Primer group and kit for MNS blood type genetic typing detection and application

Abstract

The invention relates to a primer group for MNS blood type genotyping detection, a kit and application, and belongs to the field of biomedicine clinical molecular detection. The primer group comprises one pair of amplification primers designed according to a GYPA gene specific sequence and five sequencing primers, and one pair of amplification primers designed according to a GYPB gene specific sequence and five sequencing primers. The specific amplification primer can be used for accurately and specifically amplifying the GYPA and the GYPB respectively, and the specific sequencing primer can be used for carrying out high-resolution sequencing reaction on the GYPA and GYPB gene subtypes. The invention has the following technical effects: the specificity of the GYPA and GYPB gene specific amplification primers designed by adopting a mismatch and double specific base combination method is high, and the GYPA and GYPB gene subtypes can be accurately distinguished by matching with the specific sequencing primers provided by the invention. The kit provided by the invention can be used for accurately judging the MNS blood type genetic typing of an experimental sample. The method is quick and simple to operate and low in cost, and has wide application prospect and clinical reference value.

Description

TECHNICAL FIELD

[0001] The application relates to a primer set, a kit and application for MNS blood group genotyping detection, and belongs to the field of biomedical clinical molecular detection. BACKGROUND

[0002] As an important red blood cell blood group system (ISBT 002) of human, the MNS blood group system is located in the GYPA, GYPB and GYPE highly homologous gene cluster on the long arm of chromosome 4 (4q28-q31). Among them, GYPA is responsible for encoding M / N antigen (29th amino acid: M is serine, and N is glycine), and GYPB encodes S / s antigen (47th amino acid: S is methionine, and s is threonine). Moreover, the gene cluster is prone to unequal crossing over and gene conversion, thereby forming hybrid genes (such as Mi series, Mur and other rare antigen related hybrids), which makes the MNS blood group antigen show extremely high diversity.

[0003] In 1927, Landsteiner and Levine discovered M and N antigens on human red blood cells by immunizing rabbits with human red blood cells and prompting the rabbits to produce antibodies. In 1947, Walsh and Monotgomey discovered the S antigen closely related to M and N. In 1951, Levine discovered the s antigen in a pair relationship with the S antigen. At present, researchers have confirmed that the MNS blood group system contains more than 50 blood group antigens, and the complexity of the antigen and gene structure ranks first among known human blood groups, showing high polymorphism.

[0004] In the field of blood transfusion medicine, accurate identification of the MNS blood group system is closely related to blood transfusion safety. In the past, ABO and Rh blood group systems were the main focus of pre-transfusion blood group detection, but the MNS blood group system cannot be ignored. If the matching of the MNS blood group is ignored during blood transfusion, it is likely to cause serious consequences. The immune system in the recipient may recognize the MNS antigen on the donor red blood cells as a foreign substance, and then initiate an immune response. The combination of anti-M, anti-N, anti-S and anti-s antibodies with the corresponding antigens will cause red blood cells to agglutinate and be destroyed, causing hemolytic transfusion reactions.

[0005] Molecular biology methods, especially PCR-SSP (sequence-specific primer polymerase chain reaction) or gene chip technology, have been applied to MNS blood group typing. However, these methods can usually only detect known specific single nucleotide polymorphisms (SNPs), and it is difficult to find new gene mutations, and there are certain limitations in detection throughput. As the "gold standard" for genotyping, first-generation sequencing technology can directly read DNA sequences, providing the most accurate genetic information, and also has the ability to discover new alleles. However, at present, the method for efficient and specific amplification and sequencing of the key region of the MNS blood group system still needs to be improved.

[0006] In addition, there is a very high sequence similarity between GYPA, GYPB genes and the highly homologous pseudogene GYPE in the MNS blood group system, which makes it easy to cause cross-reaction and non-specific amplification when amplifying the target gene, thereby causing serious deviation in the typing results. At the same time, as a hot spot region of genome recombination, the frequent unequal crossing over and gene conversion events produce complex hybrid genes, which requires the primer design to avoid common breakpoints and have the ability to recognize these rare variations. In addition, the particularity of the distribution of key polymorphic sites and the possible high GC content and complex secondary structure of the target region pose a comprehensive and severe challenge to the specificity of the primer, the amplification efficiency and the reliability of the sequencing read length. In view of the inherent molecular genetic complexity of the MNS system, there is an urgent need to develop a kit that can overcome the primer design problem, integrate all necessary components and provide a standardized process.

[0007] The Chinese patent "202010323008.7 A method for analyzing human blood group genotype based on high-throughput sequencing and its application" uses second-generation sequencing method, which directly randomly breaks the genomic DNA, constructs a library, and performs second-generation sequencing, without the need for specific amplification primers and sequencing primers. However, its process is complex, and the accuracy of second-generation sequencing is not high enough. First-generation sequencing method can obtain high-resolution typing results, but it first needs to design specific amplification primers for PCR amplification. Considering the high sequence similarity between the MNS blood group genes GYPA, GYPB and the highly homologous pseudogene GYPE, false positives are likely to occur, thereby affecting its accuracy. Therefore, there is an urgent need in the art for a detection method that is simple, fast and accurate. SUMMARY

[0008] The purpose of the present application is to overcome the shortcomings of the prior art and provide a primer set, kit and application for MNS blood group genotyping detection.

[0009] The principle of designing the primer set for detecting MNS blood group genotyping is as follows: according to the known MNS encoding gene GYPA / GYPB gene sequence, based on the amplification refractory mutation system (ARMS) analysis method, the innovative mismatch binding double-specific base method is used to design the GYPA and GYPB gene specific amplification primers. When the primer sequence can be 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 the specific primer completely same gene sequence exists in the sample, and vice versa. The PCR reaction result is detected and analyzed by using agarose gel electrophoresis method. After the electrophoresis gel is dyed and analyzed by a gel imaging system, the nucleic acid fragments are distinguished due to different sizes. 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 GYPA and GYPB.

[0010] There is a very high sequence similarity between GYPA, GYPB gene and highly homologous pseudogene GYPE in MNS blood group system, which easily causes cross reaction and non-specific amplification phenomenon when amplifying the target gene, and further causes serious deviation of the typing result. The ordinary primer design method has certain limitation for distinguishing GYPA / GYPB gene subtypes, and the accuracy is not high. The inventors first use the mismatch binding double-specific base primer design method based on the ARMS analysis method, first find the specific base of GYPA / GYPB gene in the database, the upstream specific amplification primer is located at the end of the first intron, and the downstream specific amplification primer is located at the front end of the fifth intron, so as to accurately and specifically amplify the GYPA / GYPB main exon specific fragment in the first step. And according to the preliminary detection result, a mismatch base is introduced on the primer, which improves the specificity of the primer. The primer set has high accuracy, simple operation and wide application prospect.

[0011] In the first aspect of the present application, a primer set for MNS blood group genotyping detection is provided, the primer set comprises a specific PCR amplification primer set and a sequencing primer set, the specific PCR amplification primer set comprises two pairs of primers designed according to the specific sequences of GYPA and GYPB genes respectively; the two pairs of primers are respectively used for amplifying all subtypes of GYPA and all subtypes of GYPB;

[0012] The nucleotide sequences of the two pairs of specific PCR amplification primers are shown in the following table:

[0013]

[0014] The PCR amplification primer set described above is based on the modified Amplification Restriction Mutation System (ARMS) analysis method. It employs an innovative mismatch binding double-specific base method to design specific primers. The 3' ends of both the forward and reverse amplification primers are gene-specific bases, and an additional mismatch base is introduced into the primers to improve the specificity of detection.

[0015] The sequencing primer set includes 10 specific sequencing primers, of which 5 are GYPA-specific and 5 are GYPB-specific. It enables high-resolution forward and reverse sequencing of exons 2, 3, 4, and 5 of the GYPA and GYPB genes.

[0016] The nucleotide sequences of the 12 sequencing primers are shown in the table below:

[0017]

[0018] In a second aspect of the invention, the use of the primer set for MNS blood group genotyping detection as described in the first aspect in the preparation of a kit for detecting MNS blood group genotyping is provided.

[0019] In a third aspect of the invention, a kit is provided containing a primer set for MNS blood group genotyping as described in the first aspect, the kit further comprising PCR reaction reagents.

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

[0021] Furthermore, the PCR reaction solution comprises: 0.5 mM deoxynucleotide dNTP, 40 mM magnesium chloride MgCl2, 100 mM potassium chloride KCl, 80 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.

[0022] In a fourth aspect of the invention, a method is provided for detecting MNS blood group genotyping for non-disease diagnostic purposes using a primer set for MNS blood group genotyping as described in the first aspect, the method comprising an amplification reaction and sequencing.

[0023] The amplification reaction system is as follows: total volume 12.4 μL, including 6 μL PCR reaction solution, 0.4 μL enzyme, 3 μL amplification primer mixture, and 3 μL DNA template; the PCR amplification reaction program is as follows: 95℃ for 2 minutes; 93℃ for 10 seconds, 68℃ for 4 minutes, 30 cycles; 72℃ for 5 minutes, 4℃ until removed.

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

[0025] 1) The GYPA and GYPB genes in the MNS blood group system share extremely high sequence similarity with the highly homologous pseudogene GYPE. This makes it highly susceptible to cross-reactions and non-specific amplification during target gene amplification, leading to serious deviations in typing results. Conventional primer design methods have limitations in distinguishing GYPA / GYPB gene subtypes and are not highly accurate. This invention, based on ARMS analysis, utilizes an innovative mismatch-binding double-mutant primer design method for the first time. Specific bases for the GYPA / GYPB genes are first identified in the database. The upstream specific amplification primer is located at the end of intron 1, and the downstream specific amplification primer is located at the beginning of intron 5, thus accurately and specifically amplifying the GYPA / GYPB major exon-specific fragment in the first step. Furthermore, based on preliminary detection results, a mismatched base is introduced into the primers, improving their specificity. This primer set is highly accurate, easy to operate, and has broad application prospects.

[0026] 2) The Sanger sequencing method used in this invention designs long-fragment amplification primers for the highly polymorphic regions of exons 2 to 5, and designs specific sequencing primers to perform specific first-generation sequencing on the amplification products, thereby improving the accuracy of the typing results.

[0027] 3) Kits containing the specific amplification primers and universal sequencing primers of this invention can accurately determine the MNS blood type genotyping of experimental samples. Attached Figure Description

[0028] Figure 1 This is an electrophoresis image of the specific amplification primers of this invention. The amplification products are completely accurate in terms of positive and negative results, have good specificity, and contain no extraneous bands.

[0029] Figure 2 These are mismatch-free, double-specific base set amplification primers, but the amplification products have poor specificity and contain many impure bands.

[0030] Figure 3 Primers with mismatched single-specific base sets result in poor amplification product specificity and numerous extraneous bands.

[0031] Figures 4-1 to 4-8 The sequencing results for samples S1-S8 are shown. The sequencing peaks of the amplified products are good and correctly interpreted.

[0032] Figure 5 This is an example of a sequencing chromatogram of the amplification product from primers used for amplifying a mismatched double-base mutant genome. The sequencing chromatogram contains many impurity peaks, making the result uninterpretable.

[0033] Figure 6This example shows the sequencing chromatogram of the amplification product from primers used to amplify a mismatched single-base mutant genome. The sequencing chromatogram contains numerous impurity peaks, making the results uninterpretable. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0035] The main raw materials involved in this invention are listed below:

[0036]

[0037]

[0038] Example 1

[0039] 1. Raw materials and equipment:

[0040] 1.1 Reagent kit components:

[0041] 1.1.1 The specific amplification primer set and specific sequencing primer set of the present invention:

[0042] The specific amplification primer set of the present invention comprises two pairs of primers designed based on the specific sequences of the GYPA / GYPB genes, respectively. The two pairs of primers are used to amplify all GYPA subtypes and all GYPB subtypes, with sequences SEQ ID NO: #01~#04;

[0043] The specific sequencing primer set of the present invention includes 10 specific sequencing primers with sequences SEQ ID NO: #05 to #14.

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

[0045]

[0046] The nucleotide sequences of the 10 sequencing primers are shown in the table below:

[0047]

[0048] 1.1.2 Two sets of conventional amplification primers were designed.

[0049] The first set is a specific amplification primer set without mismatched double-specific base sets, and the sequences are as follows:

[0050]

[0051] The second set consists of specific amplification primers with mismatched single-specific base sets, the sequences of which are as follows:

[0052]

[0053]

[0054] 1.1.3 Primer preparation scheme

[0055] ① Prepare each primer solution to a concentration of 12 OD / mL; ② Prepare the amplification primer solution (AMP MIX).

[0056]

[0057] ③ Preparation of sequencing primer solution (SEQ MIX)

[0058]

[0059]

[0060] 1.1.4 PCR Reaction Reagents

[0061] DNA polymerase: a high-fidelity Taq polymerase;

[0062] PCR reaction solution: 0.5 mM deoxynucleotide dNTP, 40 mM magnesium chloride MgCl2, 100 mM potassium chloride KCl, 80 mM tris-hydroxymethylaminomethane hydrochloride Tris-HCl, 1 mM tetramethylammonium chloride TMAC, glycerol 0.6% v / v, cresol red 0.02% v / v and betaine 5% v / v.

[0063] 1.2 Source of Samples

[0064] 1.2.1 Blood Sample Collection

[0065] Blood samples can be collected using blood collection tubes containing the anticoagulant sodium citrate and ethylenediaminetetraacetic acid (EDTA). Fresh or frozen whole blood samples that have not been repeatedly frozen and thawed can be used as experimental samples.

[0066] 1.2.2 Nucleic acid sample extraction

[0067] Nucleic acid can be extracted from samples containing nucleated cells, such as whole blood or white blood cell layer, using precipitation, column, or magnetic bead methods to obtain sufficient quantity and quality of nucleic acid for polymerase chain reaction.

[0068] 1.2.3 Nucleic acid sample quantification

[0069] The extracted nucleic acid sample must be dissolved in sterile water or other suitable solution (such as TE buffer) at a concentration between 10-40 ng / μl. The nucleic acid sample must not be dissolved in a solution containing more than 0.5 mM chelates such as ethylenediaminetetraacetic acid (EDTA).

[0070] 1.2.4 Nucleic Acid Sample Quality Standards

[0071] The A260 / A280 ratio of nucleic acid samples should be between 1.6 and 2.1.

[0072] 1.3 Required Experimental Equipment

[0073] PCR instrument, sequencer, pipettes of different capacities, and small benchtop centrifuge (including 8-tube horizontal head).

[0074] 2. Genotyping process

[0075] Eight EDTA-anticoagulated whole blood samples were selected and amplified using the primer sets of this invention, the specific amplification primer sets without mismatched double-specific base sets, and the specific amplification primer sets with mismatched single-specific base sets. Each sample was amplified using six primer sets: GYPA, GYPB, GYPA-1, GYPB-1, GYPA-2, and GYPB-2. The GYPA amplification products were subjected to five sequencing reactions: GYPA-2R, GYPA-3R, GYPA-4F, GYPA-4R, and GYPA-5F. The GYPB positive bands were subjected to five sequencing reactions: GYPB-2R, GYPB-3R, GYPB-4F, GYPB-4R, and GYPB-5F.

[0076] 2.1 Preparation of the reaction system: The reaction system is shown in the table below:

[0077] PCR reaction system

[0078] Component name Addition μL / tube PCR reaction solution 6 Amplification primer mixture 3 Taq enzyme 0.4 Nucleic acid sample 3 Total volume 12.4

[0079] Cap the reaction tube, centrifuge briefly, and then place it in a real-time PCR instrument.

[0080] 2.2 PCR reaction procedure: as shown in the table below:

[0081] PCR reaction procedure

[0082]

[0083] 2.3 Electrophoresis

[0084] Run the gel at 8–10 volts / cm, 200V, for approximately 10–20 minutes. Photograph the PCR products using a UV transilluminator to confirm their quality.

[0085] 2.4 Purification of PCR Products

[0086] Add 4 μL of ExoSAP to the reaction wells intended for sequencing to remove excess primers and DNA.

[0087] Refer to the table below to set the procedure and begin the purification steps. The total reaction time is approximately 1 hour.

[0088] ExoSap PCR reaction program settings

[0089]

[0090]

[0091] 2.5 Sequencing reaction

[0092] Add 1.5 μL of BDT sequencing reagent to each reaction well;

[0093] Add 2.5 μL of sequencing primers to each reaction well;

[0094] Add 1 μL of purified PCR product to each reaction well.

[0095] 2.6 Sequencing Product Purification

[0096] Excess BDT was removed by ethanol precipitation.

[0097] 2.7 Sequencing

[0098] Before sequencing, you can choose to add 10 μL of HiDi formamide, heat it in the PCR instrument, and then put it into the sequencer.

[0099] 3. Analysis of Experimental Results

[0100] 3.1 The specific amplification primers of this invention produce amplification products with high specificity, no impurities, and completely accurate positive and negative phenotypes. See electrophoresis diagram below. Figure 1 .

[0101] 3.2 Primers without mismatched double-specific base sets resulted in amplification products with poor specificity and numerous extraneous bands. See the electrophoresis diagram below. Figure 2 .

[0102] 3.3 Primers with mismatched single-specific base sets resulted in amplification products with poor specificity and numerous extraneous bands. See the electrophoresis diagram below. Figure 3 .

[0103] 3.3 The specific amplification primers of this invention produce sequencing results of completely accurate amplification products, which can accurately determine the GYPA / GYPB genotype of experimental samples. See the sequencing chromatogram below. Figures 4-1 to 4-8 The sequencing results are shown in Table 1.

[0104] 3.4 Primers without mismatched double-base mutant genome amplification produce sequencing chromatograms that are messy or lack signal, making it impossible to identify the GYPA / GYPB genotype. A typical example of such a chromatogram can be found in [reference needed]. Figure 5 .

[0105] 3.5 Primers for amplifying mismatched single-base mutant genomes produce sequencing chromatograms of disordered or absent signals, making it impossible to identify the GYPA / GYPB genotype. A typical example of such a chromatogram can be found in [link to example]. Figure 6 .

[0106] Table 1

[0107]

[0108]

[0109] Conclusion: The specific amplification primers of this invention have high specificity, and when used in conjunction with the specific sequencing primers of this invention, they can accurately distinguish GYPA / GYPB genotypes. The kit of this invention can accurately determine the GYPA / GYPB genotype of experimental samples.

[0110] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or variations made in accordance with the spirit of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A primer set for MNS blood group genotyping, characterized by, The primer set comprises a specific PCR amplification primer set and a sequencing primer set; The specific PCR amplification primer set comprises two pairs of primers designed according to the specific sequences of GYPA and GYPB genes; the two pairs of primers are respectively used for amplifying all subtypes of GYPA and all subtypes of GYPB; The nucleotide sequences of the two pairs of specific PCR amplification primers are shown in the following table: The sequencing primer set comprises 10 specific sequencing primers, wherein 5 are GYPA specific sequencing primers and 5 are GYPB specific sequencing primers, which can perform forward and reverse high-resolution sequencing on the 2nd, 3rd, 4th and 5th exons of GYPA and GYPB genes; The nucleotide sequences of the 12 sequencing primers are shown in the following table:

2. Use of the primer set for MNS blood group genotyping detection according to claim 1 in the preparation of a kit for detecting MNS blood group genotyping.

3. A kit comprising the primer set for MNS blood group genotyping according to claim 1, characterized in that, The kit further comprises PCR reaction reagents.

4. The kit of claim 3, wherein The PCR reaction reagents comprise PCR reaction solution and 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, 100 mM potassium chloride KCl, 80 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 detecting MNS blood group genotyping for non-diagnostic purposes using the primer set according to claim 1 for MNS blood group genotyping detection, characterized in that, The method comprises an amplification reaction and sequencing.

7. The method of claim 6, wherein, The amplification reaction system is as follows: a total volume of 12.4 μL, comprising 6 μL of PCR reaction solution, 0.4 μL of enzyme, 3 μL of amplification primer mixture and 3 μL of DNA template; the PCR amplification reaction is performed at 95℃ for 2 minutes, 93℃ for 10 seconds, 68℃ for 4 minutes for 30 cycles, 72℃ for 5 minutes and 4℃ until removed.

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