Construction method and kit of high-throughput multiplex fluorescence PCR-based HLA-C REG and HPA gene typing synchronous detection system

Abstract

The application discloses a method and a kit for constructing a high-throughput multiplex fluorescence PCR-based HLA-CREG and HPA gene typing synchronous detection system. The application is based on common specific SNP sites, and two types of different primers are combined and designed to obtain a system for HLA cross-reactive group and HPA gene typing detection, which can realize rapid, simple, low-cost and high-throughput HLA epitope and HPA gene typing detection, so as to more effectively prevent platelet transfusion invalidity and immunological platelet transfusion invalidity.

Description

Technical Field

[0001] This invention relates to the field of gene detection, specifically to a method and kit for constructing a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR. Background Technology

[0002] Currently, there are 35 HPA antigens, named HPA-1 to HPA-35 according to their discovery time, and their genetic characteristic is codominant biallelic alleles. HLA is a non-specific platelet antigen present on the platelet surface, shared with other cells or tissues. Among them, HLA-I antigens are the main HLA antigens expressed on the platelet surface. For HLA-I antigens, transfusing platelets with HLA-A and HLA-B genotypes can significantly improve platelet transfusion ineffectiveness. However, due to the high polymorphism of HLA, the probability of finding a perfectly matching HLA allele for a patient is only one in 100,000. Therefore, establishing a large platelet donor bank is difficult to implement due to both economic investment and donor sourcing. Research has found that recipients produce anti-donor HLA antibodies targeting only certain epitopes of their own HLA antigens, not the entire HLA antigen of the donor. Further research has confirmed that matching the HLA epitopes between donor and recipient can avoid the production of donor-specific antibodies. Clinical studies have shown that for patients already sensitized by HLA alloimmunization and those unresponsive to platelet transfusions, transfusion of HLA epitope-matched platelets based on HLA antigen composition cross-reactivity groups (CREGs) is equivalent to transfusion of HLA antigen-matched platelets, providing direct evidence that HLA epitope matching can replace HLA antigen-matched transfusions. Therefore, targeted testing of donors and recipients for HPA gene and HLA-AB epitopes, and transfusion of HPA gene and HLA epitope-matched compatible platelets, has significant clinical implications.

[0003] The main methods currently used for HLA and HPA genotyping include: polymerase chain reaction-sequence-specific primers (PCR-SSP), polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSO), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) DNA microarray method, sequencing-based genotyping (SBT), and next-generation sequencing (NGS).

[0004] PCR-SSP is a typing method based on PCR technology. It utilizes the highly specific amplification of PCR and DNA sequence-specific hybridization to type the target DNA. However, PCR-SSP requires analysis by gel electrophoresis, which is complex and prone to causing laboratory contamination.

[0005] The principle of PCR-SSO is based on the base sequence of various HLA alleles. By artificially synthesizing complementary oligonucleotides, namely SSO probes, they hybridize with HLA in the genomic DNA of the sample to be tested. By observing whether hybridization occurs, the HLA type of the sample can be determined. Although this method is suitable for typing large batches of samples, its disadvantages are that it requires high-end equipment, is complex to operate, and is difficult to complete the typing of all HLA alleles in a short time.

[0006] PCR-RFLP typing offers significantly higher accuracy than serological methods, but it involves numerous steps and complex result interpretation, especially in cases of heterozygotes. Furthermore, this method requires enzyme digestion analysis with multiple restriction endonucleases, resulting in substantial time and expense.

[0007] DNA microarray technology is based on anchored oligonucleotides. A series of oligonucleotide probes are introduced onto the microarray. These probes can bind to highly specific DNA sequences of different variant types on the HLA / HPA genes. When the probe successfully hybridizes with the target DNA, the probe at a specific position on the microarray will show a fluorescent signal, indicating that the DNA in the sample is a perfect match for the probe. However, this method is technically complex, expensive, and requires professional experimental techniques and data analysis capabilities, making it difficult to widely apply in clinical practice.

[0008] Sanger sequencing and NGS are currently the gold standard for clinical applications. They use PCR to amplify HLA / HPA gene-specific sequences, and then perform Sanger sequencing on the amplified products, or extract whole-genome DNA from samples and use high-throughput sequencing technology to sequence and genotype HLA genes. However, Sanger sequencing has a relatively low throughput, and a single experiment can only sequence tens to hundreds of target sequences. It also requires cloning the target sequences individually and cannot identify multiple HLA alleles simultaneously. NGS, on the other hand, requires a high level of operational skills, generates large amounts of data, requires specialized instruments and equipment, and professional data analysis techniques. It is expensive and not suitable for large-scale clinical implementation.

[0009] Currently, the HLA typing kits approved by the National Medical Products Administration include those developed by One Lambda based on the principles of PCR-SSP and PCR-SSO, and those developed by Debigene based on the SBT principle. One Lambda's kit requires specialized testing instruments, a high level of technical expertise, and professional data analysis skills, making it expensive and thus difficult to widely apply in clinical practice. Debigene's kit also requires specialized analysis software and has low throughput, allowing only one sample to be tested on a 96-well plate; therefore, it is not suitable for large-scale clinical HLA testing.

[0010] In addition, existing literature also discloses techniques for detecting and typing HLA. For example, Chinese patent document CN107190088A provides a kit for detecting HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 genotyping using a fluorescence PCR melting curve method. This kit contains 262 primers for amplification in a 96-well optical reaction plate. The double-stranded DNA generated by PCR amplification binds to SYBR Green I. The low-resolution genotyping of HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 is determined by analyzing the different melting curves generated by different products. However, the binding of SYBR Green I to the double-stranded DNA product is not specific, resulting in poor specificity and accuracy. Multiplex reactions are difficult to perform in a single-well PCR tube, leading to extremely low throughput.

[0011] Chinese patent document CN101845520A uses a specific MGB probe to genotype the alleles of HPA1-5 and 15 genes. The allele genotype is determined by the distribution of two different fluorescence curves and scatter plots after amplification with ordinary primers. However, the MGB probe used in this method is expensive and costly, making it unsuitable for widespread application. Furthermore, it only detects one allele per well, resulting in low efficiency.

[0012] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0013] To address at least some of the technical problems existing in the prior art, this invention groups HLA genes based on SNP sites according to the HPA / HLA gene sequences published in the IPD-IMGT / HLA and Versiti databases, resulting in multiple HLA subgroups. Simultaneously, it targets HPA genes, especially low-frequency genes with high frequency in the population, namely HPA... Specific primers and probes were designed for the two different subtypes (a and b) of genes 1-6, 9, 12, 13, 15, 21, 27, 30, and 31. The probes were labeled with fluorescent dyes such as FAM / VIC / ROX / CY7 / ATTO425 to detect HLA and HPA specific targets. Each well also included a pair of internal control primers and probes labeled with CY5 to monitor for false negatives caused by instrument malfunction, reagent factors, polymerase activity, or inhibitors in the sample. An additional negative control well was included to eliminate false positives caused by sample contamination. This method enables rapid, simple, low-cost, and high-throughput detection of HLA epitopes and HPA genotyping, which can be widely applied to platelet bank establishment, detection, and treatment of patients unresponsive to platelet transfusions. Specifically, this invention includes the following:

[0014] In a first aspect, the present invention provides a method for constructing a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR, comprising the following steps: (1) For each HLA gene in the cross-reactive group, analyze its epitope gene sequence, screen for common specific SNP sites, and group the HLA genes according to the common specific SNP sites to obtain multiple HLA subgroups, of which at least some HLA subgroups contain multiple HLA genes. (2) For each HPA allele, analyze its gene sequence and screen for specific SNP sites of low-frequency and high-frequency HPA genes; (3) Primers and probes are designed for the common specific SNP sites and frequency-specific SNP sites, respectively. When designing primers, a mutation-repressive amplification system is preferred for the presence of a single specific SNP site, and a U-shaped anchoring primer is preferred when there are linked mutant bases near the specific SNP site. The probes are labeled with different fluorescent groups; and (4) The probes are divided into tubes according to their fluorescent group type and primer position. After division, each of the multiple independently set subsystems contains primers and probes for specific SNP sites. The information of primers and probes in the subsystems is shown in Table 3.

[0015] In some embodiments, according to the method for constructing a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR according to the first aspect of the present invention, the HLA genes include HLA-A and HLA-B.

[0016] In some embodiments, according to the method for constructing a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR according to the first aspect of the present invention, the multiple HLA subgroups obtained after grouping in step (1) include: The A1C1 subgroup contains A01, A11, A36, and A80; Subgroup A1C2 containing A03 and A30; The A1C3 subgroup containing A29; Subgroup A1C4 contains A30 and A31; The A2C-A subgroup contains A02, A23, A24, A68, and A69; Subgroup A10C1 contains A32 and A74; The A10C2 subgroup contains A25, A26, A34, A66, and A43; The A10C3 subgroup contains A33; The B2C subgroup contains B57 and B58; The B5C1 subgroup contains B35, B51, B52, B53, B58, and B78; Subgroup B5C2 contains B15 and B46; The B5C3 subgroup containing B18; The B5C4 subgroup containing B49; The B5C5 subgroup containing B50; The B5C7 subgroup containing B73; The B7C1 subgroup contains B7, B42, B54, B55, B56, B67, B81, and B82; The B7C2 subgroup contains B54, B55, B56, and B59; The B7C3 subgroup contains B8, B41, and B42; The B7C4 subgroup contains B7, B13, B27, B40, B47, B48, and B81; The B8C1 subgroup contains B8, B14, B38, B39, and B67; The B12C1 subgroup containing B13, B40, B41, B44, B45, B47, B49, and B50; and The B12C2 subgroup containing B37.

[0017] In some embodiments, according to the method for constructing a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR according to the first aspect of the present invention, when designing primers using a mutation arrest amplification system, a mismatched base is introduced at the 3' end of the primer, including: a. First, introduce single base mismatches and determine the specificity of the amplification product, i.e., whether there is a specific peak, based on the melting curve of real-time fluorescence PCR; b. When the melting curve shows no specific peak, introduce another base mismatch and again determine whether there is a specific peak based on the melting curve of real-time fluorescence PCR; Optionally, step b may be repeated until a specific peak is obtained.

[0018] In some embodiments, according to the method for constructing a simultaneous HLA-CREG and HPA genotyping detection system based on high-throughput multiplex fluorescent PCR according to the first aspect of the present invention, the U-shaped anchoring primer includes a 5' end binding region, a 3' end binding region, and a vacuolar structure region located between the two, and the design of the U-shaped anchoring primer includes the following steps: a. Adjust the number of binding bases at the 3' end of the primers and the vacuolar structure of the linker, and select primers with better specificity as preliminary primers based on the results of the SYBR dye method; b. Further optimize the number of bases in the linker of the preliminary primers to obtain optimized primers; c. Adjust the length of the 5' binding region of the U-shaped anchor primer according to the Taqman probe real-time fluorescence PCR method.

[0019] In a second aspect, the present invention provides a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR, which is obtained by the construction method described in the first aspect of the present invention.

[0020] In some embodiments, the HLA-CREG and HPA genotyping simultaneous detection system based on high-throughput multiplex fluorescent PCR according to the second aspect of the present invention includes primers with sequences as shown in SEQ ID No. 1-100; and probes with sequences as shown in SEQ ID No. 101-150; Preferably, the detection system includes multiple independently configured subsystems, each of which contains primers and probes targeting specific SNP sites. Information on the primers and probes in the subsystems is shown in Table 3. Preferably, the probe further comprises a fluorescent group; Preferably, the system further comprises Mg 2+ dNTPs and Taq hot-start enzyme; Preferably, the system includes an internal reference primer and a probe, wherein the internal reference primer and the probe have the sequences shown in SEQ ID No. 151 and 152, and the internal reference probe has the sequence shown in SEQ ID No. 153.

[0021] A third aspect of the present invention provides a high-throughput multiplex fluorescent PCR-based HLA-CREG and HPA genotyping simultaneous detection kit, comprising the detection system. Preferably, the detection system comprises multiple independently configured subsystems, each subsystem containing primers and probes targeting specific SNP sites. Information on the primers and probes in the subsystems is shown in Table 3. Optionally, it further comprises internal standard primers and internal standard probes.

[0022] In some embodiments, according to the kit described herein, the kit further includes a reading rule specification.

[0023] A fourth aspect of the present invention provides a method for HLA-CREG and HPA genotyping or detection based on high-throughput multiplex fluorescent PCR, comprising the step of performing multiplex PCR using the detection system described in the second aspect; and The steps involve interpreting fluorescence signals of different colors.

[0024] The detection system of this invention can effectively identify HLA epitopes and HPA antigen-specific SNP sites. Combined with TaqMan probes, it establishes a rapid, simple, low-cost, and high-throughput multiplex real-time fluorescence PCR detection method, thereby providing a convenient and effective detection method for the prevention of platelet transfusion ineffectiveness and the treatment of patients with immune platelet transfusion ineffectiveness. Attached Figure Description

[0025] Figure 1 A: Schematic diagram of HLA and HPA alleles and HLA CREG grouping; BD: Schematic diagram of the specific locations of HLA-A related detection sites in CREG grouping.

[0026] Figure 2 A diagram showing the specific locations of HLA-B-related specific detection sites in the CREG group.

[0027] Figure 3 Examples of ARMS and UAP primer optimization are shown in the figure, illustrating the primer optimization process for the HLA-A site specific to the 1C1 target sequence. The blue curve represents the melting curve results when screening primers using the SYBR method for real-time fluorescent PCR; the orange curve represents the amplification curve when the screened specific primers are used for real-time fluorescent PCR using the TaqMan probe method.

[0028] Figure 4 Negative quality control system testing.

[0029] Figure 5 A schematic diagram of amplification curves for HLA-A, B epitopes, and HPA allele typing of clinical samples. AL: corresponds to the test results of tubes 1-12 respectively. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] In this article, the term "shared specific SNP" refers to an SNP site that is shared among multiple HLA genes in a specific HLA genome, particularly within a cross-reactivity group (CREG), but not by any HLA gene in other CREGs. A shared specific SNP site is typically a single base, but sometimes includes combinations of two or more bases. The distance between the two or more bases is not limited, but is typically less than 10 nt, such as less than 9 nt, less than 8 nt, less than 7 nt, less than 6 nt, less than 5 nt, less than 4 nt, less than 3 nt, less than 2 nt, or 1 nt, or the two or more bases may be adjacent.

[0034] In this article, the term "binding region" refers to a portion of an oligonucleotide that can specifically bind to a continuous sequence of a target gene; it is a sequence consisting of multiple consecutive nucleotides.

[0035] In this article, the term "specific binding," also known as "specific hybridization," includes the following meanings: (1) There is only one target gene or target DNA region that hybridizes with the oligonucleotide in a template, and each base in the oligonucleotide pairs with the corresponding base of the target gene or target DNA. That is, the oligonucleotide and the target DNA are completely matched; (2) Under conditions suitable for PCR reaction, after the first oligonucleotide and the second oligonucleotide hybridize with the same template, only one detectable fragment with a length between 25-350 bp can be obtained.

[0036] In this article, the terms "high-frequency HPA genes" or "type a HPA" refer to HPA SNP loci with a frequency greater than 50% in a specific population. Similarly, "low-frequency HPA genes" or "type b HPA" refer to HPA SNP loci with a frequency less than 50% in a specific population. Specifically, low-frequency genes in HPA-1, 2, 3, 4, 5, 6, 9, 12, 13, 15, 21, 27, 30, and 31 have a frequency greater than 50% in the population. .

[0037] In the method of the present invention, the type of sample used for detection is not particularly limited. Preferably, it is a blood sample, such as serum or plasma.

[0038] In this invention, the reaction system includes: 10-50 mM (preferably 20-40 mM) of Tris, with a pH of 8.0-9.0; 50-500 mM (preferably 100-200 mM) of KCl; 1-50 mM (preferably 5-20 mM) of MgCl2; 0.01-10 mM (preferably 0.1-2 mM) of dNTPs; 0.01-10 mM (preferably 0.1-2 mM) of 50% trehalose; and 1-10 U / Test (preferably 1-5 U / Test) of Taq hot-start enzyme.

[0039] Primers and probes for HLA-A and HLA-B epitopes, HPA alleles, and random sequences, along with the above-mentioned system and the internal reference gene HLA-B primers and probes, were placed in a 12-tube PCR system according to Table 9. The final concentration of each primer in each well was 50-500 nM, preferably 100-200 mM, and the final concentration of the probe was 50-500 nM, preferably 50-150 mM.

[0040] In a preferred embodiment, the detection system of the present invention includes first to twelfth reaction systems. The first reaction system includes primers shown in SEQ ID No. 1-6, 11-12 and probes shown in SEQ ID No. 101-103, 106; the second reaction system includes primers shown in SEQ ID No. 7-8, 13-14, 41-42, 53-54 and probes shown in SEQ ID No. 104, 107, 121, 127; the third reaction system includes primers shown in SEQ ID No. 9-10, 19-20, 31-32, 55-56 and probes shown in SEQ ID No. 105, 110, 116, 146; the fourth reaction system includes primers shown in SEQ ID No. 15-16, 21-22, 27-28, 89-90 and probes shown in SEQ ID No. 108, 111, 114, 127; and the fifth reaction system includes SEQ ID No. The first reaction system includes primers No. 23-26, 43-44, 91-92 and probes No. 112-113, 122, 127; the second reaction system includes primers No. 17-18, 33-34, 39-40, 77-78, 82-83 and probes No. 109-110, 120, 139, 141; the third reaction system includes primers No. 29-30, 35-38, 79-81, 85 and probes No. 115, 118-119, 139, 141; the fourth reaction system includes primers No. 45-46, 57-58, 65-66, 71-74 and probes No. 123, 129, 133, 135, 137; the fifth reaction system includes primers No. 112-113, 122, 127; the sixth reaction system includes primers No. 17-18, 33-34, 39-40, 77-78, 82-83 and probes No. 109-110, 120, 139, 141; the sixth reaction system includes primers No. 29-30, 35-38, 79-81, 85 and probes No. 115, 118-119, 139, 141; the seventh reaction system includes primers No. 29-30, 35-38, 79-81, 85 and probes No. 115, 118-119, 139, 141; the eighth reaction system includes primers No. 45-46, 57-58, 65-66, 71- The tenth reaction system includes primers shown in SEQ ID Nos. 47-48, 59-60, 67-70, and 75-76, and probes shown in SEQ ID Nos. 123, 129, 133, 135, and 137; the eleventh reaction system includes primers shown in SEQ ID Nos. 49-50, 61-62, 85-86, 93-94, and 97-98, and probes shown in SEQ ID Nos. 125, 131, 143, 147, and 149; the eleventh reaction system includes primers shown in SEQ ID Nos. 52-53, 63-64, 87-88, 95-96, and 99-100, and probes shown in SEQ ID Nos. 125, 131, 143, 147, and 149; the twelfth reaction system includes a negative control.

[0041] In this invention, in addition to the probes and primers mentioned above, the system further includes internal reference primers and probes, wherein the internal reference primers and probes have sequences shown in SEQ ID No. 151, 152 and 153, respectively.

[0042] The detection system or reaction solution of the present invention can be made into a kit. Therefore, the kit containing it is also within the protection scope of the present invention. The kit may further include instructions on how to perform the detection method and interpretation rules of the present invention.

[0043] This invention provides a method for typing or detecting HLA cross-reactivity groups and HPA genes, sometimes referred to herein as "the typing or detection method of this invention," which includes commercial experimental tests for non-diagnostic purposes as well as tests for diagnostic purposes. The diagnostic test method can be understood as a diagnostic tool.

[0044] Example 1. HLA and HPA sequence analysis and plasmid standard construction HLA-A and HLA-B sequences are polymorphic. Therefore, the complete sequences of HLA-A and HLA-B were downloaded from the AFND and GenBank databases, respectively, and aligned using Mega software. Primers were designed in conserved regions to amplify the full-length genomes of HLA-A and HLA-B. HPA sequences are relatively conserved compared to HLA. Based on the HPA genes 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 (low-frequency antigen genes with a frequency higher than 5 in the population) in the Versiti and GenBank databases, primers were designed to amplify the full-length genomes of HLA-A and HLA-B. Sequence design was used to design primers for amplifying corresponding gene fragments. Human gDNA with known HLA-A and B gene subtypes was used as templates. The designed primers were used to amplify various HLA-A, HLA-B, and HPA gene fragments via PCR. For samples with extremely low gene frequencies or no corresponding gene subtypes, site-directed mutagenesis was used for construction, or the fragments were synthesized by a gene biotechnology company. Successfully amplified products were subjected to agarose gel electrophoresis. Bands with the expected fragment length were excised and recovered from the gel. These bands were then ligated into T-vectors or B-vectors using T4 DNA, transformed into DH5α cells, and plasmids were extracted for enzyme digestion identification. Positive plasmids were sent to Shanghai Sangon Biotech for sequencing analysis. Plasmids with correct sequencing results were retained as detection templates for methodology establishment. Table 1 shows the information of HLA-A, HLA-B, and HPA plasmids with correct sequencing results.

[0045] Table 1. Sanger sequencing genotyping results of constructed standard plasmids 2. HLA-A, HLA-B, and HPA sequence analysis, specific primer design and optimization scheme MUSCLE iterative sequence analysis was performed on all HLA-A and HLA-B sequences in the IPD-IMGT / HLA database. Simultaneously, sequences from HLA subtypes with the same epitope in the CREG table were grouped for analysis. The groups in the CREG table were further subdivided according to epitope-specific SNP sites, resulting in 8 groups for HLA-A and 14 groups for HLA-B. Specific UAP and / or ARMS primers were designed based on the SNP sites in each group. Specific epitope locations within the groupings are shown in Table 2 and [Table 2 is missing from the original text]. Figure 1 As shown; Table 2 Examples of CREG groupings, corresponding group antigens, and SNP sequences For HPA, different HPA loci are located on different chromosomes or distant gene segments on the same chromosome. The genotyping of different HPA subtypes is mainly due to the difference of a single SNP. Therefore, UAP and / or ARMS primers are designed based on the specific SNP loci in the HPA-1-6, 9, 12, 13, 15, 21, 27, 30, and 31 genes.

[0046] Primers can only extend when the 3' bases of a conventional primer are perfectly complementary to the template. When a mismatch occurs between the 3' bases of the primer and the template, primer extension is inhibited or even terminated completely. However, similar sequences can lead to non-specific amplification. Therefore, the design structure of ARMS primers, based on the mutation arrest amplification system, can eliminate this type of non-specific amplification. This is achieved by artificially introducing mismatched bases at the 3' end of the primer. Both the type and number of mismatches introduced affect the amplification effect. Taking the design and optimization of primers targeting the 1C1 target sequence in the HLA-A locus as an example, the design and optimization scheme of ARMS primers is as follows: Figure 1The upper half of the diagram shows: In the first round, a single-base mismatch was used. The specificity of the amplified products was determined based on the melting curve of the SYBR Green dye-based real-time fluorescent PCR. The Tm value of the peak of the melting curve was determined using the X-axis. Increased temperature causes the DNA double strand to unwind. When the temperature reaches a specific value, all double-stranded DNA unwinds into single-stranded DNA. The SYBR Green dye dissociates from the unwinded DNA double strands, and the fluorescence signal reaches its peak. The peak value of the melting curve corresponds to the Tm value of the specific amplified product. The fluorescence signal intensity of the fluorescent dye bound to the DNA strand was determined using the Y-axis. The more products, the stronger the fluorescence intensity. The difference between specific and non-specific peaks can be determined based on the fluorescence intensity of the melting curve peak. Typically, a melting curve peak above 80℃ is a specific peak. The figure shows that the ARMS primer with a single-base mismatch produces a non-specific peak above 80℃ after amplification. Therefore, further optimization of the primer structure is needed. In the second round of optimization, a second and third mismatched base were artificially introduced. Although the introduction of multiple mismatches affected… The amplification efficiency of primers is affected, but mismatches of 2-3 bases can increase primer complexity and suppress non-specific amplification. As shown in the figure, the non-specific melting curve peak of the AC and TC base mismatch primers is weaker than that of the single base C mismatch, proving that double base mismatches can effectively suppress the amplification of non-specific products. Furthermore, by interpreting the melting curves, primers with better specificity are screened for TaqMan probe real-time fluorescence PCR detection. The specificity of the designed primers is confirmed by directly reading the ΔCT value between the specific and non-specific amplification curves. In addition, the 5' end primer length of the screened ARMS primers is optimized and fine-tuned to further screen primers with better specificity.

[0047] Similarly, due to the high polymorphism and linkage disequilibrium between HLA-A and B site subtypes—meaning that the differences in some gene segments are not just single bases—designing ARMS primers for specific SNPs can be challenging due to the linkage mutations before and after the SNP. Therefore, UAP primers are designed simultaneously to screen for specific primers. Since UAP primers have two relatively independent primer fragments connected by a deoxyadenine nucleotide linker, they can effectively avoid other base mutations near the SNP. Because UAP primers have two binding regions, the primer structure and binding sequence at the 5' and 3' ends can be adjusted separately. Again, taking the design and optimization of primers for the 1C1 target specific sequence at the HLA-A site as an example, the specific optimization scheme for UAP primers is as follows: Figure 2As shown in the lower section, in the first round of UAP primer optimization, the number of binding bases at the 3' end of the primer and the linker method (vacuum bridging and substitution bridging) were adjusted. Based on the results of the SYBR dye method, primers with better specificity were selected for further optimization. In the second round of optimization, the number of bases in the linker (3-9 deoxyadenine nucleotides) was optimized. Finally, primers with better specificity were detected by TaqMan probe real-time fluorescence PCR, and the length of the 5' end of the UAP primer was further adjusted. Ultimately, UAP primers with better specificity were selected.

[0048] For each SNP site, the primers with the best specificity were selected for multiplex PCR by designing and optimizing ARMS and UAP primers.

[0049] Table 3 shows the primer and probe sequences for different CREG table groups specific SNP sites and HPA specific SNP sites after screening and optimization according to the present invention.

[0050] Table 3 CREGs and HPA primer and probe sequences 3. Optimization of reaction conditions To balance the specificity of ARMS and UAP primer systems, this invention optimizes the reaction procedure and annealing parameters. By comparing two-step and three-step methods and optimizing the annealing temperature and time using a gradient, the most suitable reaction conditions for ARMS and UAP primers were selected, resulting in the strongest specificity for both primers. The results are shown in Table 4. Experimental results show that under the same annealing conditions, the two-step method exhibits better specificity than the three-step method, with the non-specific amplification Ct value of the three-step method being <35. Gradient optimization of the annealing temperature shows that lower annealing temperatures (56, 58℃) ​​lead to significant non-specific amplification, while higher annealing temperatures (62, 64℃) inhibit the reaction. Optimized annealing time results show that longer annealing times lead to primer binding to the non-specific template; when the annealing time is ≥45 s, the non-specific amplification Ct value is <35, while shorter annealing times result in incomplete target amplification, with a specific amplification Ct value >30. Therefore, the two-step amplification program annealing condition of 60℃ for 30 s was adopted. Under this condition, the system has both high specificity and stable amplification efficiency, and it serves as the reaction condition for subsequent primer screening and multiplex real-time fluorescence PCR.

[0051] Table 4: Effects of different reaction conditions on the specific amplification of ARMS and UAP primer systems 3. Screening of internal reference gene primers and probes This invention designs internal reference gene primers and probes based on the conserved region of HLA-B. Primer-BLAST was performed against all HLA-B allele sequences in the IMGT / HLA database to verify sequence conservation. BLAST alignment in the NCBI database verified the absence of interference from other homologous genes in the amplicon, confirming that this region is not conserved in non-human primates and other species, thus avoiding false detections due to contaminated samples. Because the internal reference primer sequence is conserved, its amplification efficiency is higher than other targets, consuming dNTPs and Taq DNAase activity in earlier amplification cycles. This leads to delayed or inhibited amplification of other targets in multiplex real-time fluorescence PCR. Therefore, 1-2 base mismatches were introduced at the 3' end of each bidirectional primer to adjust the primer amplification efficiency. The designed primers are shown in Table 5. Multiple designed primers were paired with normal upstream and downstream primers to detect gDNA samples. The results are shown in Tables 6 and 7. When HLA-B-2427-2449-F-TA and HLA-B-2427-2449-F-TT were used as upstream primers (both with HLA-B-2568-2592-R as the downstream primer), the amplification curves were S-shaped with Ct values ​​between 28 and 30. Repeat experiments were performed on 10 gDNA samples using each primer pair. When HLA-B-2427-2449-F-TT was used as the upstream primer, the intra-assay and inter-assay standard deviations were less than 0.5, and the CV was less than 5%, indicating good precision. Therefore, the upstream ARMS primer HLA-B-2427-2449-F-TT and the downstream normal primer HLA-B-2568-2592-R were selected as internal standard primers for establishing a multiplex real-time fluorescence PCR system. The final selected internal standard primers and probe sequences are shown in Table 8.

[0052] Table 5: Internal Standard Primer Design Sequence The underlined a symbol indicates a mismatched base designed for the internal standard primer sequence. Table 6: Results of gDNA sample detection using internal standard primers Table 7: Results of Repeated Experiments for Screening Internal Standard Primers Table 8 Primer and probe sequences of the screened internal reference genes 4. Establishment of negative quality control system This invention prepares an adenovirus solution coated with an irrelevant sequence as a negative control. This solution participates in the extraction process as a negative control. The negative control wells simultaneously contain primers and probes for both the internal reference gene and the irrelevant sequence. This is used to control the sample extraction process and to prevent false positives during the detection process. In the negative control wells, the internal reference gene does not amplify, but the Ct value for the irrelevant sequence detection is <35, indicating that the detection results of the entire detection system are valid. When the primers and probes for the irrelevant sequence are used for dual detection with the primers and probes for the internal reference gene, their Ct values ​​are not affected by each other. Figure 3 ).

[0053] 5. Detection of HLA-A, B epitopes and HPA alleles using a multiplex PCR system. (1) The Magnetic Bead Blood Genomic DNA Extraction Kit (DP329) produced by Beijing Tiangen Company was used to extract gDNA from the sample to be tested according to the instructions. The concentration was >20 ng / μL and the OD260nm / OD280nm ratio was 1.7-2.0.

[0054] (2) 2×Eagle Premix (PCR) reaction solution (brand: Beijing Jin Nuomei, catalog number: SJ-PCR002) was used. This premix solution contains all the components required for the reaction except for primers, Taqman probes and templates: 35 mM Tris (pH=8.5), 150 mM KCl, 14 mM MgCl2, 0.6 mM dNTP, 0.25 mM 50% trehalose, and 2 U / Test Taq hot-start enzyme. The primers and probes for HLA-A and HLA-B epitopes, HPA alleles and random sequences were placed in a 12-tube PCR system with the premix solution and the internal reference gene HLA-B primers and probes according to Table 9 below. The final concentration of each primer in each well was 120 nM, and the final concentration of the probe was 100 nM. Finally, ddH2O was added to make up to 40 μl.

[0055] Table 9. Primer and probe combinations for HLA-A and B epitopes, HPA alleles, random sequences, and internal reference genes in a 12-tube multiplex PCR system. (3) Amplification was performed using an 8-channel PCR amplification instrument (Hongshi 96H, China), under the following reaction conditions: (4) Interpretation criteria A. Quality Control ① If the internal reference gene in the negative control well (well 12) shows no curve rise or the Ct value is ≥32, and the FAM amplification curve of the irrelevant sequence is normal and the Ct value is <32, then this experiment is valid and there are no false positives; ② If the amplification curve of the internal reference gene CY5 in wells 1-11 shows a standard "S" shape and the Ct value is ≤32, then the amplification results are valid; Detection results that meet the above conditions are valid. For reactions that do not meet the above conditions, repeat experiments or re-extraction and amplification should be performed according to the specific circumstances. The specific situations and solutions are shown in Table 10 below.

[0056] Table 10 Problems and Solutions in Multiplex Real-Time Fluorescent PCR Detection B. Result Interpretation Experimental results that meet quality control standards are analyzed for HLA-A, B epitope, and HPA genotyping. All target detection results are interpreted using the same criteria: if the probe amplification curve of any marker for different targets is normal and the Ct value is ≤35, it is considered a positive result; if no amplification curve appears or the Ct value is >35, it is considered a negative reaction.

[0057] 6. Limit of Detection (LOD) Based on the gDNA sample detection results, the target with the worst amplification effect in each tube was analyzed for LOD (Limit of Detection), representing the lowest detection limit for each tube. The extracted gDNA samples were serially diluted, and multiplex PCR was used to repeatedly test the diluted gDNA samples, seven times a day for three consecutive days. The results were analyzed using the Probit method to determine the lowest detection limit. The specific results are shown in Table 11. The results show that the LOD of tubes 1-11 are 0.444 ng / μL, 0.389 ng / μL, 0.548 ng / μL, 0.223 ng / μL, 0.305 ng / μL, 0.121 ng / μL, 0.754 ng / μL, 0.838 ng / μL, 0.298 ng / μL, 0.195 ng / μL, and 0.431 ng / μL, respectively. Therefore, the LOD of the 12-tube multiplex real-time fluorescence PCR detection system is 0.838 ng / μL.

[0058] Table 11. Limit of Detection (LOD) Results for 12-tube Multiplex PCR System Detection of gDNA Samples 7. Reagent precision verification Two concentrations of gDNA samples (low and high) were selected to verify the precision of 11 multiplex PCR detection systems. All samples were tested continuously for 3 days, with 7 replicate experiments per day, and inter-batch and intra-batch precision were calculated. The specific results are shown in Table 12. The results show that the intra-batch and inter-batch precision of the target gene in each tube was less than 5%, indicating that the multiplex PCR of the present invention has good precision.

[0059] Table 12 Precision results of multiplex PCR system for detecting gDNA samples 8. Clinical sample validation Genomic DNA was extracted from 211 clinical samples with known subtypes using the Tiangen reagent kit. The 211 samples were then tested and analyzed according to the methods and judgment rules provided in this invention. All samples were consistent with the known subtypes, with a consistency rate of 100%. Figure 4 The CREGs grouping and HPA genotyping amplification curves for one sample were analyzed. Based on the interpretation of the amplification curve results, the CREGs grouping results for this sample were 2C-A (HLA-A), 5C1 (HLA-B), and 5C2 (HLA-B), and the HPA genotyping results were HPA-1a1a, HPA-2a2a, HPA-3a3b, HPA-4a4a, HPA-5a5a, HPA-6a6a, HPA-9a9a, HPA-12a12a, HPA-13a13a, HPA-15a15b, HPA-21a21a, HPA-27a27a, HPA-30a30a, and HPA-31a31a.

[0060] Technical advantages: This invention mainly designs specific ARMS and UAP primers based on specific SNP sites in different HLA-AB epitopes and different HPA subtypes. It screens and optimizes a large number of primers, and finally selects the primers with the best amplification effect to combine with TaqMan probes. In addition, the combination method of multiplex PCR has also been optimized many times to ensure that the primers and probes amplified in each tube do not affect each other, and finally can detect HLA-AB epitopes and HPA genes.

[0061] Advantages at the application level: The effect of transfusing HLA epitope-matched platelets is equivalent to transfusing HLA antigen-matched platelets. Therefore, HLA epitope matching can replace HLA antigen matching for transfusion. However, currently there are no kits for detecting HLA epitopes in clinical practice; all tests target HLA antigen genes, resulting in reagent waste. Furthermore, the complex operation process, expensive testing instruments, and high technical requirements hinder its widespread clinical application. This invention not only provides a method for HLA epitope detection but also a method for HPA detection, allowing for the testing of a single sample in just 12 tubes. This invention will greatly simplify the platelet HLA / HPA typing process and significantly reduce its testing costs, enabling more small and medium-sized laboratories to use conventional testing instruments to detect HLA epitopes and HPA antigens in platelet transfusion donors and recipients, thereby improving the effectiveness of platelet transfusions. In addition, the HLA epitope detection results can not only be applied to patients who are not responding to platelet transfusions, but also, since the concept and principle of HLA epitope matching are also used in organ transplantation to select HLA-matched donors, it can be applied to HLA epitope matching between some organ transplant patients and donors, thus having broad application prospects.

[0062] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for constructing a simultaneous HLA-CREG and HPA genotyping detection system based on high-throughput multiplex fluorescent PCR, characterized in that, Includes the following steps: (1) For each HLA gene in the cross-reactive group, analyze its epitope gene sequence, screen for common specific SNP sites, and group the HLA genes according to the common specific SNP sites to obtain multiple HLA subgroups, of which at least some HLA subgroups contain multiple HLA genes. (2) For each HPA allele, analyze its gene sequence and screen for specific SNP sites of low-frequency and high-frequency genes of different HPA subtypes. (3) Primers and probes are designed for the common specific SNP sites and frequency-specific SNP sites, respectively. When designing primers for a single specific SNP site, a mutation-repressive amplification system is preferred, and when there are linked mutant bases near the specific SNP site, U-shaped anchoring primers are preferred. The probes are labeled with different fluorescent groups; and (4) Separate tubes according to the type of fluorescent group of the probe and the position of the primer.

2. The method for constructing a simultaneous HLA-CREG and HPA genotyping detection system based on high-throughput multiplex fluorescent PCR according to claim 1, characterized in that, The HLA genes include HLA-A and HLA-B.

3. The method for constructing a simultaneous HLA-CREG and HPA genotyping detection system based on high-throughput multiplex fluorescent PCR according to claim 2, characterized in that, The multiple HLA subgroups obtained after grouping in step (1) include: The A1C1 subgroup contains A01, A11, A36, and A80; Subgroup A1C2 containing A03 and A30; The A1C3 subgroup containing A29; Subgroup A1C4 contains A30 and A31; The A2C-A subgroup contains A02, A23, A24, A68, and A69; Subgroup A10C1 contains A32 and A74; The A10C2 subgroup contains A25, A26, A34, A66, and A43; The A10C3 subgroup contains A33; The B2C subgroup contains B57 and B58; The B5C1 subgroup contains B35, B51, B52, B53, B58, and B78; Subgroup B5C2 contains B15 and B46; The B5C3 subgroup containing B18; The B5C4 subgroup containing B49; The B5C5 subgroup containing B50; The B5C7 subgroup containing B73; The B7C1 subgroup contains B7, B42, B54, B55, B56, B67, B81, and B82; The B7C2 subgroup contains B54, B55, B56, and B59; The B7C3 subgroup contains B8, B41, and B42; The B7C4 subgroup contains B7, B13, B27, B40, B47, B48, and B81; The B8C1 subgroup contains B8, B14, B38, B39, and B67; The B12C1 subgroup containing B13, B40, B41, B44, B45, B47, B49, and B50; and The B12C2 subgroup containing B37.

4. The method for constructing a simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR according to claim 1, characterized in that, When designing primers using a mutation arrest amplification system, mismatched bases are introduced at the 3' end of the primer, including: a. First, introduce single base mismatches and determine the specificity of the amplification product, i.e., whether there is a specific peak, based on the melting curve of real-time fluorescence PCR; b. When the melting curve shows no specific peak, introduce another base mismatch and again determine whether there is a specific peak based on the melting curve of real-time fluorescence PCR; Optionally, step b may be repeated until a specific peak is obtained.

5. The method for constructing a simultaneous HLA-CREG and HPA genotyping detection system based on high-throughput multiplex fluorescent PCR according to claim 1, characterized in that, The U-shaped anchoring primer includes a 5' binding region, a 3' binding region, and a vacuolar structure region located between the two. The design of the U-shaped anchoring primer includes the following steps: a. Adjust the number of binding bases at the 3' end of the primers and the vacuolar structure of the linker, and select primers with better specificity as preliminary primers based on the results of the SYBR dye method; b. Further optimize the number of bases in the linker of the preliminary primers to obtain optimized primers; c. Adjust the length of the 5' binding region of the U-shaped anchor primer according to the Taqman probe real-time fluorescence PCR method.

6. A simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR, characterized in that, Obtained by the construction method described in any one of claims 1-5.

7. The simultaneous detection system for HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR according to claim 6, characterized in that, It includes primers with sequences as shown in SEQ ID No. 1-100; and probes with sequences as shown in SEQ ID No. 101-150; Preferably, the detection system includes multiple independently configured subsystems, each of which contains primers and probes targeting specific SNP sites. Information on the primers and probes in the subsystems is shown in Table 3. Preferably, the probe further comprises a fluorescent group; Preferably, the system further comprises Mg 2+ dNTPs and Taq hot-start enzyme; Preferably, the system includes an internal reference primer and a probe, wherein the internal reference primer and the probe have the sequences shown in SEQ ID No. 151 and 152, and the internal reference probe has the sequence shown in SEQ ID No.

153.

8. A kit for simultaneous detection of HLA-CREG and HPA genotyping based on high-throughput multiplex fluorescent PCR, characterized in that, Includes the detection system described in claim 6 or 7; Preferably, the detection system includes multiple independently configured subsystems, each of which contains primers and probes targeting specific SNP sites. Information on the primers and probes in the subsystems is shown in Table 3.

9. The reagent kit according to claim 8, characterized in that, The kit further includes an interpretation rule instruction manual.

10. A method for HLA-CREG and HPA genotyping or detection based on high-throughput multiplex fluorescent PCR, characterized in that, Includes the step of performing multiplex PCR using the detection system according to claim 6 or 7; and The steps involve interpreting fluorescence signals of different colors.

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