Human rhce blood group genotyping primer set and kit based on sanger sequencing

Abstract

The present application relates to a kind of human RHCE blood group genotyping primer group and kit based on Sanger sequencing, belong to biomedical clinical molecular detection field.The primer group of the present application includes 9 pairs of primers designed according to the specific sequence of RHCE gene;The 9 pairs of primers are used to amplify RHCE 1 to 10 exons.The primer group also includes 10 RHCE specific sequencing primers corresponding to amplification primer.The specific amplification primer designed by ARMS combined with homologous sequence specific base method is high in specificity, cooperates with the RHCE specific sequencing primer of the present application, detects the gene sequence of the sample to be detected by Sanger sequencing method, interprets the RHCE genotype, and new mutation point can be found.The identification result of RHCE blood group gene 1 to 10 exons can be obtained by the kit of the present application, so as to accurately determine the RHCE high-resolution genotyping of experimental sample.

Description

TECHNICAL FIELD

[0001] The present application relates to a human RHCE blood group genotyping primer set and kit based on Sanger sequencing, belonging to the field of biomedical clinical molecular detection. BACKGROUND

[0002] The RHCE gene and RHD gene are located on chromosome 1 and have highly similar sequence structures. The RHCE gene is mainly composed of 10 exons, and the protein it encodes 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 expression of C / c, E / e, and other antigens. The RHCE gene has high polymorphism, which is one of the important reasons for the complexity of the Rh blood group system. Common polymorphic forms include SNPs (single nucleotide polymorphisms) and gene rearrangements. The allele frequency of the RHCE gene varies significantly among different populations, which leads to different expression patterns of C / c and E / e antigens.

[0003] In recent years, there have been more studies on the structure of the RHCE gene in China, but few studies on the genotyping characteristics of the RHCE gene. The RHCE gene encodes Rh blood group antigens on the surface of red blood cells, including C, c, and E, e antigens. These antigens are of great importance 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 alloimmunization. Some rare RHCE variations may lead to specific clinical manifestations, such as Rh-negative individuals carrying certain specific alleles may exhibit a lack or weak expression of certain Rh antigens, which requires special attention in clinical blood transfusions. In RHCE detection, C / c and E / e genotypes are the focus of attention. This is because C, c, E, e antigens are more antigenic in the Rh blood group system and are more likely to trigger immune responses in clinical blood transfusions and maternal-fetal blood type incompatibility. For example, for Rh-immunized patients with anti-C or anti-e antibodies, genotyping can help predict potential antibody reactions. C antigen and c antigen are products encoded by a pair of alleles. If the recipient is cc genotype (no C antigen on the surface of red blood cells), and blood containing C antigen (such as Ce genotype blood) is input, anti-C antibodies may be produced. Similarly, the same applies to E and e antigens, and for ee genotype individuals, input of blood containing E antigen may trigger the production of anti-E antibodies.

[0004] The RHCE gene typing can be achieved by various molecular biology techniques, including PCR-SSO method, PCR-SSP method, PCR-fluorescent probe method, and next-generation sequencing (NGS) technology. PCR-SSO is a high-efficiency and accurate genotyping technology, which is suitable for gene detection scenarios requiring high resolution and high specificity. The whole technology is complicated and time-consuming, and due to the complexity of operation, operation errors are prone to occur in the process, thereby affecting the accuracy of the results; the principle of PCR-SSP method is to use primers that can specifically recognize specific alleles to detect sequence polymorphism by PCR amplification to produce corresponding specific amplification product bands, and the PCR product is detected by agarose gel electrophoresis, and then the alleles are typed according to the presence or absence of the PCR product, also known as allele-specific primer PCR method. This method has low cost, but the operation is complex, and the result cannot be automatically obtained, and the accuracy needs to be improved; the emergence of Taqman probe method solves the problem of non-specificity of fluorescent PCR dye method, but Taqman probe method also has defects, although it is simple and fast, but it cannot achieve high-resolution typing results; only sequencing method in the above methods can obtain high-resolution results, but specific amplification primers need to be designed first, and PCR amplification is carried out; NGS technology has the characteristics of high throughput and high sensitivity, and the powerful analysis capability helps to identify gene variations, but the high cost and technical threshold also limit its wide application.

[0005] Patent CN 114507724A, entitled "Primer set, kit and application for detecting human red blood cell Rh blood group gene typing", the primer set includes amplification primers for RHCE gene 1-10 exons and 2 introns, and the invention is based on gene chip technology. This technology is a high-throughput and high-sensitivity detection technology. However, due to the large amount of data generated by gene chips, professional data analysis tools and knowledge are needed for processing and interpretation, and the result analysis is difficult; and the cost of gene chip technology equipment and reagents is high, which limits the popularization and application of this technology. Therefore, in practical application, other detection methods and technical means need to be combined to make up for their shortcomings and fully play their advantages.

[0006] In summary, in view of the limitations of the prior art, there is an urgent need for a faster, more accurate and simpler human red blood cell RHCE gene typing detection method to meet the clinical needs, improve accuracy, simplify operation process, reduce cost, and adapt to the needs of different fields. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a human RHCE blood group genotyping primer set and kit based on Sanger sequencing to solve the shortcomings of the prior art.

[0008] The principle of the present application is: according to the RHCE gene sequence published in the GENBANK database, the amplification refractory mutation system (ARMS) analysis method is combined with the homologous sequence specific base method to design RHCE subtype specific amplification primers. When the primer sequence is completely matched with the target sequence to be detected, the polymerase chain reaction (PCR) is carried out. In the reaction process, the target nucleic acid fragment will be copied and amplified, which indicates that there is a gene sequence completely identical with the specific primer in the sample, and vice versa. The PCR reaction result is detected and analyzed by agarose gel electrophoresis method. After the electrophoresis gel is stained and analyzed by a gel imaging system, the nucleic acid fragments are distinguished by size. The reaction amplification identified by electrophoresis is purified for next step of sequencing analysis to identify the sequence of each allele, so as to realize 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 human red blood cell blood group systems. RHD and RHCE are two highly homologous genes located on the short arm of human chromosome 1, which encode RHD and RHCE antigens in the Rh blood group system, respectively. The two genes are very similar in structure, with the same number and sequence of exons, but they are different in function and encode different blood group antigens. The high homology of RHD and RHCE makes it extremely difficult to accurately identify the RHCE subtype. The ordinary primer design method has certain limitations in distinguishing the RHCE gene subtypes, and the accuracy is not high, or specific probes need to be designed, which is high in cost.

[0010] Due to the high polymorphism of RHCE gene and the high similarity with RHD sequence, the sequences of various RHCE gene variants are highly similar, and most of them only differ by one base. In actual detection, false positives are easily generated. In order to more accurately detect and avoid false positives, the inventors improved the ARMS primer design method based on the traditional ARMS, and designed RHD gene specific position specific primers by combining ARMS with homologous sequence specific base method, which improved the accuracy of detection. First, find the specific position of RHD that is different from RHCE, and design forward and reverse primers so that the specific position is at the end of the forward and reverse primers; according to the detection result, add mismatched bases at different positions of the above primers. This design method has high specificity and sensitivity, and is low in cost, which can quickly and accurately detect specific single nucleotide polymorphism or insertion and deletion of DNA mutation.

[0011] The technical solution of the present application to solve the technical problem is as follows:

[0012] In the first aspect of the present application, a Sanger sequencing-based human RHCE blood group genotyping primer set is provided, which comprises a specific PCR amplification primer set containing 9 pairs of primers designed according to the specific sequences of the RHCE gene; the 9 pairs of primers are respectively used for amplifying the 1st to 10th exons of the RHCE gene.

[0013] The nucleotide sequences of the 9 pairs of specific PCR amplification primers 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 the homologous sequence-specific base method, and the amplification primer introduces a mismatched base on the basis of the homologous sequence-specific base primer, thereby improving the specificity of detection.

[0016] The primer set for detecting human red blood cell RHCE gene typing further comprises 10 RHCE sequencing primers corresponding to the amplification primers; the nucleotide sequences of the 10 RHCE sequencing primers are shown in the following table:

[0017]

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

[0019] In the third aspect of the present application, a kit containing the Sanger sequencing-based human RHCE blood group genotyping primer set in the first aspect is provided, and the kit further comprises a PCR reaction reagent.

[0020] Further, the PCR reaction reagent comprises a PCR reaction solution and a high-fidelity Taq enzyme.

[0021] Further, the PCR reaction solution comprises 0.5 mM deoxynucleotides dNTP, 40 mM magnesium chloride MgCl2, 80 mM potassium chloride KCl, 60 mM 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 application, a method for detecting RHCE gene typing for non-disease diagnosis purposes using the Sanger sequencing-based human RHCE blood group genotyping primer set in the first aspect is provided, which comprises an amplification reaction and sequencing.

[0023] The amplification reaction system is as follows: a total volume of 12.14 muL, including 6 muL of PCR reaction solution, 0.14 muL of enzyme, 3 muL of amplification primer mixture and 3 muL of DNA template; the PCR amplification reaction is carried out at 95 DEG C for 5 minutes, 93 DEG C for 30 seconds, 60 DEG C for 40 seconds, 72 DEG C for 2 minutes for 36 cycles, 72 DEG C for 5 minutes, and 4 DEG C until taking out.

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

[0025] 1) The present application adopts the RHCE gene specific amplification primer designed by ARMS combined with the homologous sequence specific base method, which is more specific and accurate; in combination with the RHCE specific sequencing primer of the present application, the gene sequence of the sample to be detected is detected by Sanger sequencing method, the RHCE genotype is judged, and new mutation points can be found. The identification results of the first to tenth exons of the RHCE blood group gene can be obtained by the kit of the present application, so as to accurately judge the RHCE gene typing of the experimental sample. The operation is fast and simple, the cost is low, and it has wide application prospect and clinical reference value.

[0026] 2) The kit containing the Sanger sequencing method specific amplification primer and the specific sequencing primer of the present application can accurately judge the RHCE gene typing of the experimental sample, and can detect the single nucleotide polymorphism or insertion and deletion of each exon of the RHCE variant sample. It can be used for detecting human red blood cell RHCE gene typing. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is an electrophoresis map of the amplification product of the specific amplification primer of the present application. The amplification product is completely correct in positive and negative, and has good specificity without impurity band.

[0028] Figure 2 It is an electrophoresis map of the amplification product of the specific amplification primer of the ordinary non-mismatch group. The amplification product has more non-specific amplification, and the positive and negative of part of the sample is incorrect.

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

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

[0031] Figure 3-3 It is a sample S3 sequencing map, and the genotype is RHCE*cCE. The sequencing peak map result is good and correct.

[0032] Figure 3-4 The sequencing graph of sample S4 is shown in the following table. The genotype is RHCE*cCe. The sequencing peak graph result is good, and the interpretation is correct.

[0033] Figure 4 The sequencing graph of the amplification product of the common mismatch-free group-specific amplification primer is shown in the following table. The sequencing map has many miscellaneous peaks, and the result cannot be interpreted. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. Obviously, the described examples are only some of the embodiments of the present application, but not all.

[0035] In the present application, 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 primer of the present application comprises 9 pairs of primers designed according to the specific sequence of RHCE gene; the 9 pairs of primers are respectively used for amplifying RHCE 1 to 10 exons. The primer set further comprises 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 application, common RHCE amplification primers are also designed for comparison. The sequences are as follows:

[0048]

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

[0050] Preparation of the mixture of amplification primers:

[0051]

[0052]

[0053]

[0054] Preparation of the solution of sequencing primers:

[0055]

[0056] 2) PCR reaction reagents

[0057] DNA polymerase: high fidelity Taq polymerase; (purchased from Aikongrui Biology)

[0058] PCR reaction solution: including 0.5 mM deoxynucleotides dNTP, 40 mM magnesium chloride MgCl2, 80 mM potassium chloride KCl, 60 mM Tris-HCl, 1 mM tetramethylammonium chloride TMAC, glycerol 0.6% v / v, cresol red 0.02% v / v and betaine 5% v / v.

[0059] 1.2 Source of the sample

[0060] 1) Blood sample collection

[0061] The blood sample can be collected by blood collection tube containing anticoagulant sodium citrate and ethylenediaminetetraacetic acid (EDTA), and the fresh or frozen whole blood sample without repeated freeze-thawing is used as the experimental sample.

[0062] 2) Nucleic acid sample extraction

[0063] The nucleic acid extraction can be performed by precipitation method, column method or magnetic bead method from whole blood or white blood cell layer containing nucleated cells, and a sufficient amount of qualified nucleic acid is obtained for polymerase chain reaction.

[0064] 3) Nucleic acid sample quantification

[0065] The extracted nucleic acid sample must be dissolved in sterile water or other appropriate solution (such as TE Buffer), and the concentration should be between 10-40 ng / μl. The nucleic acid sample should not be dissolved in a solution containing more than 0.5 mM chelate 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 machine, sequencer, pipettor of different scales, small desktop centrifuge (containing 8 connecting tube horizontal head).

[0070] 2. Genotyping process

[0071] Select 4 EDTA anticoagulant whole blood samples, respectively, using the primer group and the ordinary primer group for amplification. The amplified reaction product identified by electrophoresis will be purified to remove excess primers and DNA. Then do the sequencing reaction, the sequencing product is purified by ethanol precipitation, and at the same time remove the excess BDT. The final reaction product is read on the sequencer after software analysis to obtain the final detection result.

[0072] 2.1 Preparation of reaction system: the reaction system is shown in the table below:

[0073] PCR reaction system

[0074] Component Name Addition μL / tube PCR reaction solution 6 Amplification primer mixture 3 Taq enzyme 0.14 Nucleic acid sample 3 Total volume 12.14

[0075] Put the reaction tube cover, centrifuge for a while, and then put it into the fluorescence quantitative PCR instrument.

[0076] 2.2 PCR reaction program: as shown in the table below:

[0077] PCR reaction program

[0078]

[0079]

[0080] 2.3 Electrophoresis

[0081] Run the gel at 8-10 volts / cm, 200V, about 10-20 minutes. Take a picture on the ultraviolet transmission instrument to confirm the quality of the PCR product.

[0082] 2.4 PCR product purification

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

[0084] Refer to the table below to set the program and start the purification step. The total reaction time is about 45 min.

[0085] ExoSap PCR reaction program setting

[0086]

[0087] 2.5 Sequencing reaction

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

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

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

[0091] 2.6 Purification of sequencing product

[0092] Remove excess BDT by ethanol precipitation.

[0093] 2.7 Sequencing

[0094] Before sequencing, 15 μL of HiDi formamide can be added, and the PCR instrument is heated and then sequenced.

[0095] 3 Analysis of experimental results

[0096] 3.1 The specific amplification primer of the present application has high specificity of the amplification product, no banding, and completely correct positive and negative types. The electrophoresis chart is shown in Figure 1 .

[0097] 3.2 The specific amplification primer of the ordinary non-mismatch group has poor specificity of the amplification product, many bands, and incorrect positive and negative types of some samples. The electrophoresis chart is shown in Figure 2 .

[0098] 3.3 The specific amplification primer of the present application has completely correct sequencing results of the amplification product, and can accurately determine the RHCE gene typing of the experimental sample. The sequencing chart is shown in Figure 3-1 to Figure 3-4 .

[0099] 3.4 The specific amplification primer of the ordinary non-mismatch group has disordered or no signal of the sequencing chart of the amplification product, and cannot read the RHCE genotype. Typical chart is shown in Figure 4 .

[0100]

[0101] Conclusion: The specific amplification primer of the present application has high specificity, and can obtain the identification results of the 1st to 10th exons of the RHCE blood group gene, thereby accurately determining the RHCE high-resolution gene typing of the experimental sample.

[0102] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent modification or change according to the spirit of the present application should be covered in the protection scope of the present application.

Claims

1. A primer set for human RHCE blood group genotyping based on Sanger sequencing, characterized in that, The primer set includes a specific PCR amplification primer set, which contains 9 pairs of primers designed based on the specific sequence of the RHCE gene; the 9 pairs of primers are used to amplify exons 1 to 10 of the RHCE gene; The nucleotide sequences of the nine primer pairs are shown in the table below: The aforementioned Sanger sequencing-based human RHCE blood group genotyping primer set also includes 10 RHCE sequencing primers corresponding to the amplification primers; the nucleotide sequences of the 10 RHCE sequencing primers are shown in the table below:

2. The application of the Sanger sequencing-based human RHCE blood group genotyping primer set as described in claim 1 in the preparation of a kit for detecting RHCE genotyping.

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

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

5. The reagent kit according to claim 4, 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 diagnostic purposes using the Sanger sequencing-based human RHCE blood group genotyping primer set as described in claim 1, characterized in that, The method includes amplification reaction and sequencing.

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

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