Sanger sequencing method primer group and kit for human RHD blood group genotyping detection

Abstract

The invention relates to a Sanger sequencing method primer group and a kit for human RHD blood group genotyping detection, and belongs to the field of biomedicine clinical molecular detection. The Sanger sequencing method primer group for human RHD blood type genotyping detection comprises a specific PCR (Polymerase Chain Reaction) amplification primer group, and the specific PCR amplification primer group comprises 9 pairs of amplification primers designed according to an RHD gene specific sequence; and the primer group also comprises 10 RHD specific sequencing primers corresponding to the amplification products. The RHD gene specific amplification primer has the following technical effects that the RHD gene specific amplification primer designed by adopting an ARMS combined homologous sequence specific base method is higher in specificity and accuracy; in cooperation with the RHD specific sequencing primer, the gene sequence of a sample to be detected is detected through a Sanger sequencing method, the RHD genotype is interpreted, and a new mutation point can be found. According to the kit disclosed by the invention, the identification results of the exons from the RHD blood group genes 1 to 10 can be obtained, so that the RHD genetic typing of an experimental sample can be accurately judged.

Description

Technical Field

[0001] The invention relates to a Sanger sequencing primer set and a kit for human RHD blood type gene typing detection, and belongs to the field of biomedical clinical molecular detection. Background Art

[0002] The Rh blood group system is the second largest blood group system after the ABO blood group system, and it has complex polymorphism. Among the five most important antigens in the Rh blood group system, D, E, C, c, and e, the D antigen has the strongest immunogenicity. It is encoded by the RHD gene, consists of 10 exons, and the total length of the coding region is 1251bp, encoding a glycoprotein composed of 417 amino acids. From a clinical point of view, the D antigen is the most immunogenic and important of the 54 blood group antigens in the Rh system. Therefore, the Rh blood type is divided into Rh positive and Rh negative according to whether the D antigen is contained on the surface of red blood cells. The Rh blood group system is the most complex, polymorphic, and immunogenic blood group system among the more than 30 red blood cell blood group systems currently confirmed by the International Society of Blood Transfusion (ISBT). Its importance in clinical transfusion is second only to the ABO blood group. It is highly valued in clinical practice because it can cause hemolytic transfusion reactions (HTR), hemolytic disease of the newborn (HDFN), and autoimmune hemolytic anemia (AIHA).

[0003] At present, serological tests are used for the initial screening of clinical blood recipients in my country. Anti-D antibodies (IgM) are routinely used for preliminary screening of Rh blood types in clinical practice, and negative results are usually regarded as Rh-negative blood types. However, a series of D variants are present at low frequencies in these initially negative samples. They are not completely negative for D antigen in the true sense, and the expression of D antigen on red blood cells has quantitative or qualitative changes. Subsequently, weak D, partial D and Del phenotypes can be identified by IAT and absorption emission method. Since serological tests have many steps, the process is cumbersome, and the result judgment is greatly affected by human factors, false detection and missed detection often occur. If the phenotype of D variants is not clearly identified, it is very likely to lead to the occurrence of anti-D alloimmunization; or, if high-titer monoclonal antibodies are used in the initial test, some D-type samples will show positive reactions and be misjudged as RhD positive.

[0004] Correct identification of RhD blood type is the key to ensuring clinical safe blood transfusion. Incorrect blood type identification may cause serious transfusion reactions or even lead to the patient's death. Clinically difficult RhD blood types cannot be distinguished and determined only by serological means and must be identified through molecular biological testing. RHD gene typing is more accurate than serological methods. Molecular biological testing can significantly reduce the false positive rate of RhD blood type identification in patients, improve the ability to solve difficult blood matching, improve the safety of transfusion therapy, and provide guarantees for accurate blood transfusion.

[0005] In terms of RHD genotyping, the commonly used detection methods currently include PCR-SSP method, fluorescence quantitative PCR (SYBRGreen dye method), fluorescence quantitative PCR (Taqman probe method), sequencing method, etc. The most commonly used detection method in practice is PCR-SSP (sequence specific primer, SSP) method, the principle of which is to use primers that can specifically identify specific alleles to detect sequence polymorphisms through PCR amplification, produce corresponding specific amplification product bands, and detect PCR products by agarose gel electrophoresis, and then type alleles according to the presence or absence of PCR products, which is also called allele-specific primer PCR method. This method is low-cost, but the operation is complicated, the results cannot be obtained automatically, and the accuracy needs to be improved; SYBR Green I is a dye with a green excitation wavelength that binds to all dsDNA double helix minor groove regions. Its binding to DNA is non-specific. Although this method is simple and fast to operate, it lacks specificity and has poor accuracy; the emergence of TaqMan probes has solved the problem of non-specificity of the dye method, but the TaqMan probe method also has defects. Although the operation is simple and fast, it cannot achieve high-resolution typing results; Among the above methods, only sequencing can obtain high-resolution results, but first it is necessary to design specific amplification primers for PCR amplification. Sequencing is not widely used at present, and in existing studies it is only used as an auxiliary means for the identification of difficult samples.

[0006] In the paper entitled "Application of Multiple Ligation-dependent Probe Amplification (MLPA) Technology in Genotyping Research of RhD-negative Blood Donors in Guangzhou", published in the Chinese Journal of Blood Transfusion, a detailed RHD gene sequencing method was provided. This method is based on the Sanger sequencing principle and PCR amplifies exons 1 to 10 of the RHD gene. It is used in the paper as a supplement when other methods do not produce clear genotyping results. This experimental method has certain limitations. The amplification primers used in the paper for exons 1 to 10 of the RHD gene require different annealing times and temperatures, which increases the complexity of the experimental process. In addition, sequencing primers mostly use amplification primers without additional design. The functions and applicability of sequencing primers are different from those of amplification primers. Using amplification primers for direct sequencing may result in low specificity and biased results.

[0007] Therefore, in view of the limitations of existing technologies, there is an urgent need for a more accurate and simple human RHD blood type genotyping method to meet clinical needs, improve accuracy, simplify operating procedures, reduce costs, and be able to meet the needs of different fields. Summary of the invention

[0008] The purpose of the present invention is to improve the existing technology and provide a Sanger sequencing primer set and a kit for human RHD blood type genotyping detection to solve the shortcomings of the existing technology.

[0009] The principle of the present invention is: according to the RHD gene sequence published in the GENBANK database, the specific amplification primers for each RHD exon are designed by using the Amplification Refractory Mutation System (ARMS) analysis method combined with the homologous sequence specific base method. When the primer sequence and the target sequence to be tested can completely match, a polymerase chain reaction (PCR) reaction is performed. During the reaction, the target nucleic acid fragment will be copied and amplified, indicating that there is a gene sequence in the sample that is exactly the same as the specific primer, otherwise not. The PCR reaction results are detected and analyzed using agarose gel electrophoresis. When the electrophoresis gel is stained and analyzed by a gel imaging system, the nucleic acid fragments will be distinguished due to different sizes. The reaction amplification product preliminarily identified by electrophoresis will be purified for the next step of sequencing analysis to identify the sequence of each allele, thereby achieving high-resolution genotyping of RHD.

[0010] The Rh blood group system is one of the most important blood group systems in clinical practice, and it is also the most complex and polymorphic system in the human red blood cell blood group system. There are more than 50 known antigens in the Rh system, and 5 common antigens are C, c, D, E, and e, among which the D antigen has the strongest immunogenicity. The high homology between RHD and RHCE makes it extremely difficult to accurately identify RHD subtypes. Ordinary primer design methods have certain limitations in distinguishing RHD gene subtypes, with low accuracy, or require the design of specific probes, which is costly.

[0011] Since the RHD gene is highly polymorphic and highly similar to the RHCE sequence, most of them only differ by one base. In actual detection, false positives are very likely to occur. In order to detect more accurately and avoid false positives, the inventors have improved the ARMS primer design method based on the traditional ARMS, and used ARMS combined with the homologous sequence specific base method to design specific primers for specific positions of the RHD gene, thereby improving the accuracy of detection. First, find out the specific position of RHD that is different from RHCE, and design forward and reverse primers so that the specific position is located at the end of the forward and reverse primers; according to the test results, add mismatched bases at different positions of the above primers. This design method is highly specific and sensitive, and is low-cost, and can quickly and accurately detect specific single nucleotide polymorphisms or DNA mutations such as insertions and deletions.

[0012] Compared with other traditional methods, the technology used in the present invention has high sensitivity and specificity, realizes high-resolution genotyping of RHD, can provide an important molecular biological detection tool for clinical blood typing and blood transfusion matching, and has broad application prospects in clinical transfusion and genetic research.

[0013] The technical solution of the present invention to solve the technical problem is as follows:

[0014] In the first aspect of the present invention, a Sanger sequencing primer set for human RHD blood type genotyping detection is provided, wherein the primer set includes a specific PCR amplification primer set, wherein the specific PCR amplification primer set includes 9 pairs of primers designed according to the RHD gene specific sequence; the 9 pairs of primers are respectively used to amplify RHD exons 1 to 10.

[0015] The nucleotide sequences of the 9 pairs of specific PCR amplification primers are shown in the following table:

[0016]

[0017] The PCR amplification primer set is a specific primer designed based on the optimized ARMS combined with the homologous sequence specific base method. The amplification primer introduces mismatched bases on the basis of the homologous sequence specific base primer, thereby improving the specificity of the detection;

[0018] The Sanger sequencing primer set for human RHD blood type genotyping detection also includes 10 RHD-specific sequencing primers corresponding to the amplified products; the nucleotide sequence of each of the 10 RHD-specific sequencing primers is shown in the following table:

[0019]

[0020] In the second aspect of the present invention, there is provided a use of the Sanger sequencing primer set for human RHD blood type genotyping detection as described in the first aspect in the preparation of a kit for detecting RHD genotyping.

[0021] In the third aspect of the present invention, a kit is provided containing the Sanger sequencing primer set for human RHD blood type genotyping detection as described in the first aspect, and the kit also includes a PCR reaction reagent.

[0022] Furthermore, the PCR reaction reagents include PCR reaction solution and high-fidelity Taq enzyme.

[0023] Furthermore, the PCR reaction solution includes: 0.5 mM deoxyribonucleotide dNTP, 40 mM magnesium chloride MgCl 2, 80 mM potassium chloride KCl, 60 mM tris-HCl Tris-HCl, 1 mM tetramethylammonium chloride TMAC, glycerol 0.6% v / v, cresol red 0.02% v / v and betaine 5% v / v.

[0024] In the fourth aspect of the present invention, a method for detecting RHD genotyping for non-disease diagnosis purposes using the Sanger sequencing primer set for human RHD blood type genotyping detection as described in the first aspect is provided, wherein the method comprises an amplification reaction and sequencing.

[0025] 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; 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 removed.

[0026] The present invention includes the following technical effects:

[0027] 1) The present invention adopts ARMS combined with homologous sequence specific base method to design RHD gene specific amplification primers, which have higher specificity and accuracy; in combination with the RHD specific sequencing primers of the present invention, the gene sequence of the sample to be tested is detected by Sanger sequencing, the RHD genotype is determined, and new mutation points can be found. The kit of the present invention can obtain the identification results of exons 1 to 10 of the RHD blood type gene, thereby accurately determining the RHD genotyping of the experimental sample. The operation is fast and simple, the cost is low, and it has broad application prospects and clinical reference value.

[0028] 2) The kit containing the Sanger sequencing specific amplification primer and the specific sequencing primer of the present invention can accurately determine the RHD genotype of the experimental sample, and can detect the single nucleotide polymorphism or insertion and deletion of each exon of the RHD variant sample. It can be used to detect the RHD genotype of human red blood cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The electrophoresis diagram of the amplification product using the specific amplification primer of the present invention is shown in Figure 1. The amplification product has completely correct positive and negative properties, good specificity, and no mixed bands.

[0030] Figure 2 This is the electrophoresis diagram of the amplification product using common non-mismatched group-specific amplification primers. The amplification product has a lot of non-specific amplification, and the positive and negative of some samples are incorrect.

[0031] Figure 3-1This is the sequencing graph of sample S1, the genotype is RHD*15, RHD*15. The sequencing peak graph result is good and the interpretation is correct.

[0032] Figure 3-2 This is the sequencing graph of sample S2, the genotype is RHD*01EL.01, RHD*01EL.01. The sequencing peak graph result is good and the interpretation is correct.

[0033] Figure 3-3 This is the sequencing graph of sample S3, the genotype is RHD*01EL.01, RHD*15. The sequencing peak graph result is good and the interpretation is correct.

[0034] Figure 3-4 This is the sequencing graph of sample S4, the genotype is RHD*01EL.32, RHD*01EL.32. The sequencing peak graph result is good and the interpretation is correct.

[0035] Figure 4 This is an example of the sequencing graph of the amplification product of a common non-mismatched group-specific amplification primer. The sequencing graph has many impurities and the results cannot be interpreted. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0037] In the present invention, the list of main raw materials involved is as follows:

[0038]

[0039]

[0040] Example 1

[0041] 1. Raw materials and equipment:

[0042] 1.1 Kit components:

[0043] 1) The specific amplification primers of the present invention include 9 pairs of primers designed according to the RHD gene subtype specific sequence. The 9 pairs of primers are used to amplify RHD exons 1 to 10 respectively. It also includes 10 RHD sequencing primers.

[0044] The nucleotide sequences of 9 pairs of specific PCR amplification primers are shown in the following table:

[0045]

[0046]

[0047] The nucleotide sequences of the 10 sequencing primers are shown in the following table:

[0048]

[0049]

[0050] In the present invention, for comparison, common RHD amplification primers were also designed.

[0051]

[0052]

[0053] The primer dry powder was dissolved in TE#1 (10 mmol / L Tris-HCl, 0.1 mmol / L EDTA) and diluted into an aqueous solution of 12 OD / mL.

[0054] Preparation of amplification primer mix:

[0055]

[0056]

[0057] Preparation of sequencing primer solution:

[0058]

[0059] 2) PCR reaction reagents

[0060] DNA polymerase: high-fidelity Taq polymerase (purchased from Acre Bio)

[0061] PCR reaction solution: including 0.5mM deoxynucleotide dNTP, 40mM magnesium chloride MgCl 2 , 80 mM potassium chloride KCl, 60 mM tris-HCl Tris-HCl, 1 mM tetramethylammonium chloride TMAC, glycerol 0.6% v / v, cresol red 0.02% v / v and betaine 5% v / v.

[0062] 1.2 Source of samples

[0063] 1) Blood sample collection

[0064] Blood samples can be collected using blood collection tubes containing anticoagulants sodium citrate and ethylenediaminetetraacetic acid (EDTA), and fresh or frozen whole blood samples that have not been repeatedly frozen and thawed can be used as experimental samples.

[0065] 2) Nucleic acid sample extraction

[0066] 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 nucleic acids of acceptable quality for polymerase chain reaction.

[0067] 3) Quantification of nucleic acid samples

[0068] The extracted nucleic acid sample must be dissolved in sterile water or other appropriate solutions (such as TE Buffer), and the concentration should be between 10-40ng / μl. The nucleic acid sample should not be dissolved in a solution containing more than 0.5mM chelating agents such as ethylenediaminetetraacetic acid (EDTA).

[0069] 4) Nucleic acid sample quality standards

[0070] The A260 / A280 ratio of nucleic acid samples should be between 1.6 and 2.1.

[0071] 1.3 Required experimental equipment

[0072] PCR instrument, sequencer, pipettes of different ranges, small desktop centrifuge (including 8-tube horizontal head).

[0073] 2 Genotyping process

[0074] Four EDTA anticoagulated whole blood samples were selected and amplified using the primer set of the present invention and the common primer set, respectively. The reaction amplification products preliminarily identified by electrophoresis were purified to remove excess primers and DNA. Then a sequencing reaction was performed, and the sequencing products were purified by ethanol precipitation, while removing excess BDT. The final reaction product was read on the sequencer and analyzed by software to obtain the final test result.

[0075] 2.1 Preparation of reaction system: The reaction system is shown in the following table:

[0076] PCR reaction system

[0077] Component name Add μL / tube PCR reaction solution 6 Amplification Primer Mix 3 Taq enzyme 0.14 Nucleic acid samples 3 Total volume 12.14

[0078] Cover the reaction tube, centrifuge briefly, and then place it in the fluorescence quantitative PCR instrument.

[0079] 2.2 PCR reaction procedure: as shown in the following table:

[0080] PCR reaction procedure

[0081]

[0082] 2.3 Electrophoresis

[0083] Run the gel at 8-10 volts / cm, 200 V, for about 10-20 minutes. Take a photo on a UV transilluminator to confirm the quality of the PCR product.

[0084] 2.4 PCR product purification

[0085] Add 4 μL of ExoSAP to the wells you wish to sequence to remove excess primers and DNA.

[0086] Set up the program according to the table below and start the purification step. The total reaction time is about 45 minutes.

[0087] ExoSap PCR reaction program settings

[0088]

[0089] 2.5 Sequencing reaction

[0090] Add 1.5 μL of BDT sequencing reagent to each reaction well;

[0091] Add 2.5 μL of sequencing primer to each reaction well;

[0092] Add 1 μL of purified PCR product to each reaction well.

[0093] 2.6 Sequencing product purification

[0094] Excess BDT was removed by ethanol precipitation.

[0095] 2.7 Sequencing

[0096] Before sequencing, you can choose to add 15 μL of HiDi formamide, heat it in the PCR instrument, and then put it on the sequencer.

[0097] 3 Experimental results analysis

[0098] 3.1 The specific amplification primers of the present invention have high specificity for the amplified product, no mixed bands, and the positive and negative types are completely correct. Figure 1 .

[0099] 3.2 Ordinary non-mismatched group-specific amplification primers, the amplification product has poor specificity, many mixed bands, and some samples have incorrect positive and negative types. The electrophoresis diagram is shown in Figure 2 .

[0100] 3.3 The specific amplification primers of the present invention have completely correct sequencing results of their amplification products, and can accurately determine the RHD genotype of the experimental samples. The sequencing diagram is shown in Figure 3-1 to Figure 3-4 .

[0101] 3.4 The sequencing graph of the amplification product of the common non-mismatched group-specific amplification primer is messy or has no signal, and the RHD genotype cannot be read. For example, a typical graph is shown in Figure 4 .

[0102]

[0103] Conclusion: The RHD gene-specific amplification primers designed by the ARMS combined with the homologous sequence-specific base method of the present invention have high specificity. When combined with the sequencing primers of the present invention, the identification results of exons 1 to 10 of the RHD blood type gene can be obtained, thereby accurately determining the RHD high-resolution genotyping of the experimental samples.

[0104] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used 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 included in the protection scope of the present invention.

Claims

1. A Sanger sequencing primer set for human RHD blood type genotyping detection, 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 RHD gene; the 9 pairs of primers are respectively used to amplify exons 1 to 10 of the RHD gene; The nucleotide sequences of the 9 pairs of specific PCR amplification primers are shown in the following table: The Sanger sequencing primer set for human RHD blood type genotyping detection also includes 10 RHD-specific sequencing primers corresponding to the amplified products; the nucleotide sequence of each of the 10 RHD-specific sequencing primers is shown in the following table:

2. Use of the Sanger sequencing primer set for human RHD blood type genotyping detection as claimed in claim 1 in the preparation of a kit for detecting RHD genotyping.

3. A kit comprising the Sanger sequencing primer set for human RHD blood type genotyping detection 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 RHD genotyping for non-disease diagnosis purposes using the Sanger sequencing primer set for human RHD blood type genotyping detection 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.

Images