Primer combination, kit and method for detecting human neutrophil antigen genotyping

Abstract

The present invention relates to the field of molecular biology detection technology, and in particular to a primer combination, a kit and a method for detecting human neutrophil antigen genotyping. The present invention designs a forward primer, a reverse primer and an extension primer for all 11 SNP sites of the human granulocyte antigen HNA 1-5 system, and the sequence is shown in SEQ ID No: 1-22. The method for detecting human neutrophil antigen genotyping using the primer combination can achieve batch detection of 96 or 384 samples per batch, and high-throughput detection will bring great convenience to related clinical diagnosis and scientific research work, greatly save experimental time, and the detection method is accurate and stable.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological detection, and in particular to a primer combination, a kit and a method for detecting human neutrophil antigen genotyping. Background Art

[0002] Human Neutrophil Antigens (HNA) are a group of glycoproteins expressed on the surface of human neutrophil granulocytes, playing an important role in alloimmunity and autoimmunity. Differences in individual HNA antigens can lead to the production of granulocyte antibodies after blood transfusion. It has been confirmed that HNA antigens and antibodies play an important role in a variety of clinical diseases, including neonatal alloimmune neutropenia (NIN), autoimmune neutropenia, febrile non-hemolytic transfusion reactions, transfusion-related acute lung injury (TRALI), alloimmune neutropenia after bone marrow transplantation, transfusion-related alloimmune neutropenia, drug-induced neutropenia and ineffective granulocyte transfusion. Therefore, the study of HNA antigens has important clinical significance.

[0003] The definition and related nomenclature of human neutrophil antigens are standardized by the HNA nomenclature of the International Society of Blood Transfusion Granulocyte Immunology Working Group (ISBT GIWP), and are based on serologically defined epitopes on related glycoproteins. The symbol HNA is used to represent human neutrophil antigens, and different antigen systems are represented by numbers. Different polymorphisms of the same glycoprotein are represented by lowercase English letters according to the order of discovery, such as HNA-la, HNA-lb, HNA-1c, etc. ISBT GIWP has officially assigned 14 HNA alleles to 5 HNA antigen systems.

[0004] At present, the main methods for HNA specific identification are serological methods and genotyping methods. Serological methods for identifying HNA antigens or antibodies mainly include granulocyte agglutination test, granulocyte immunofluorescence test (GIFT), monoclonal antibody specific granulocyte antigen capture test (Monoclonal Antibody Immobilization of Granulocyte Antigen, MAIGA), flow cytometry and ELISA. The genotyping methods of HNA antigen system mainly include PCR-sequence specific primers (PCR-SSP), real-time quantitative PCR (RT-qPCR), PCR-based sequencing (PCR-SBT), etc.

[0005] Although the serological method for identifying HNA antigen specificity is simple, cheap, and closely related to clinical practice, it has disadvantages such as being time-consuming, low throughput, and dependence on antiserum. Among the currently used HNA antigen system genotyping methods: the PCR-SSP method requires multi-tube amplification. Although it is simple to operate and the reagents are cheap, it has disadvantages such as low throughput and large experimental operation workload; RT-qPCR has the advantage of multiple amplification, but it has disadvantages such as low detection throughput and high experimental detection reagent costs; the PCR-SBT method has the advantages of being able to directly detect sequences and discover new mutations, but it has disadvantages such as high detection costs, relatively complex analysis, and low throughput. Summary of the invention

[0006] The object of the present invention is to provide a primer combination, a kit and a method for detecting human neutrophil antigen genotyping in view of the defects of the prior art.

[0007] In the first aspect, the present invention provides a primer combination for detecting human neutrophil antigen genotyping, and the sequences of the detected gene loci and the corresponding forward primers, reverse primers and extension primers are shown in the following table:

[0008]

[0009]

[0010] In a second aspect, the present invention provides a kit for detecting human neutrophil antigen genotyping, comprising the primer combination.

[0011] In a third aspect, the present invention provides a method for detecting human neutrophil antigen genotyping using the primer combination, comprising the following steps:

[0012] S1, extracting DNA from the sample to be tested;

[0013] S2, using the DNA of S1 as a template, using multiplex PCR technology to amplify the DNA sequence containing the target SNP, and the amplification primers are the forward primer and reverse primer mentioned above;

[0014] S3, after SAP treatment of the amplified PCR product, single base extension is performed using the above-mentioned extension primer;

[0015] S4. The obtained single-base extension product is transferred to the SpectroCHIP chip and subjected to mass spectrometry detection to determine its genotype.

[0016] Furthermore, the amplification system in S2 was: 0.8 μL ultrapure water containing 20 mM Mg 2+0.5μL of 10× PCR Buffer, 0.4μL of 25mM MgCl2, 0.1μL of 25mM dNTP, 1.0μL of 0.5μM PCR primer mixture, 0.2μL of 5U / μL HotstarTaq enzyme, 2.0μL of 20-50ng / μL human genomic DNA solution.

[0017] Furthermore, the amplification program in S2 was: 95°C, 2 min; 45 cycles of 95°C, 30 sec, 56°C, 30 sec, 72°C, 1 min; 72°C, hold for 5 min, and keep warm at 4°C.

[0018] Furthermore, the SAP treatment system in S3 was: 1.53 μL of ultrapure water, 0.17 μL of SAP buffer, and 0.30 μL of SAP enzyme.

[0019] Furthermore, the SAP treatment procedure in S3 was: 37°C for 40 min; 85°C for 5 min; and insulation at 4°C.

[0020] Furthermore, the extension system in S3 is: ultrapure water 0.62 μL, PLEX Buffer 0.2 μL, iPLEXTermination Mix 0.2 μL, extension primer mixture 0.94 μL, and iPLEX Pro Enzyme 0.04 μL.

[0021] Furthermore, the extended procedure in S3 is:

[0022] 94℃, 30sec;

[0023] 40 cycles: 94°C, 5 sec; (52°C, 5 sec; 80°C, 5 sec) 5 cycles;

[0024] 72℃, 3min;

[0025] Keep warm at 4℃.

[0026] The present invention has the following beneficial effects:

[0027] By using the detection method, primers and kit of the present invention, all 11 SNP sites of the human granulocyte antigen HNA1-5 system can be detected in a single PCR reaction well, and the time-of-flight mass spectrometry platform is used. Based on the fact that the flight time of nucleic acid molecules in an electric field is inversely proportional to the ion mass, the accurate molecular weight of the sample analyte is obtained by detecting the flight time of nucleic acid molecules in a vacuum, thereby detecting the SNP site information. Combining multiple PCR technology, single base extension technology, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis mass spectrometry technology for typing detection, the experimental steps are simple, the software analysis of the experimental results is fast and convenient, and the batch detection of 96 or 384 samples per batch can be achieved by a nucleic acid mass spectrometer. High-throughput detection will bring great convenience to related clinical diagnosis and scientific research work, greatly saving experimental time, and the detection method is accurate and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the peak diagram of 11 SNP nucleic acid mass spectrometry detection of the HNA1-5 system (sample number: 2-19053037).

[0029] Figure 2 This is the peak diagram of nucleic acid mass spectrometry detection of HNA1_G108C SNP in sample 2-19053037.

[0030] Figure 3 This is the peak diagram of nucleic acid mass spectrometry detection of HNA1_C114T SNP in sample 2-19053037.

[0031] Figure 4 This is the peak diagram of nucleic acid mass spectrometry detection of HNA1_A194G SNP in sample 2-19053037.

[0032] Figure 5 This is the peak diagram of nucleic acid mass spectrometry detection of HNA1_C233A SNP in sample 2-19053037.

[0033] Figure 6 This is the peak diagram of nucleic acid mass spectrometry detection of HNA1_G244A SNP in sample 2-19053037.

[0034] Figure 7 This is the peak diagram of nucleic acid mass spectrometry detection of HNA1_G316A SNP in sample 2-19053037.

[0035] Figure 8 This is the peak diagram of nucleic acid mass spectrometry detection of HNA2_A787T SNP in sample 2-19053037.

[0036] Fig. 9 This is the peak diagram of nucleic acid mass spectrometry detection of HNA3_C451T SNP in sample 2-19053037.

[0037] Fig.10 This is the peak diagram of nucleic acid mass spectrometry detection of HNA3_G455A SNP in sample 2-19053037.

[0038] Fig.11 This is the peak diagram of nucleic acid mass spectrometry detection of HNA4_G230A SNP in sample 2-19053037.

[0039] Fig.12 This is the peak diagram of nucleic acid mass spectrometry detection of HNA5_G2372C SNP in sample 2-19053037.

[0040] Fig.13 This is a cluster diagram of the results of HNA1_G108C SNP nucleic acid mass spectrometry detection for 48 samples.

[0041] Fig.14 This is a cluster diagram of the results of HNA1_C114T SNP nucleic acid mass spectrometry detection for 48 samples.

[0042] Fig.15 This is a cluster diagram of the results of HNA1_A194G SNP nucleic acid mass spectrometry detection for 48 samples.

[0043] Fig.16 This is a cluster diagram of the results of HNA1_C233A SNP nucleic acid mass spectrometry detection for 48 samples.

[0044] Fig.17 This is a cluster diagram of the results of HNA1_G244A SNP nucleic acid mass spectrometry detection for 48 samples.

[0045] Fig.18 This is a cluster diagram of the results of HNA1_G316A SNP nucleic acid mass spectrometry detection for 48 samples.

[0046] Fig.19 This is a cluster diagram of the results of HNA2_A787T SNP nucleic acid mass spectrometry detection for 48 samples.

[0047] Fig. 20 This is a cluster diagram of the results of HNA3_C451T SNP nucleic acid mass spectrometry detection for 48 samples.

[0048] Fig.21 This is a cluster diagram of the results of HNA3_G455A SNP nucleic acid mass spectrometry detection for 48 samples.

[0049] Fig. 22 This is a cluster diagram of the results of HNA4_G230A SNP nucleic acid mass spectrometry detection for 48 samples.

[0050] Fig.23 This is a cluster diagram of the results of HNA5_G2372C SNP nucleic acid mass spectrometry detection for 48 samples.

[0051] Fig.24 Comparison of HNA nucleic acid mass spectrometry and Sanger sequencing test results for 16 samples. DETAILED DESCRIPTION

[0052] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0053] The following experiment conducted multiple rounds of primer design and testing, completed the testing experiment of 11 SNP sites in 48 blood genomic DNA samples, and performed first-generation sequencing verification. The detection method and results are as follows:

[0054] 1. Sample preparation:

[0055] The results of genomic DNA extraction from 48 blood samples are shown in Table 1 . The samples were diluted to a concentration of 20-50 ng / μL and used in subsequent detection experiments.

[0056] Table 1 DNA concentration and purity of experimental samples

[0057]

[0058]

[0059] 2. Primer preparation:

[0060] The primer design results are shown in Table 2.

[0061] Table 2 Sequences of forward primer, reverse primer and extension primer

[0062]

[0063]

[0064]

[0065] 3. Testing process:

[0066] 3.1 Sample preparation

[0067] (1) DNA sample to be tested, concentration 20-50 ng / μL.

[0068] (2) Positive quality control samples, concentration 20-50 ng / μL, samples with known HNA typing.

[0069] (3) Negative blank, RNase-free ultrapure water sample.

[0070] (4) Prepare a sample table and layout the sample information to be tested in a 96-well plate. After the sample tubes are shaken and mixed, centrifuged briefly, 2 μL of each sample is transferred to the 96-well plate.

[0071] 3.2 PCR amplification reaction

[0072] (1) Prepare the PCR reaction system according to Table 3 below. You can prepare 10%-20% more of the actual volume.

[0073] Table 3 Preparation of PCR amplification reaction mixture

[0074]

[0075] (2) After preparation, vortex thoroughly to mix and centrifuge briefly.

[0076] (3) Dispense the PCR reaction system into 8 strip tubes as needed. Use a dispenser to add the mixture into 96 reaction wells, adding 3 μL of PCR amplification reaction mixture to each well.

[0077] (4) Seal the 96-well plate with sealing film, vortex to mix, and centrifuge briefly.

[0078] (5) Place the 96-well plate on the PCR instrument, close the cover of the PCR instrument, and run the PCR amplification program.

[0079] The PCR amplification procedure is as follows:

[0080] 95°C, 2 min;

[0081] 45 cycles: 95°C, 30 sec, 56°C, 30 sec, 72°C, 1 min;

[0082] 72°C, maintain for 5 min;

[0083] Keep warm at 4℃.

[0084] Select the reaction system 5 μL and start the reaction program.

[0085] (6) After the reaction is completed, take out the 96-well PCR reaction plate and centrifuge at 3000 rpm for 1 min.

[0086] 3.3 Alkaline phosphatase (SAP) reaction

[0087] (1) Prepare the SAP reaction system according to Table 4 below, and add 10%-20% more of the actual volume.

[0088] Table 4 Alkaline phosphatase (SAP) reaction mixture preparation table

[0089]

[0090] (2) After preparation, vortex thoroughly to mix and centrifuge briefly.

[0091] (3) Dispense the SAP reaction system into 8 strip tubes as needed. Use a dispenser to add it to 96 reaction wells, adding 2 μL of alkaline phosphatase (SAP) reaction mixture to each well.

[0092] (4) Seal the 96-well plate with sealing film, vortex to mix, and centrifuge briefly.

[0093] (5) Place the 96-well plate on the PCR instrument, close the cover of the PCR instrument, and run the SAP program.

[0094] The SAP program was as follows: 37°C, 40 min; 85°C, 5 min; 12°C, Hold.

[0095] Select the reaction system 7μL and start the reaction program.

[0096] (6) After the reaction is completed, take out the 96-well PCR reaction plate and centrifuge at 3000 rpm for 1 min.

[0097] 3.4 Single base extension reaction

[0098] (1) Prepare the single base extension reaction system according to Table 5 below. You can prepare 10%-20% more of the actual volume.

[0099] Table 5 Single base extension reaction mixture preparation table

[0100] Reagents Reagent volume per sample [μL] RNase-free ultrapure water 0.62 iPLEX Buffer 0.20 iPLEX Termination mix 0.20 Extension Primer Mix 0.94 iPLEX Pro Enzyme 0.04 Total volume 2.00

[0101] (2) After preparation, vortex thoroughly to mix and centrifuge briefly.

[0102] (3) Dispense the extension reaction system into 8 strip tubes as needed. Use a dispenser to add the mixture to 96 reaction wells, adding 2 μL of the extension reaction mixture to each well.

[0103] (4) Seal the 96-well plate with sealing film, vortex to mix, and centrifuge briefly.

[0104] (5) Place the 96-well plate on the PCR instrument and close the cover of the PCR instrument.

[0105] The single base extension reaction procedure is as follows:

[0106] 94℃, 30sec;

[0107] 40 cycles: 94°C, 5 sec; (52°C, 5 sec; 80°C, 5 sec) 5 cycles;

[0108] 72℃, 3min;

[0109] Keep warm at 4℃.

[0110] (6) Select 9 μL of reaction system and start the reaction procedure.

[0111] After the reaction was completed, the 96-well PCR reaction plate was taken out and centrifuged at 3000 rpm for 1 min.

[0112] (7) Centrifuge the 96-well plate briefly, add 41 μL of RNase-free ultrapure water to each well, seal the reaction plate with sealing film, and centrifuge briefly at 3000 rpm.

[0113] 3.5 Resin purification and spot detection

[0114] (1) Place the 96-sample plate and chip in the designated position of the nucleic acid mass spectrometer, set the parameters, run the resin purification and spot detection procedures, and conduct the mass spectrometry experiment.

[0115] (2) Result analysis: Use the Typer Analyzer module in the Typer software to analyze the experimental results and export the PlateData data after the interpretation is completed.

[0116] 4. Experimental results:

[0117] (1) 48 genomic DNA samples were tested and the experiment was repeated once. The results of the HNA1-5 system 11 SNP nucleic acid mass spectrometry test are as follows Figure 1 As shown, the results of nucleic acid mass spectrometry detection of 11 SNPs in sample 2-19053037 are as follows: Figure 2-12 As shown in the figure, the clustering results of nucleic acid mass spectrometry detection results of 11 SNPs in 48 samples are as follows: Figure 13-23 shown.

[0118] Success rate of one-time detection of 48 samples:

[0119] 528 data results were all detected, with a 100% success rate in one experiment.

[0120] (2) Clinical sensitivity and specificity:

[0121] The first-generation Sanger sequencing was performed on 48 samples.

[0122] A total of 16 samples were sequenced, and 11 SNP sites were detected and analyzed for each sample, with a total of 176 results, including 88 wild-type results, 4 mutant results, and 84 heterozygous results. The results are shown in Tables 6 and Fig.24 .

[0123] Table 6 Experimental detection sensitivity and specificity analysis table

[0124]

[0125]

[0126] True positive results: The number of results with both non-wild type results from mass spectrometry and non-wild type results from sequencing was 88.

[0127] False negative results: The number of results in which the mass spectrometry result was non-wild type and the sequencing result was wild type was 0.

[0128] True negative results: There were 88 cases in which both the mass spectrometry results and the sequencing results were wild-type.

[0129] False positive results: The number of results in which the mass spectrometry result was wild type and the sequencing result was non-wild type was 0.

[0130] Sensitivity = true positive result / (true positive result+false negative result)×100%=88 / (88+0)×100%=100%.

[0131] Specificity = number of true negatives / (number of true negatives + number of false positives)×100%=88 / (88+0)×100%=100%.

[0132] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0133] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A primer combination for detecting human neutrophil antigen genotyping, characterized in that: The primer combination consists of eleven primer pairs, primer pairs 1 to 11, each of which consists of a forward primer, a reverse primer and an extension primer, wherein: Primer pair 1: The gene locus to be detected is HNA-1 G108C, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 11 respectively; Primer pair 2: The gene locus detected is HNA-1 C114T, and the sequences of the reverse primer, forward primer and extension primer are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 12 respectively; Primer pair 3: the gene locus detected is HNA-1 A194G, and the sequences of the reverse primer, forward primer and extension primer are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 13 respectively; Primer pair 4: the gene locus detected is HNA-1 C233A, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 14 respectively; Primer pair 5: the gene locus detected is HNA-1 G244A, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 15 respectively; Primer pair 6: the gene locus detected is HNA-1 G316A, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 16 respectively; Primer pair 7: the gene locus detected is HNA-2 A787T, the sequences of the reverse primer and the forward primer are shown in SEQ ID No: 3 and SEQ ID No: 4 respectively, and the sequences of the extension primers are shown in SEQ ID No: 17 and SEQ ID No: 18; Primer pair 8: the gene locus detected is HNA-3 C451T, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 19 respectively; Primer pair 9: the gene locus detected is HNA-3 G455A, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 20 respectively; Primer pair 10: the gene locus detected is HNA-4 G230A, and the sequences of the reverse primer, the forward primer and the extension primer are shown in SEQ ID No: 7, SEQ ID No: 8 and SEQ ID No: 21 respectively; Primer pair 11: The gene locus detected is HNA-5 G2372C, and the sequences of the reverse primer, forward primer and extension primer are shown in SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 22 respectively.

2. A kit for detecting human neutrophil antigen genotyping, characterized in that: Comprising the primer combination described in claim 1.

3. A method for detecting human neutrophil antigen genotyping for non-diagnostic and therapeutic purposes using the primer combination of claim 1, characterized in that: The following steps are involved: S1, extracting DNA from the sample to be tested; S2, using the DNA of S1 as a template, using multiplex PCR technology to amplify the DNA sequence containing the target SNP, and the amplification primers are the forward primer and reverse primer in claim 1; S3, after SAP treatment of the amplified PCR product, single base extension is performed using the extension primer in claim 1; S4. The obtained single-base extension product is transferred to the SpectroCHIP chip and subjected to mass spectrometry detection to determine its genotype.

4. The method according to claim 3, characterized in that The amplification system in S2 is: 0.8 μL ultrapure water, containing 20 mM Mg 2 + 0.5μL of 10× PCR Buffer, 0.4μL of 25mM MgCl2, 0.1μL of 25mM dNTP, 1.0μL of 0.5μM PCR primer mixture, 0.2μL of 5U / μL HotstarTaq enzyme, 2.0μL of 20-50ng / μL human genomic DNA solution.

5. The method according to claim 4, characterized in that The amplification program in S2 was: 95°C, 2 min; 45 cycles of 95°C, 30 sec, 56°C, 30 sec, 72°C, 1 min; 72°C, hold for 5 min, and keep warm at 4°C.

6. The method according to claim 5, characterized in that The SAP treatment system in S3 is: 1.53 μL ultrapure water, 0.17 μL SAP buffer, and 0.30 μL SAP enzyme.

7. The method according to claim 6, characterized in that The SAP treatment procedure in S3 was: 37°C for 40 min; 85°C for 5 min; and insulation at 4°C.

8. The method according to claim 7, characterized in that The extension system in S3 is: ultrapure water 0.62μL, PLEXBuffer buffer 0.2μL, iPLEX Termination Mix 0.2μL, extension primer mixture 0.94μL, iPLEX ProEnzyme enzyme 0.04μL.

9. The method according to claim 8, characterized in that The extension procedure in S3 is: 94℃, 30sec; 40 cycles: 94°C, 5 sec; 52°C, 5 sec, 80°C, 5 sec, 5 cycles; 72℃, 3min; Keep warm at 4℃.

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