Construction method of detection system for HLA cross reaction group and kit

Abstract

The invention discloses a construction method of a detection system for an HLA cross reaction group and a kit. The HLA cross reaction groups are grouped, the primers and the specific probes are designed, a reasonable detection system is constructed, the groups of the cross reaction groups can be directly detected instead of independently detecting each subtype in the groups, so that the number of the required primers and probes is greatly reduced, the detection efficiency is improved, and the cost is reduced. Combined interpretation is carried out by means of multiple fluorescence channels and melting curves, grouping information of samples can be obtained only through three reaction systems, and the method has the advantages of being rapid, easy and convenient to operate, high in flux and the like and has wide application prospects and clinical value in the fields of clinical blood transfusion medicine, organ transplantation and the like.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to a method for constructing a detection system for an HLA cross-reaction group and a kit. Background Art

[0002] Human leukocyte antigens (HLA) are a highly polymorphic gene complex located on the short arm of chromosome 6. They encode cell surface molecules that present and recognize self- and non-self-peptides and are the primary mediators of antigen-antibody reactions and organ transplant rejection. HLA comprises class I, class II, and class III antigens. HLA class I antigens (including HLA-A, HLA-B, and HLA-C) are crucial in mediating immune responses and the development of a variety of diseases, including tumors and autoimmune diseases. Although HLA possesses high genetic polymorphism at the locus, making it the most complex human gene complex known to date, the amino acid residues in the proteins produced by HLA class I and class II antigens differ only slightly. Different HLA antigens structurally share some immunogenic epitopes, enabling cross-reactivity with HLA antibodies. Therefore, HLA antigens carrying shared epitopes constitute the HLA cross-reactive group (CREG).

[0003] Currently, methods and reagents capable of directly detecting HLA-CREG are relatively scarce. Most reagents require separate typing of both HLA-A and HLA-B alleles before confirming CREG grouping, which is generally limited by high cost and time consumption. Currently available CREG detection kits require 13 reaction tubes per sample and require electrophoresis after amplification. These complex, time-consuming, and low-throughput procedures significantly limit the widespread application of CREG typing. Therefore, there is an urgent need to develop a rapid, easy-to-use, and high-throughput HLA-CREG detection method to better meet clinical needs.

[0004] The information in the background technology is only intended to illustrate the general background of the invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to a person skilled in the art. Summary of the Invention

[0005] To address at least some of the technical issues existing in the prior art, the present invention establishes a multiplex detection system for HLA CREG based on multicolor probe melting curve analysis. This is an endpoint detection technique that utilizes asymmetric PCR (in which the amounts of forward and reverse primers differ significantly, referred to as limiting or excess primers) to generate large amounts of single-stranded DNA. This DNA then binds to a dual-labeled, self-quenching fluorescent probe to produce partially double-stranded hybrids. Different mutations in the target gene form different hybrids, resulting in different melting temperatures (Tm) during melting analysis, enabling simultaneous detection of multiple mutations within a single channel. The introduction of Tm values ​​significantly increases the number of mutations detectable within a single channel. Combining multiple channels enables "N×N" amplification, effectively increasing detection throughput and eliminating the need for electrophoresis, simplifying operation. Currently, no research or registered reagents are available for CREG detection using this approach. Therefore, the present invention establishes a multiplex detection system based on multicolor probe melting curve analysis that can detect HLA CREG groups in just three reaction tubes, offering the advantages of rapidity, simplicity, high throughput, and low cost. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, a method for constructing a detection system for an HLA cross-reaction panel is provided, comprising:

[0007] (1) grouping the HLA antigens in the HLA cross-reaction group to obtain HLA cross-reaction subgroups, wherein the index used for grouping includes the SNP coordinate position of the HLA antigen;

[0008] (2) Designing and synthesizing primers and specific probes based on the SNP sites in each cross-reaction subgroup after grouping;

[0009] (3) Separate the primers and probes to obtain the detection system.

[0010] In certain embodiments, the method for constructing a detection system for an HLA cross-reactive panel according to the present invention further comprises: (4) a step of validating and optimizing the detection system using samples with known HLA-A and HLA-B genotyping.

[0011] In certain embodiments, according to the method for constructing a detection system for HLA cross-reaction groups of the present invention, the cross-reaction groups obtained after grouping in step (1) include:

[0012] (a) Subgroup 1: A1C1 containing HLA antigens A01, A03, A11, A30, and A36; A1C2 containing HLA antigen A31; and A1C3 containing HLA antigen A29.

[0013] (b) Subgroup II: A2C1 containing HLA antigens A02, A23, A24, A68, and A69; A2C2 containing HLA antigens B57 and B58;

[0014] (c) Subgroup III: A10C including HLA antigens A25, A26, A32, A33, A34, A66, and A74;

[0015] (d) Subgroup IV: includes B5C1 containing HLA antigens B35, B51, B52, B53, B58, and B78; B5C2 containing HLA antigens B15, B45, B46, B49, and B50; B5C3 containing HLA antigen B18; and B5C4 containing HLA antigen B73;

[0016] (e) Subgroup V: includes B7C1 containing HLA antigens of B7, B8, B40, B41, B42, B48, B54, B55, B56, B59, and B81; B7C2 containing HLA antigens of B13, B54, B55, B56, B59, and B82; and B7C3 containing HLA antigens of B27, B40, and B47;

[0017] (f) Subgroup VI: B8C1, which includes HLA antigens of B8; B8C2, which includes HLA antigens of B14, B18, B38, B39, and B67;

[0018] (g) Subgroup VII: includes B12C1 containing HLA antigens B13, B40, B41, B44, B45, B47, B49, and B50; and B12C2 containing HLA antigen B37.

[0019] In certain embodiments, according to the method for constructing a detection system for an HLA cross-reaction panel according to the present invention, in step (2), the primers include sequences shown in SEQ ID Nos. 1-12; in step (2), the probes include sequences shown in SEQ ID Nos. 13-30.

[0020] In certain embodiments, according to the method for constructing a detection system for an HLA cross-reactive panel according to the present invention, the detection system after step (3) is divided into:

[0021] A first detection system for detecting HLA-A genes, comprising primers having sequences shown in SEQ ID No. 1-6;

[0022] A second detection system for detecting HLA-B genes, comprising primers having sequences shown in SEQ ID Nos. 7-10;

[0023] The third detection system for detecting HLA-B gene includes primers with sequences shown in SEQ ID No. 7-12.

[0024] The second aspect of the present invention provides a detection system for HLA cross-reaction groups, which is obtained according to the construction method described in the first aspect.

[0025] In certain embodiments, according to the detection system for HLA cross-reactive panel of the present invention, the first detection system further comprises probes of sequences shown in SEQ ID No. 13-18;

[0026] The second detection system further includes probes with sequences shown in SEQ ID No. 22-24, 27-29;

[0027] The third detection system further includes probes with sequences shown in SEQ ID No. 19-21, 25-26, and 30.

[0028] The third aspect of the present invention provides a detection kit for HLA cross-reaction group, which comprises the detection system described in the second aspect;

[0029] Preferably, it further comprises an internal standard primer and an internal standard probe.

[0030] A fourth aspect of the present invention provides a method for detecting HLA cross-reactive groups, comprising the steps of performing asymmetric PCR using the detection system of the present invention;

[0031] Preferably, the method further comprises the step of performing joint interpretation based on the fluorescence channel and the melting curve.

[0032] In a fifth aspect, the present invention provides a reagent for use in preparing an HLA cross-reaction panel detection kit, wherein the reagent comprises the detection system described in the second aspect.

[0033] The present invention provides a detection system and a kit containing the same for detecting HLA cross-reaction groups based on multicolor probe melting curve analysis. The detection system or kit comprises the required primers and a dual-labeled self-quenching probe combination. By analyzing HLA-A or HLA-B gene sequences within the same CREG group to identify common sites, the present invention designs primers and specific probes based on these sites. This allows for direct detection of CREG groups rather than individually detecting each subtype within the group, significantly reducing the number of primers and probes required, improving detection efficiency, and lowering costs. By combining interpretation with multiple fluorescence channels and melting curves, only three reaction systems are required to obtain CREG grouping information for a sample. This method offers advantages such as rapidity, ease of operation, and high throughput, and has broad application prospects and clinical value in fields such as clinical transfusion medicine and organ transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The single-plex system verifies that the probes that meet the performance requirements detect the melting curve of some heterozygous templates.

[0035] Figure 2 Melting curve results of A-Probe 2 before and after optimization.

[0036] Figure 3 Melting curve results of A-Probe 3 before and after optimization.

[0037] Figure 4 Melting curve results of B-Probe H before and after optimization.

[0038] Figure 5 Melting curve results of B-Probe I before and after optimization.

[0039] Figure 6 Examples of results from preliminary validation of three multiplex systems using HLA plasmids.

[0040] Figure 7 Examples of multiplex system optimization results (A: dNTP concentration optimization. B: Addition of DMSO improves the low melting curve signals of samples detected in channels 4 and 6 of the HLA-A system. C: Addition of DMSO significantly improves the low specific melting peak signals and the appearance of nonspecific peaks in samples detected in channel 5 of the HLA-B-1 system. D: Addition of DMSO significantly improves the poor differentiation of melting peaks with similar Tm values ​​in samples detected in channel 6 of the HLA-B-2 system. E: Example results of melting curves of samples detected in three optimized multiplex systems).

[0041] Figure 8 Examples of clinical sample test results and sequencing results. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0045] Currently, most methods first perform HLA-A and HLA-B allele typing and then confirm the CREG grouping. HLA genotyping methods include a range of PCR-based methods and next-generation sequencing (NGS). The former includes single-strand conformation polymorphism (PCR-SSCP), restriction fragment length polymorphism (PCR-RFLP), sequence-specific primers (PCR-SSP), sequence-specific oligonucleotide probes (PCR-SSO), and sequence-based typing (PCR-SBT). PCR-SSCP and PCR-RFLP distinguish different HLA types by electrophoresis based on differences in DNA conformation or restriction enzyme cleavage sites, respectively. While these two techniques offer good accuracy, they suffer from low throughput and complex procedures, limiting their clinical application. PCR-SSP and PCR-SSO utilize specific primers or oligonucleotide probes, respectively, for typing, significantly improving sensitivity and specificity compared to the former two methods. However, they still require electrophoresis or hybridization, making them less convenient. PCR-SBT involves sequencing DNA fragments after PCR amplification. While accurate and capable of identifying novel alleles, it is expensive and requires a long testing cycle. NGS utilizes high-throughput sequencing technology to sequence the entire HLA genome or specific regions, performing HLA genotyping by comparison with an HLA reference sequence. This method offers the advantages of high throughput and resolution. However, the resulting data volume is enormous, making data processing and analysis complex, and equipment maintenance and reagent costs high.

[0046] Most existing technologies cannot directly detect HLA CREGs. They require separate genotyping of HLA-A and HLA-B before confirming their CREG groups. This logic is equivalent to a two-step detection process, which not only increases process complexity but, more importantly, introduces significant difficulty and cost for accurate HLA-A and HLA-B genotyping. Existing methods for direct CREG detection rely on PCR-SSP technology. This not only requires numerous reaction tubes, low throughput, and a complex preparation system, but also requires electrophoresis for identification after amplification, increasing workload, detection time, and the potential for human error, making it unsuitable for large-scale clinical application. The present invention adopts a strategy for direct HLA CREG detection. By analyzing different HLA gene sequences within the same CREG group, identifying shared sites and designing primers and specific probes based on these shared sites, a single probe can simultaneously detect multiple HLA types. This significantly reduces the number of probes required and avoids the complexity of separate typing of each HLA gene, effectively improving detection efficiency and reducing costs. On the technical level, based on multi-color probe melting curve analysis, it is possible to directly detect 7 CREG groups with only 3 reaction tubes. The results are interpreted by combining fluorescence channels and melting curve Tm values, without the need for electrophoresis identification. It has the significant advantages of simple operation, high throughput, and short time consumption, and has good clinical application value.

[0047] Construction method

[0048] The present invention first provides a method for constructing a detection system for an HLA cross-reaction panel, comprising:

[0049] (1) grouping the HLA antigens in the HLA cross-reaction group to obtain HLA cross-reaction subgroups, wherein the index used for grouping includes the SNP coordinate position of the HLA antigen;

[0050] (2) Designing and synthesizing primers and specific probes based on the SNP sites in each cross-reaction subgroup after grouping;

[0051] (3) Separate the primers and probes to obtain the detection system.

[0052] In step (1), grouping according to the SNP coordinate position of the HLA antigen includes analyzing different HLA gene sequences located in the same CREG group, and grouping HLA antigens with common sites into the same subgroup, wherein, when the physical positions of the adjacent SNPs do not exceed 5 bp, the HLA antigens containing the above SNP sites are grouped into the same subgroup.

[0053] In step (2), primers and specific probes were designed and synthesized based on the SNP sites in each cross-reaction subgroup after grouping. The primers were designed to cover multiple probe binding sites through one amplification product. To further improve typing efficiency, SNP sites with SNP physical locations greater than 500 bp were selected for probe design based on the common SNP sites.

[0054] In step (3), the designed primers and probes are divided into groups to obtain the detection system, which takes into account multiple factors such as the signal intensity of the fluorescent marker, the melting curve, the primer position, the size of the amplified product, etc., so that the three reaction tubes can directly detect the seven CREG groups and 17 subgroups accurately.

[0055] Detection system or reaction solution

[0056] After developing specific primers and probes, the present inventors conducted in-depth research and discovered that, on the one hand, a single-plex asymmetric PCR system is not directly suitable for multiplex detection. On the other hand, the detection primers and probes need to be grouped and further optimized to accurately detect HLA cross-reactive groups. Regarding the probes, the present inventors discovered that shortening the probe length can actually enhance the resolution of certain subgroups (e.g., the B8C2 and B12C1 subgroups). Furthermore, the present inventors optimized different fluorescent labels and the position of the fluorescent label (i.e., not at the 5' end of the probe, but in the middle).

[0057] After constructing the detection system or reaction solution, the present invention further carried out a lot of optimization suitable for the detection method of the present invention, including the concentration of each added component (such as dNTP, Mg 2+ , forward primer and reverse primer, probe, DMSO concentration, etc.), the concentration of which can be controlled within a reasonable concentration range, thereby realizing the detection of HLA cross-reaction group.

[0058] In a preferred embodiment, the detection system includes or consists of a first detection system, a second detection system and a third detection system, wherein the first detection system includes:

[0059]

[0060]

[0061] The second detection system includes:

[0062]

[0063] The third detection system includes:

[0064]

[0065]

[0066] In the present invention, the final concentration of MgCl2 in the reaction system is 0.1-10 mM, preferably 1-5 mM, further preferably 2-4 mM, and further preferably 2.5-3.5 mM.

[0067] In the present invention, the final concentration of dNTP in the reaction system is 100-500 μM, preferably 200-450 μM, and further preferably 200-400 μM.

[0068] In the present invention, the concentration of DMSO added to the third reaction system is not less than 4%, for example, it can be 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0069] In the present invention, the final concentration of the probe in the reaction system is 0.01-10 μM, preferably 0.01-5 μM, and further preferably 0.05-5 μM.

[0070] In the present invention, the concentration of the reverse primer is greater than that of the forward primer. The final concentration of the forward primer in the reaction system is 0.01-1 μM, preferably 0.01-0.5 μM, more preferably 0.01-0.1 μM, and further preferably 0.01-0.08 μM, for example 0.02-0.08 μM, 0.02-0.06 μM, or 0.04-0.06 μM. The final concentration of the reverse primer in the reaction system is 0.1-5 μM, preferably 0.2-1 μM, more preferably 0.2-0.8 μM, and further preferably 0.4-0.6 μM.

[0071] In the present invention, the melting curve analysis reaction solution can use a commercially available reaction solution for melting curve analysis, such as the GNM multiple asymmetric amplification and multi-color melting curve PCR reaction solution with the product number RXD03GS.

[0072] The detection system or reaction solution of the present invention can be prepared into the form of a kit. Therefore, the kit containing the same is also within the scope of protection of the present invention. The kit may further include instructions on how to perform the detection method and interpretation rules of the present invention.

[0073] Methods for detecting HLA cross-reactive groups

[0074] The present invention provides a method for detecting HLA cross-reactive groups, sometimes referred to herein as the "detection method of the present invention," which includes commercial experimental detection for non-diagnostic purposes as well as detection for diagnostic purposes. Detection methods for diagnostic purposes can be understood as diagnostic uses.

[0075] The detection method of the present invention is not particularly limited to the specific process or steps as long as the detection system or kit described herein is used. In an exemplary method, the method of the present invention includes a PCR amplification step, particularly an asymmetric PCR amplification step. The instrument used for the above amplification process can be a real-time fluorescence PCR reaction instrument known in the art.

[0076] In the method of the present invention, the type of sample used for detection is not particularly limited, but is preferably a blood sample, for example, a serum or plasma sample.

[0077] Example

[0078] 1. HLA sequence analysis and primer probe design

[0079] Sequence information for HLA-A and HLA-B genes was collected from the HLA Real-Time Database (http: / / ftp.ebi.ac.uk / pub / databases / ipd / imgt / hla / and http: / / hla.alleles.org / nomenclature / index.html). A total of 7,712 HLA-A gene sequences and 9,640 HLA-B gene sequences were collected, respectively. These sequences were imported into Geneious and MEGA 7.0 software for sequence alignment. Given the large number of sequences, 5-10 representative sequences were selected for each low-resolution HLA-A and HLA-B gene subtype for primer and probe design. These sequences were then aligned with all subtype sequences from the corresponding low-resolution subtype to ensure that they could detect at least 99% of the sequences in that subtype.

[0080] Primers and probes are designed as follows: Within each CREG group, specific probes are selected to cover as many SNPs as possible, rather than designing probes for each genotype individually. This design strategy significantly reduces the number of probes required, reduces system complexity, and effectively improves detection efficiency. For example, within the A1C group, a single probe can detect all genotypes except A31 and A29 within that group, designated the A1C1 subgroup. Similarly, detection within the A2C1, A10C, B7C1, B8C2, B12C1, and A10C groups can be achieved with just a single probe. Furthermore, in addition to detecting more genotypes with fewer probes, primer design aims to maximize coverage of multiple probe binding sites within a single amplification product, significantly reducing the number of primer pairs required and further reducing system complexity. Detection of all 17 CREG subgroups requires only six primer pairs. The HLA CREG grouping test design and the required primers and probes are shown in Tables 1 and 2.

[0081] Table 1. HLA CREG group design

[0082]

[0083]

[0084] *SNPs are color-coded. The A1C2 panel detects two SNPs that are far apart; the B7C1 panel detects three closely spaced SNPs (covered by a single probe). The HLA antigens detected by the different colored SNPs are coded accordingly.

[0085] 2. Preliminary screening of probes with satisfactory performance by single-plex asymmetric PCR and probe optimization

[0086] A melting curve analysis reaction solution from Beijing Jinnuomei was used. Predesigned primers and probes for different CREG groups / subgroups were added to the reaction system. Singleplex asymmetric PCR was performed using previously constructed HLA plasmid standards as templates to determine the Tm values ​​of the melting curves for each probe when detecting different templates. In addition, equal volumes of different plasmid templates with similar Tm values ​​were mixed (hybrid templates) to simulate HLA alleles found in real samples. The reaction procedure was as follows: 90-98°C for 1-5 min, 90-98°C for 5-30 s, 55-65°C for 0.5-3 min, and 45-55 cycles. Denaturation was performed at 90-98°C for 0.5-3 min, hybridization was performed at 40-55°C for 1-10 min, and a gradual temperature increase from 45-90°C. The results showed that among the 18 probes designed, except for A-Probe 2, A-Probe 3, B-Probe H and B-Probe I, the melting curve peaks of the remaining probes for detecting heterozygous templates were good and the Tm values ​​were distinguishable, which can be used in the subsequent establishment of multiplex systems ( Figure 1 ).

[0087] We further optimized the above four probes, including A-Probe 2, which had poor melting curve peaks or were difficult to distinguish when detecting heterozygous templates. First, when the A-Probe 2 probe amplified different plasmid single templates, the baseline of the melting curve peak was uneven and showed a clear upward trend; the Rm value of the melting peak was generally low and the difference in Rm between replicates was also large; when detecting heterozygous templates, the Rm of the melting peak was further reduced, and one replicate could not even distinguish two types due to low signal ( Figure 2Left). Based on this, the fluorescent group was moved four bases from the original 5' end to the 3' end to become an intermediate modification. Since there is no T base at the corresponding position in the sequence, the modification was done on the backbone (the backbone can only label CY5). After the intermediate modification, the melting curve peaks obtained by detecting different plasmid single templates are more regular, the baseline is flat, the melting peak height is significantly improved, and the consistency of the multiple wells is better; when detecting heterozygous templates, the difference in the Tm values ​​of the two melting peaks is significantly increased compared to before, and the Rm value is higher, which is more conducive to software interpretation and differentiation of different types ( Figure 2 right).

[0088] The A-Probe 3 probe is used to detect the A1C3 subgroup. When amplifying different plasmid single templates, a large depression appeared in the melting curve, and except for the fully matched A*29 plasmid, no obvious melting peaks appeared in the other HLA plasmids; when detecting the heterozygous template, only the melting peak of A*29 was found, and different types could not be distinguished. Further analysis of the sequence revealed that the GC content of the probe binding region was higher than 80%, and there was little room for adjustment within the same region. Therefore, other sites with relatively low GC content were replaced and the probes were redesigned. It can be seen that after optimization, A*29 and other plasmids can produce melting peaks. When detecting the heterozygous template, the difference in Tm of the two melting peaks was 5.37°C, which can be interpreted by the software and can distinguish the two types (the unequal peak heights are related to the unequal concentrations of the two plasmids) ( Figure 3 ).

[0089] The B-Probe H probe is used to detect the B8C2 subgroup and is labeled with CY7 fluorescence. When the probe covers different plasmid single templates with the same sequence in the region, the heterogeneity of the melting curve Tm and Rm is large, and the Tm and Rm of the melting peaks of the duplicate wells are quite different. The peak shape of the melting curve is relatively chaotic, and some non-specific peaks appear at the front end. When detecting heterozygous templates, a single peak appears in one of the duplicate wells, and different typing cannot be stably distinguished. The fluorescent group of the probe was replaced with Atto 425, and the probe length was shortened by 3 bases. The above experiment was repeated. The results showed that after changing the fluorescence and shortening the length, the consistency of the detection of different plasmid single templates with the same sequence in the region covered by the probe was significantly improved, the melting curve peak shape was more regular, and the melting peak Rm value was significantly improved; when detecting heterozygous templates, the difference in Tm of the two melting peaks was significantly increased, and the software can stably distinguish the two typing ( Figure 4 ).

[0090] The B-Probe I probe is used to detect the B12C1 subgroup and is fluorescently labeled with CY7. Its optimization method is similar to that of B-Probe H. Before optimization, the B-Probe I probe had certain differences in the Tm and Rm values ​​of the duplicate melting peaks when detecting different plasmid single templates (the maximum Tm difference reached 1°C); when detecting heterozygous templates, no two peaks were produced, and different types could not be distinguished. After the fluorescent group of the probe was replaced with Atto425 and shortened by 2 bases, the consistency of the duplicate Tm and Rm values ​​when detecting plasmid single templates was significantly improved, and the melting curve peak shape was more neat; when detecting heterozygous templates, two melting peaks were produced, which could clearly distinguish different types ( Figure 5 ).

[0091] The final primer and probe sequences are shown in Figure 2.

[0092] Table 2 Primer and probe sequences used for HLA CREG detection

[0093]

[0094] 3. Internal standard sequence analysis and primer probe design

[0095] The housekeeping gene GAPDH was selected as the internal standard gene for the multiplex system. First, multiple human GAPDH gene sequences were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Sequence alignment was performed using MEGA 7.0 software, and highly conserved regions were selected for multiple primer and probe design. Amplification was performed using extracted human gDNA templates. Ultimately, primers and probes with a single melting peak, a well-defined peak shape, and a relatively high cycle threshold (Ct) value were selected. The specific sequences are shown in Table 3.

[0096] Table 3 Internal standard GAPDH primer and probe sequences

[0097]

[0098] 4. Establishment and optimization of a multiplex detection system for HLA CREG based on multicolor probe melting curve analysis

[0099] (1) Establishment of multiple detection system and verification of plasmid template

[0100] The probes that met the performance requirements screened in the early stage were combined (in order to coordinate the fluorescence channels, the fluorescent group of the B-Probe L probe in the same tube as the optimized B-Probe H was replaced with CY7, and the fluorescent group of the B-Probe K in the same tube as the optimized B-Probe I was replaced with CY7) so that they were placed in as few PCR reaction tubes as possible. Finally, they were combined into three multiplex reaction systems. The first reaction system was used to detect the A1C and A2C1 groups in CREG (involving HLA-A genes), and the other two systems were used to detect the remaining groups in CREG (involving HLA-B genes). First, each multiplex system was preliminarily verified using the HLA plasmid as a template. Taking the detection of heterozygous templates as an example, it was found that the peak shape, Tm and Rm values ​​of the melting curves of each channel in the three multiplex systems were basically consistent with those of the single-plex detection, and no interference between the channels was found. This result preliminarily confirmed the feasibility of the three multiplex systems ( Figure 6 ).

[0101] (2) Sample verification and reaction system optimization of multiple detection systems

[0102] The three multiplex detection systems were further validated using samples. Human genomic DNA (gDNA) was extracted from whole blood samples using a magnetic bead-based blood genomic DNA extraction kit from Beijing Tiangen Company according to the kit instructions.

[0103] First, 19 known HLA-A and HLA-B genotyping samples were amplified using the same multiplex system used to amplify the plasmid template to preliminarily verify and optimize the multiplex system. The optimization primarily involved adjusting the dNTP concentration in the multiplex system and adding dimethyl sulfoxide (DMSO). Using system 2 as an example, it can be seen that increasing the dNTP concentration significantly improved the melting curves of each channel ( Figure 7 A). In addition, some channels of the multiplex system showed low melting curve signals, non-specific peaks, or a single peak when testing samples. After adding a certain concentration of DMSO to the multiplex system, the peak shape and peak height of the melting curve were significantly improved, the specific peak was significantly enhanced, and two melting peaks with similar Tm were well distinguished ( Figure 7 BD). The final melting curves of the three multiplex system detection samples are shown in the following example. Figure 7 E, the final three multiple systems after optimization are shown in Table 4, and the reaction procedures are the same as before.

[0104] Table 4 Multiplex reaction system for detecting HLA CREG

[0105]

[0106]

[0107]

[0108] (3) Interpretation of results

[0109] If any subgroup in each CREG group is positive, the CREG group is considered positive. Only when all subgroups in the CREG group are negative is the CREG group considered negative. Table 5 shows the CREG subgroup determination rules.

[0110] Table 5 Test result determination method

[0111]

[0112] (4) Validation based on clinical samples

[0113] In this example, 100 clinical samples that had been sequenced to identify HLA-A and HLA-B genotypes were collected. The gDNA in the samples was extracted using the magnetic bead method, and the gDNA was used as a template to perform three multiplex amplifications according to the above reaction conditions. The results showed that the Tm values ​​of the internal standards in all three systems of all samples met the positive judgment rules, and the samples were valid; the results of the CREG grouping test by multi-color probe melting curve analysis were consistent with the grouping results corresponding to sequencing, and the positive and negative sample coincidence rates were both 100% (Table 6). The results of the clinical sample test are shown in the following example. Figure 8 shown.

[0114] Table 6 The degree of consistency between the sample detection results of the method of the present invention and the sequencing results

[0115]

[0116]

[0117] Note: The frequency of B5C4-positive samples is very low, approximately 0.008% in the Chinese population, so it is not included in these 100 samples.

[0118] On the technical level, the technology of the present invention has significant advantages in detecting HLA CREG, such as rapidity, ease of operation, and high throughput. Only three PCR reaction tubes are needed to detect seven HLA CREG groups and 17 subgroups, significantly improving detection throughput. In terms of operation, the results are interpreted by combining fluorescence channels and melting curves, eliminating the need for electrophoresis, making the operation simple, effectively shortening experimental time, and improving detection efficiency.

[0119] At the application level, multiple studies have confirmed the reliability and significant advantages of CREG-based HLA epitope matching in the prevention and treatment of immune PTRs and organ transplantation. PTRs are extremely common in clinical practice, and the transfusion efficacy of CREG-based epitope-matched platelets is no different from that of HLA antigen-matched platelets. This not only improves PTRs but also greatly reduces the difficulty of finding donors. In kidney transplantation, CREG-based epitope matching not only significantly improves the matching rate between donors and recipients, but also achieves matching results comparable to antigen matching. Therefore, the present method for detecting CREG has broad clinical application in the fields of immune PTRs and kidney transplantation.

[0120] Although the present 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 modifications and variations may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims is to be given the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A method for constructing a detection system for HLA cross-reaction groups, characterized in that: The construction method comprises: (1) grouping the HLA antigens in the HLA cross-reaction group to obtain HLA cross-reaction subgroups, wherein the index used for grouping includes the SNP coordinate position of the HLA antigen; (2) Designing and synthesizing primers and specific probes based on the SNP sites in each cross-reaction subgroup after grouping; (3) Separate the primers and probes to obtain the detection system.

2. The method for constructing a detection system for HLA cross-reaction groups according to claim 1, characterized in that: Further including: (4) Steps to validate and optimize the detection system using samples with known HLA-A and HLA-B genotyping.

3. The method for constructing a detection system for HLA cross-reaction groups according to claim 2, characterized in that: The cross-reaction groups obtained after grouping in step (1) include: (a) Subgroup 1: A1C1 containing HLA antigens A01, A03, A11, A30, and A36; A1C2 containing HLA antigen A31; and A1C3 containing HLA antigen A29. (b) Subgroup II: A2C1 containing HLA antigens A02, A23, A24, A68, and A69; A2C2 containing HLA antigens B57 and B58; (c) Subgroup III: A10C including HLA antigens A25, A26, A32, A33, A34, A66, and A74; (d) Subgroup IV: includes B5C1 containing HLA antigens B35, B51, B52, B53, B58, and B78; B5C2 containing HLA antigens B15, B45, B46, B49, and B50; B5C3 containing HLA antigen B18; and B5C4 containing HLA antigen B73; (e) Subgroup V: includes B7C1 containing HLA antigens of B7, B8, B40, B41, B42, B48, B54, B55, B56, B59, and B81; B7C2 containing HLA antigens of B13, B54, B55, B56, B59, and B82; and B7C3 containing HLA antigens of B27, B40, and B47; (f) Subgroup VI: B8C1, which includes HLA antigens of B8; B8C2, which includes HLA antigens of B14, B18, B38, B39, and B67; (g) Subgroup VII: includes B12C1 containing HLA antigens B13, B40, B41, B44, B45, B47, B49, and B50; and B12C2 containing HLA antigen B37.

4. The method for constructing a detection system for HLA cross-reaction panel according to claim 1, characterized in that: In step (2), the primers include the sequences shown in SEQ ID No. 1-12.

5. The method for constructing a detection system for HLA cross-reaction panel according to claim 1, characterized in that: In step (2), in step (2), the probe includes the sequence shown in SEQ ID No. 13-30.

6. A detection system for HLA cross-reaction group, characterized in that: Obtained according to the construction method according to any one of claims 1 to 5.

7. The detection system for HLA cross-reaction panel according to claim 6, characterized in that: include: A first detection system for detecting HLA-A genes, comprising a primer set having sequences shown in SEQ ID Nos. 1-6 and a probe set having sequences shown in SEQ ID Nos. 13-18; A second detection system for detecting the HLA-B gene, comprising a primer set having sequences shown in SEQ ID Nos. 7-12 and a probe set having sequences shown in SEQ ID Nos. 19-21, 25-26, and 30; or The third detection system for detecting HLA-B gene includes a primer set with sequences shown in SEQ ID Nos. 7-10 and a probe set with sequences shown in SEQ ID Nos. 22-24 and 27-29.

8. The detection system for HLA cross-reaction panel according to claim 7, characterized in that: The first detection system includes: The second detection system includes: ;or The third detection system further comprises:

9. A detection kit for HLA cross-reaction group, characterized in that: Comprising the detection system according to claim 7 or 8; Preferably, it further comprises an internal standard primer and an internal standard probe.

10. A method for detecting HLA cross-reactive groups, characterized in that: The method comprises the steps of performing asymmetric PCR using the detection system according to claim 7 or 8; Preferably, the method further comprises the step of performing joint interpretation based on the fluorescence channel and the melting curve.

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