Primer set and kit for sanger sequencing method for human rhd blood group genotyping detection

Abstract

The present application relates to a kind of primer set and kit for Sanger sequencing method for human RHD blood group genotyping detection, belong to biomedical clinical molecular detection field.The primer set for Sanger sequencing method for human RHD blood group genotyping detection of the present application, including specific PCR amplification primer set, the specific PCR amplification primer set includes 9 pairs of amplification primer designed according to RHD gene specific sequence;The primer set also includes 10 RHD specific sequencing primers corresponding to amplification product.The present application has the following technical effects: the present application adopts the RHD gene specific amplification primer designed by ARMS and homologous sequence specific base method, and specificity and accuracy are higher;With the RHD specific sequencing primer of the present application, the gene sequence of the sample to be detected is detected by Sanger sequencing method, RHD genotype is interpreted, and new mutation point can be found.The identification result of RHD blood group gene 1 to 10 exons can be obtained by the kit of the present application, so as to accurately determine the RHD gene typing of experimental sample.

Description

TECHNICAL FIELD

[0001] The application relates to a Sanger sequencing method primer group and kit for human RHD blood group genotyping detection, and belongs to the field of biomedical clinical molecular detection. BACKGROUND

[0002] The Rh blood group system is the second largest blood group system next to the ABO blood group system, and has complex polymorphism. Among the five most important antigens D, E, C, c and e in the Rh blood group system, the D antigen has the strongest immunogenicity, is encoded by the RHD gene, consists of 10 exons, has a total coding region length of 1251 bp, and encodes a glycoprotein consisting of 417 amino acids. From the clinical perspective, the D antigen is the most immunogenic and important among the 54 blood group antigens in the Rh system, and therefore the Rh blood group 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 more than 30 red blood cell blood group systems currently recognized by the International Society of Blood Transfusion (ISBT), and its importance in clinical blood transfusion is only next to that of the ABO blood group. It is highly valued in the clinic because it can cause hemolytic transfusion reactions (HTR), hemolytic disease of the newborn (HDFN) and autoimmune hemolytic anemia (AIHA) and the like.

[0003] Currently, serological detection is used for the initial screening of blood recipients in China, and the Rh blood group is routinely preliminarily screened by using anti-D antibody (IgM) in the clinic, and negative results are usually regarded as Rh negative blood group. However, a series of D variants exist in these initial screening negative samples, and they are not truly D antigen completely negative, but have changes in quantity or quality of the expression of D antigen on red blood cells. Subsequently, weak D, partial D and Del phenotypes can be identified by IAT and absorption and dispersion methods. Because the serological test has many steps and a complicated process, and the result determination is greatly affected by human factors, false detection and missed detection often occur. If the D variant phenotypes are not clearly identified, the occurrence of anti-D alloimmunization is extremely likely; or if a high titer monoclonal antibody is used for initial screening, some partial D samples may present a positive reaction and be misjudged as RhD positive.

[0004] Correct identification of the RhD blood group is the key to ensuring the safety of clinical blood transfusion, and blood group identification errors may cause serious transfusion reactions and even lead to the death of patients. The clinically difficult RhD blood group cannot be distinguished and determined by serological means, and must be identified by molecular biology detection. RHD genotyping is more accurate than serological methods, and molecular biology detection can significantly reduce the misdiagnosis rate of RhD blood group identification of patients, improve the ability to solve difficult blood matching, and improve the safety of blood transfusion therapy, thereby providing protection for precise blood transfusion.

[0005] In the aspect of RHD genotyping, the currently commonly used detection methods mainly include PCR-SSP method, fluorescent quantitative PCR (SYBR Green dye method), fluorescent quantitative PCR (Taqman probe method), sequencing method and the like. The detection method most applied in practice at present is PCR-SSP (sequence specific primer, SSP) method, the principle of which is to use primers capable of specifically recognizing specific alleles to detect sequence polymorphism through PCR amplification to produce corresponding specific amplification product bands, and to detect PCR products by agarose gel electrophoresis, and then to genotype alleles according to the presence or absence of PCR products, also known as allele-specific primer PCR method. This method has low cost, but the operation is complex, the result cannot be automatically obtained, and the accuracy needs to be improved; SYBR Green I is a dye with green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its binding to DNA is non-specific. This method is simple and fast, but lacks specificity and has poor accuracy; the appearance of TaqMan probe solves the problem of non-specificity of dye method, but TaqMan probe method also has defects, and although it is simple and fast, it cannot achieve high-resolution genotyping 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 performed. Sequencing method is not widely used at present, and is only used as an auxiliary means for identification of difficult samples in existing research.

[0006] In the paper entitled "Application of Multiplex Ligation-dependent Probe Amplification (MLPA) Technology in Genotyping of RhD Negative Blood Donors in Guangzhou Area" published in Chinese Journal of Transfusion, a detailed RHD gene sequencing method is provided. The method is based on the principle of Sanger sequencing, and PCR amplification of RHD gene exons 1-10 is used as a supplement when other methods do not obtain clear genotyping results in the paper. This experimental method has certain limitations, and the annealing time and temperature required by the RHD gene exon 1-10 amplification primers used in the paper are different, which increases the complexity of the experimental process; in addition, the sequencing primers are mostly amplification primers, and no additional design is performed, the function and applicability of the sequencing primers and the amplification primers are different, and the use of amplification primers for sequencing may lead to weak specificity and deviation of the results.

[0007] Therefore, in view of the limitations of the prior art, there is an urgent need for a more accurate and simple human RHD blood group genotyping detection method to meet the clinical needs, improve accuracy, simplify the operation process, reduce cost, and meet the needs of different fields. SUMMARY

[0008] The purpose of the present application is to improve the prior art and provide a Sanger sequencing method primer set and kit for human RHD blood group genotyping detection to solve the problems in the prior art.

[0009] The principle of the present application is that according to the RHD gene sequence published in the GENBANK database, the RHD exon-specific amplification primers are designed by combining the amplification refractory mutation system (ARMS) analysis method with the homologous sequence-specific base method. 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, indicating that there is a gene sequence completely identical with the specific primer in the sample, and vice versa. The PCR reaction results are detected and analyzed by agarose gel electrophoresis method. After the electrophoresis gel is dyed and analyzed by a gel imaging system, the nucleic acid fragments are distinguished according to the size difference. The reaction amplification identified by electrophoresis is purified for the next step of sequencing analysis to identify the sequence of each allele, so as to realize high-resolution genotyping of RHD.

[0010] The Rh blood group system is one of the most important blood group systems in clinical and is also the most complex and polymorphic system in human red blood cell blood group system. There are more than 50 antigens in the known Rh system, and the common antigens are C, c, D, E and e, among which the immunogenicity of D antigen is the strongest. The high homology of RHD and RHCE makes it extremely difficult to accurately identify RHD subtypes. The ordinary primer design method has certain limitations for distinguishing RHD gene subtypes, and the accuracy is not high, or specific probes need to be designed, and the cost is high.

[0011] Due to the high polymorphism of RHD gene and the high similarity with RHCE sequence, most of which only have one base difference, false positives are easily generated in actual detection. In order to more accurately detect and avoid the occurrence of 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, the specific position of RHD different from RHCE is found, and the forward and reverse primers are designed so that the specific position is located at the end of the forward and reverse primers; according to the detection results, mismatched 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 polymorphism or insertion and deletion of DNA mutation.

[0012] The application has high sensitivity and specificity compared with other conventional methods, realizes high-resolution genotyping of RHD, can provide an important molecular biology detection tool for clinical blood type identification and blood matching, and has a wide application prospect in clinical transfusion and genetic research.

[0013] The technical solutions of the application for solving the technical problems are as follows:

[0014] In the first aspect of the application, a Sanger sequencing method primer group for human RHD blood group genotyping detection is provided, the primer group comprises a specific PCR amplification primer group, the specific PCR amplification primer group comprises 9 pairs of primers designed according to the specific sequence of the RHD gene; the 9 pairs of primers are respectively used for amplifying RHD 1 to 10 exons.

[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 group is a specific primer designed according to the optimized ARMS combined with the homologous sequence specific base method, the amplification primer introduces a mismatch base on the basis of the homologous sequence specific base primer, and the specificity of detection is improved.

[0018] The Sanger sequencing method primer group for human RHD blood group genotyping detection further comprises 10 RHD specific sequencing primers corresponding to the amplification products; the 10 RHD specific sequencing primers, and the nucleotide sequence of each primer is shown in the following table:

[0019]

[0020] In the second aspect of the application, the application of the Sanger sequencing method primer group for human RHD blood group genotyping detection as described in the first aspect in the preparation of a kit for detecting RHD genotyping is provided.

[0021] In the third aspect of the application, a kit containing the Sanger sequencing method primer group for human RHD blood group genotyping detection as described in the first aspect is provided, and the kit further comprises a PCR reaction reagent.

[0022] Further, the PCR reaction reagent comprises a PCR reaction solution and a high-fidelity Taq enzyme.

[0023] 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.

[0024] In a fourth aspect of the present application, a method for detecting RHD genotyping for non-disease diagnosis purposes is provided, which employs the primer set for Sanger sequencing method for human RHD genotyping detection according to the first aspect, and the method comprises an amplification reaction and sequencing.

[0025] 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 program is 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, 4°C until removed.

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

[0027] 1) The present application adopts RHD gene-specific amplification primers designed by ARMS combined with the same sequence-specific base method, which has higher specificity and accuracy; in combination with the RHD-specific sequencing primers of the present application, the gene sequence of the sample to be detected is detected by Sanger sequencing method, the RHD genotype is judged, and new mutation points can be found. The identification results of the first to tenth exons of the RHD blood group gene can be obtained by the kit of the present application, so as to accurately judge the RHD genotyping of the experimental sample. The operation is fast and simple, the cost is low, and it has wide application prospect and clinical reference value.

[0028] 2) The kit containing the Sanger sequencing method-specific amplification primers and specific sequencing primers of the present application can accurately judge the RHD genotyping 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 for detecting human red blood cell RHD genotyping. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is an electrophoresis map of the amplification product using the specific amplification primer of the present application. The amplification product is completely correct in positive and negative, has good specificity, and has no miscellaneous band.

[0030] Figure 2 It is an electrophoresis map of the amplification product using the specific amplification primer of the ordinary non-mismatch group. The amplification product has more non-specific amplification, and the positive and negative of some samples are incorrect.

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

[0032] Figure 3-2 The sequencing graph for sample S2 is shown, and 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 The sequencing graph for sample S3 is shown, and the genotype is RHD*01EL.01, RHD*15. The sequencing peak graph result is good, and the interpretation is correct.

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

[0035] Figure 4 The sequencing graph of the amplification product of the specific amplification primer of the common mismatch-free group is shown. The sequencing map has many miscellaneous peaks, and the result cannot be interpreted. DETAILED DESCRIPTION

[0036] 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, not all of the embodiments.

[0037] In the present application, the main raw materials are listed as follows:

[0038]

[0039]

[0040] Example 1

[0041] 1. Raw materials and equipment:

[0042] 1.1 Kit components:

[0043] 1) The specific amplification primer of the present application comprises 9 pairs of primers designed according to the specific sequences of RHD gene subtypes. The 9 pairs of primers are used to amplify RHD 1-10 exons, respectively. 10 RHD sequencing primers are also included.

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

[0045]

[0046]

[0047] 10 sequencing primer nucleotide sequences are shown in the following table:

[0048]

[0049]

[0050] In the present application, in order to make a comparison, a common RHD amplification primer is also designed.

[0051]

[0052]

[0053] The primer dry powder is dissolved with 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 the amplification primer mixture:

[0055]

[0056]

[0057] Preparation of the sequencing primer solution:

[0058]

[0059] 2) PCR reaction reagents

[0060] DNA polymerase: high-fidelity Taq polymerase; (purchased from Aikongrui Biology)

[0061] 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.

[0062] 1.2 Source of the sample

[0063] 1) Blood sample collection

[0064] The blood sample can be collected with a blood collection tube containing an anticoagulant such as sodium citrate and ethylenediaminetetraacetic acid (EDTA), and the whole blood sample without repeated freeze-thawing is used as the experimental sample, which can be stored fresh or frozen.

[0065] 2) Nucleic acid sample extraction

[0066] The nucleic acid sample can be extracted from whole blood or white blood cell layer containing nucleated cells by precipitation, column or magnetic beads method to obtain sufficient and qualified nucleic acid for polymerase chain reaction.

[0067] 3) Quantification of nucleic acid sample

[0068] The extracted nucleic acid sample must be dissolved in sterilized 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).

[0069] 4) Quality specification of nucleic acid sample

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

[0071] 1.3 Required experimental equipment

[0072] PCR instrument, sequencer, pipettor of different scales, small desktop centrifuge (containing 8 connected horizontal head).

[0073] 2 Genotyping process

[0074] Four EDTA anticoagulated whole blood samples were selected and amplified by the primer set of the present application and the common primer set, respectively. The amplified reaction products identified by electrophoresis were purified to remove excess primers and DNA. Then sequencing reaction was performed, and the sequencing products were purified by ethanol precipitation to remove excess BDT. The final reaction product was read on the sequencer, and the final detection result was obtained after software analysis.

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

[0076] PCR reaction system

[0077] 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

[0078] The reaction tube was covered with a lid, centrifuged briefly, and then placed in a fluorescent quantitative PCR instrument.

[0079] 2.2 PCR reaction program: as shown in the table below:

[0080] PCR reaction program

[0081]

[0082] 2.3 Electrophoresis

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

[0084] 2.4 PCR product purification

[0085] Add 4 μL ExoSAP to the reaction well which you want to sequence, to remove excess primers and DNA.

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

[0087] ExoSap PCR reaction program setting

[0088]

[0089] 2.5 Sequencing reaction

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

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

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

[0093] 2.6 Purification of sequencing product

[0094] Remove excess BDT by ethanol precipitation.

[0095] 2.7 Sequencing

[0096] Before sequencing, you can choose to add 15 μL of HiDi formamide, and then heat treat the PCR instrument before loading it into the sequencer.

[0097] 3 Analysis of experimental results

[0098] 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 .

[0099] 3.2 The specific amplification primer of the ordinary non-mismatch group has poor specificity of the amplification product, many bands, and partially incorrect positive and negative types. The electrophoresis chart is shown in Figure 2 .

[0100] 3.3 The specific amplification primer of the present application has completely correct sequencing results of the amplification product, and can accurately determine the RHD gene typing of the experimental sample. The sequencing chart is shown in Figure 3-1 to Figure 3-4 .

[0101] 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 RHD genotype. Typical chart is shown in Figure 4 .

[0102]

[0103] Conclusion: The ARMS of the application combines the RHD gene specific amplification primer designed by the homologous sequence specific base method, and the identification result of the 1th to 10th exon of the RHD blood group gene can be obtained by cooperating with the sequencing primer of the application, so as to accurately determine the RHD high-resolution gene typing of the experimental sample.

[0104] 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 to implement it, and cannot limit the protection scope of the present application. Any equivalent modification or change made according to the spirit of the present application should be covered within the protection scope of the present application.

Claims

1. A primer set for Sanger sequencing method for human RHD blood group genotyping, characterized in that, The primer set comprises a specific PCR amplification primer set, wherein the specific PCR amplification primer set comprises 9 pairs of primers designed according to the specific sequence of the RHD gene; and the 9 pairs of primers are respectively used for amplifying the 1st to 10th exons of the RHD gene. The nucleotide sequences of the 9 pairs of primers are shown in the following table: The primer set for the Sanger sequencing method for human RHD blood group genotyping further comprises 10 RHD-specific sequencing primers corresponding to the amplification products; and the nucleotide sequences of the 10 RHD-specific sequencing primers are shown in the following table:

2. The application of the primer set for the Sanger sequencing method for human RHD blood group genotyping in claim 1 in the preparation of a kit for detecting RHD gene typing.

3. A kit comprising the primer set for Sanger sequencing for human RHD genotyping as claimed in claim 1, characterized in that, The kit further comprises a PCR reaction reagent.

4. The kit of claim 3, wherein The PCR reaction reagent comprises a PCR reaction solution and a high-fidelity Taq enzyme.

5. The kit of claim 4, wherein 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.

6. A method for RHD genotyping for non-diagnostic purposes using the primer set for Sanger sequencing method for human RHD genotyping according to claim 1, characterized in that, The method comprises a PCR amplification reaction and sequencing.

7. The method of claim 6, wherein, The reaction system of the PCR amplification reaction is as follows: a total volume of 12.14 μL, comprising a PCR reaction solution of 6 μL, an enzyme of 0.14 μL, an amplification primer mixture of 3 μL and a DNA template of 3 μL; the reaction procedure of the PCR amplification reaction is 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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