the 9 pairs of primers are respectively used for amplifying exons 1 to 10 of the RHCE gene.

[0013] The nucleotide sequences of the 9 pairs of specific PCR amplification primer sets are shown in the following table:

[0014]

[0015] The PCR amplification primer set is a specific primer designed according to the optimized ARMS combined with homologous sequence specific base method. The amplification primer introduces a mismatched base on the basis of the homologous sequence specific base primer, which improves the specificity of detection;

[0016] The primer set for detecting human erythrocyte RHCE gene typing further includes 10 RHCE sequencing primers corresponding to the amplification primers; for the 10 RHCE sequencing primers, the nucleotide sequence of each primer is shown in the following table:

[0017]

[0018] In the second aspect of the present invention, there is provided the use of the primer set for human RHCE blood group gene typing based on Sanger sequencing as described in the first aspect in the preparation of a kit for detecting RHCE gene typing.

[0019] In the third aspect of the present invention, a kit containing the primer set for human RHCE blood group gene typing based on Sanger sequencing as described in the first aspect is provided, and the kit further includes PCR reaction reagents.

[0020] Furthermore, the PCR reaction reagents include a PCR reaction solution and a high-fidelity Taq enzyme.

[0021] Furthermore, the PCR reaction solution includes: 0.5 mM deoxynucleotide dNTP, 40 mM magnesium chloride MgCl 2 , 80 mM potassium chloride KCl, 60 mM tris(hydroxymethyl)aminomethane hydrochloride Tris-HCl, 1 mM tetramethylammonium chloride TMAC, 0.6% v / v glycerol, 0.02% v / v cresol red, and 5% v / v betaine.

[0022] In the fourth aspect of the present invention, there is provided a method for detecting RHCE gene typing for non-disease diagnosis purposes using the primer set for human RHCE blood group gene typing based on Sanger sequencing as described in the first aspect, and the method includes an amplification reaction and sequencing.

[0023] Among them, the amplification reaction system is as follows: the total volume is 12.14 μL, including 6 μL of PCR reaction solution, 0.14 μL of enzyme, 3 μL of amplification primer mixture, and 3 μL of DNA template; for the PCR amplification reaction, the reaction program is 95 °C for 5 minutes; 93 °C for 30 seconds, 60 °C for 40 seconds, 72 °C for 2 minutes, for 36 cycles; 72 °C for 5 minutes, and hold at 4 °C until taken out.

[0024] The present invention has the following technical effects:

[0025] 1) The RHCE gene-specific amplification primers designed by the present invention using the ARMS combined with homologous sequence-specific base method have higher specificity and accuracy; in combination with the RHCE-specific sequencing primers of the present invention, the gene sequence of the sample to be detected is detected by the Sanger sequencing method, the RHCE genotype is determined, and new mutation points can be found. The identification results of exons 1 to 10 of the RHCE blood group gene can be obtained through the kit of the present invention, so as to accurately judge the RHCE gene typing of the experimental sample. The operation is fast, simple, and low-cost, and has broad application prospects and clinical reference value.

[0026] 2) The kit containing the specific amplification primers and specific sequencing primers of the Sanger sequencing method of the present invention can accurately judge the RHCE gene typing of the experimental sample, and can detect single nucleotide polymorphisms or insertions and deletions and other variations in each exon of the RHCE variant sample. It can be used to detect the RHCE gene typing of human red blood cells. Description of the Drawings

[0027] Figure 1 It is the electrophoresis pattern of the amplification product using the specific amplification primers of the present invention. The positive and negative of the amplification product are completely correct, with good specificity and no non-specific bands.

[0028] Figure 2 It is the electrophoresis pattern of the amplification product using the specific amplification primers of the ordinary mismatch-free group. There are more non-specific amplifications in the amplification product, and the positive and negative of some samples are incorrect.

[0029] Figure 3-1 It is the sequencing pattern of sample S1, and the genotype is RHCE*cCeE. The result of the sequencing peak pattern is good and the interpretation is correct.

[0030] Figure 3-2 It is the sequencing pattern of sample S2, and the genotype is RHCE*Ce. The result of the sequencing peak pattern is good and the interpretation is correct.

[0031] Figure 3-3 This is the sequencing diagram of sample S3 with the genotype RHCE*cCE. The sequencing peak diagram shows good results and the interpretation is correct.

[0032] Figure 3-4 This is the sequencing diagram of sample S4 with the genotype RHCE*cCe. The sequencing peak diagram shows good results and the interpretation is correct.

[0033] Figure 4 This is an example of the sequencing diagram of the amplification product of a common mismatch-free group-specific amplification primer. There are many miscellaneous peaks in the sequencing map and the results cannot be interpreted. Detailed implementation mode

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0035] In the present invention, the main raw material list involved is as follows:

[0036]

[0037]

[0038] Example 1

[0039] 1 Raw materials and equipment:

[0040] 1.1 Reagent components:

[0041] 1) The specific amplification primers of the present invention include 9 pairs of primers designed according to the specific sequences of the RHCE gene; the 9 pairs of primers are respectively used to amplify exons 1 to 10 of RHCE. The primer set also includes 10 RHCE sequencing primers corresponding to the amplification primers.

[0042] The nucleotide sequences of the 9 pairs of specific PCR amplification primer sets are shown in the following table:

[0043]

[0044]

[0045] The nucleotide sequences of the sequencing primers are shown in the following table:

[0046]

[0047] In the present invention, for the purpose of comparison, common RHCE amplification primers were also designed. The sequences are as follows:

[0048]

[0049] The primer dry powder is dissolved and diluted with TE#1 (10 mmol / L Tris-HCl, 0.1 mmol / L EDTA) into an aqueous solution of 12 OD / mL.

[0050] Preparation of the amplification primer mixture:

[0051]

[0052]

[0053]

[0054] Preparation of the sequencing primer solution:

[0055]

[0056] 2) PCR reaction reagents

[0057] DNA polymerase: a high-fidelity Taq polymerase; (purchased from Aikrui Biotech)

[0058] PCR reaction solution: including 0.5 mM deoxynucleotide dNTP, 40 mM magnesium chloride MgCl 2 , 80 mM potassium chloride KCl, 60 mM tris(hydroxymethyl)aminomethane hydrochloride Tris-HCl, 1 mM tetramethylammonium chloride TMAC, 0.6% v / v glycerol, 0.02% v / v cresol red and 5% v / v betaine.

[0059] 1.2 Sources of samples

[0060] 1) Blood sample collection

[0061] Blood samples can be collected using blood collection tubes containing anticoagulants sodium citrate and ethylenediaminetetraacetic acid (EDTA), and fresh or frozen whole blood samples without repeated freezing and thawing are used as experimental samples.

[0062] 2) Nucleic acid sample extraction

[0063] Nucleic acids can be extracted from samples containing nucleated cells such as whole blood or buffy coat by precipitation, column or magnetic bead methods to obtain sufficient and qualified nucleic acids for polymerase chain reaction.

[0064] 3) Nucleic acid sample quantification

[0065] The extracted nucleic acid samples must be dissolved in sterile water or other appropriate solutions (such as TE Buffer), and the concentration should be between 10 - 40 ng / μl. Nucleic acid samples should not be dissolved in solutions containing more than 0.5 mM chelates such as ethylenediaminetetraacetic acid (EDTA).

[0066] 4) Nucleic Acid Sample Quality Specification

[0067] The A260 / A280 ratio of the nucleic acid sample should be between 1.6 and 2.1.

[0068] 1.3 Required Experimental Equipment

[0069] PCR instrument, sequencer, pipettes with different ranges, small desktop centrifuge (including 8-well horizontal head).

[0070] 2 Genotyping Process

[0071] Select 4 EDTA anticoagulated whole blood samples and amplify them with the primer set of the present invention and the common primer set respectively. The reaction amplification products preliminarily identified by electrophoresis will be purified to remove excess primers and DNA. Then a sequencing reaction is performed, and the sequencing products are purified by ethanol precipitation while removing excess BDT. After the data of the final reaction products are read on the sequencer, the final test results are obtained through software analysis.

[0072] 2.1 Preparation of Reaction System: The reaction system is shown in the following table:

[0073] PCR Reaction System

[0074] Component Name Addition Volume μL / tube PCR Reaction Solution 6 Amplification Primer Mix 3 Taq Enzyme 0.14 Nucleic Acid Sample 3 Total Volume 12.14

[0075] Cover the reaction tube, briefly centrifuge it, and then place it in the fluorescence quantitative PCR instrument.

[0076] 2.2 PCR Reaction Program: As shown in the following table:

[0077] PCR Reaction Program

[0078]

[0079]

[0080] 2.3 Electrophoresis

[0081] Run the gel at 8 - 10 volts / cm, 200V, for about 10 - 20 minutes. Take a photo on the ultraviolet transilluminator to confirm the quality of the PCR products.

[0082] 2.4 Purification of PCR Products

[0083] For the reaction wells to be sequenced, add 4 μL of ExoSAP to remove excess primers and DNA.

[0084] Set the program according to the following table and start the purification step. The total reaction time is about 45 min.

[0085] ExoSap PCR Reaction Program Settings

[0086]

[0087] 2.5 Sequencing Reaction

[0088] Add 1.5 μL of BDT sequencing reagent to each reaction well;

[0089] Add 2.5 μL of sequencing primer to each reaction well;

[0090] Add 1 μL of purified PCR product to each reaction well.

[0091] 2.6 Purification of Sequencing Products

[0092] Use the ethanol precipitation method to remove excess BDT.

[0093] 2.7 Sequencing on the Machine

[0094] Before sequencing, 15 μL of HiDi formamide can be optionally added, and after heat treatment in a PCR instrument, it is loaded onto the sequencer.

[0095] 3 Analysis of Experimental Results

[0096] 3.1 The specific amplification primers of the present invention have high specificity of amplification products, no non-specific bands, and the positive and negative types are completely correct. See the electrophoresis diagram in Figure 1 .

[0097] 3.2 The specific amplification primers of the ordinary mismatch-free group have poor specificity of amplification products, many non-specific bands, and incorrect positive and negative types for some samples. See the electrophoresis diagram in Figure 2 .

[0098] 3.3 The specific amplification primers of the present invention have completely correct sequencing results of their amplification products, and can accurately judge the RHCE gene typing of experimental samples. See the sequencing diagram in Figures 3-1 to 3-4 .

[0099] 3.4 The sequencing diagram of the amplification products of the specific amplification primers of the ordinary mismatch-free group is messy or has no signal, and the RHCE genotype cannot be read. See the typical diagram example in Figure 4 .

[0100]

[0101] Conclusion: The specific amplification primers of the present invention have high specificity. In combination with the sequencing primers of the present invention, the identification results of exons 1 to 10 of the RHCE blood group gene can be obtained, so as to accurately judge the high-resolution RHCE gene typing of experimental samples.

[0102] The above embodiments are only used to illustrate the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent modifications or changes made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A human RHCE blood type genotyping primer set based on Sanger sequencing, characterized in that: The primer set includes a specific PCR amplification primer set, and the specific PCR amplification primer set includes 9 pairs of primers designed according to the specific sequence of the RHCE gene; the 9 pairs of primers are respectively used to amplify exons 1 to 10 of the RHCE gene; The nucleotide sequences of the 9 pairs of specific PCR amplification primers are shown in the following table: The human RHCE blood type genotyping primer set based on Sanger sequencing also includes 10 RHCE sequencing primers corresponding to the amplification primers; the nucleotide sequence of each of the 10 RHCE sequencing primers is shown in the following table:

2. Use of the human RHCE blood type genotyping primer set based on Sanger sequencing as claimed in claim 1 in preparing a kit for detecting RHCE genotyping.

3. A kit containing the human RHCE blood type genotyping primer set based on Sanger sequencing as claimed in claim 1, characterized in that: The kit also includes PCR reaction reagents.

4. The kit according to claim 3, characterized in that The PCR reaction reagents include PCR reaction solution and high-fidelity Taq enzyme.

5. The kit according to claim 3, characterized in that The PCR reaction solution comprises: 0.5 mM deoxynucleotide dNTP, 40 mM magnesium chloride MgCl2, 80 mM potassium chloride KCl, 60 mM tris-hydroxymethylaminomethane hydrochloride Tris-HCl, 1 mM tetramethylammonium chloride TMAC, 0.6% v / v glycerol, 0.02% v / v cresol red and 5% v / v betaine.

6. A method for detecting RHCE genotyping for non-disease diagnosis purposes using the human RHCE blood type genotyping primer set based on Sanger sequencing as claimed in claim 1, characterized in that: The method includes amplification reaction and sequencing.

7. The method according to claim 1, characterized in that The amplification reaction system is as follows: a total volume of 12.14 μL, including 6 μL of PCR reaction solution, 0.14 μL of enzyme, 3 μL of amplification primer mixture, and 3 μL of DNA template; the PCR amplification reaction has a reaction procedure of 95°C for 5 minutes; 93°C for 30 seconds, 60°C for 40 seconds, 72°C for 2 minutes, 36 cycles; 72°C for 5 minutes, and 4°C until removal.

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