A method for constructing a detection system for HLA cross-reactive group and a kit thereof

Abstract

The application discloses a construction method and a kit for a detection system of an HLA cross-reactive group. The application groups the HLA cross-reactive group, designs primers and specific probes, and constructs a reasonable detection system, so that the grouping of the cross-reactive group can be directly detected instead of each typing in the grouping being detected individually, thereby greatly reducing the number of required primers and probes, improving detection efficiency, and reducing cost. With the aid of multiple fluorescence channels and melting curves, joint interpretation is realized, the grouping information of a sample can be obtained by using only three reaction systems, and the application has the advantages of rapidness, simple operation, high throughput 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] This invention relates to the field of gene detection, and more specifically to a method for constructing a detection system for HLA cross-reactivity groups and a kit. Background Technology

[0002] Human leukocyte antigens (HLA) are a highly polymorphic genetic 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 main mediators of antigen-antibody reactions and organ transplant rejection. HLA includes class I, II, and III antigens, among which HLA-I antigens (including HLA-A, HLA-B, and HLA-C) play a crucial role in mediating the body's immune response and the development of various diseases (tumors, autoimmune diseases, etc.). Although HLA exhibits high genetic polymorphism at its gene loci, making it the most complex human gene complex known to date, the amino acid residues in the protein products of HLA-I and II antigens differ only slightly. Different HLA antigens structurally share some immunogenic epitopes, enabling cross-reactions with HLA antibodies. Therefore, HLA antigens carrying common epitopes constitute the HLA cross-reactive group (CREG).

[0003] Currently, methods or reagents for directly detecting HLA CREG are scarce. Most reagents require separate detection of HLA-A and HLA-B alleles for CREG grouping, which is generally limited by high cost and long processing time. One existing CREG detection kit requires 13 reaction tubes to test one sample and necessitates electrophoresis after amplification, resulting in complex operation, long processing time, and low throughput, significantly limiting 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 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

[0005] To address at least some of the technical problems existing in the prior art, this invention establishes a multiplex detection system for HLA CREG based on multicolor probe melting curve analysis technology. This invention is an endpoint detection technique that utilizes asymmetric PCR (where the amount of forward and reverse primers differs significantly, referred to as restriction primers or excess primers) to generate a large amount of single-stranded DNA. This DNA binds to a dual-labeled self-quenching fluorescent probe, producing locally double-stranded heterozygotes. Different mutations in the target gene result in different heterozygotes, leading to different melting temperatures (Tm) during melting analysis. This allows for the simultaneous detection of multiple mutations within a single channel. The introduction of the Tm value significantly increases the number of detectable mutations within a single channel. Combining multiple channels achieves an "N×N" amplification effect, effectively increasing detection throughput. Furthermore, it eliminates the need for electrophoresis, simplifying operation. Currently, there are no related studies or registered reagents applying this method for CREG detection. Therefore, this invention establishes a multiplex detection system for HLA CREG grouping detection using only three reaction tubes based on multicolor probe melting curve analysis, offering advantages such as speed, simplicity, high throughput, and low cost. Specifically, the present invention includes the following.

[0006] A first aspect of the present invention provides a method for constructing a detection system for HLA crossreactivity groups, comprising:

[0007] (1) The HLA antigens in the HLA cross-reactive group are grouped to obtain the HLA cross-reactive subgroups. The indicators used for grouping include the SNP coordinate position of the HLA antigen.

[0008] (2) Design and synthesize primers and specific probes based on the SNP sites in each cross-reactive subgroup after grouping;

[0009] (3) The primers and probes are separated into individual tubes to obtain the detection system.

[0010] In some embodiments, the method for constructing a detection system for HLA cross-reactivity groups according to the present invention further includes: (4) a step of validating and optimizing the detection system using known HLA-A and HLA-B genotyping samples.

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

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

[0013] (b) Subgroup 2: including A2C1 containing HLA antigens A02, A23, A24, A68, and A69; and A2C2 containing HLA antigens B57 and B58;

[0014] (c) Third subgroup: including A10C containing HLA antigens of A25, A26, A32, A33, A34, A66, and A74;

[0015] (d) Subgroup 4: including B5C1 containing HLA antigens of B35, B51, B52, B53, B58, and B78; B5C2 containing HLA antigens of B15, B45, B46, B49, and B50; B5C3 containing HLA antigen of B18; and B5C4 containing HLA antigen of B73.

[0016] (e) Fifth subgroup: including 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 6: including B8C1 containing the HLA antigen B8; and B8C2 containing the HLA antigens B14, B18, B38, B39, and B67.

[0018] (g) Subgroup 7: including B12C1 containing HLA antigens of B13, B40, B41, B44, B45, B47, B49, and B50; and B12C2 containing HLA antigen of B37.

[0019] In some embodiments, according to the method for constructing a detection system for HLA cross-reactivity groups according to the present invention, in step (2), the primers include the sequences shown in SEQ ID No. 1-12; in step (2), the probes include the sequences shown in SEQ ID No. 13-30.

[0020] In some embodiments, according to the method for constructing a detection system for HLA cross-reactivity groups according to the present invention, the detection system after separation in step (3) includes:

[0021] The first detection system for detecting the HLA-A gene includes primers with the sequences shown in SEQ ID No. 1-6;

[0022] A second detection system for detecting the HLA-B gene includes primers with the sequences shown in SEQ ID No. 7-10;

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

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

[0025] In some embodiments, according to the detection system for HLA crossreactivity according to the present invention, the first detection system further includes probes of the 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 and 27-29;

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

[0028] A third aspect of the present invention provides a detection kit for HLA cross-reactivity groups, comprising the detection system described in the second aspect;

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

[0030] A fourth aspect of the present invention provides a method for detecting HLA cross-reactivity, comprising the step of performing asymmetric PCR using the detection system described in the present invention;

[0031] Preferably, the method further includes a step of jointly interpreting the fluorescence channel and the melting curve.

[0032] A fifth aspect of the invention provides the use of reagents in the preparation of an HLA cross-reactivity assay kit, wherein the reagents comprise the assay system described in the second aspect.

[0033] This invention provides a detection system and kit containing HLA cross-reactivity groups based on multicolor probe melting curve analysis. The detection system or kit includes the required primers and a dual-labeled self-quenching probe combination. This invention identifies common sites by analyzing HLA-A or HLA-B gene sequences within the same CREG group, and designs primers and specific probes accordingly. This allows for direct detection of the CREG group rather than individual genotypes within the group, significantly reducing the number of required primers and probes, improving detection efficiency, and lowering costs. By utilizing multiple fluorescence channels and melting curves for joint interpretation, CREG grouping information can be obtained from a sample using only three reaction systems. This method offers advantages such as speed, ease of operation, and high throughput, and has broad application prospects and clinical value in fields such as clinical transfusion medicine and organ transplantation. Attached Figure Description

[0034] Figure 1 The melting curve of the hybrid template was obtained by probe detection of the single-component system, which demonstrated that the probe performance met the requirements.

[0035] Figure 2 Results of melting curves before and after A-Probe 2 optimization.

[0036] Figure 3 Results of melting curves before and after A-Probe 3 probe optimization.

[0037] Figure 4 Results of melting curves before and after optimization of the B-Probe H probe.

[0038] Figure 5 Results of melting curves before and after B-Probe I probe optimization.

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

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

[0041] Figure 8 Examples of clinical sample test results and sequencing results. Detailed Implementation

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Currently, most methods first genotype HLA-A and HLA-B alleles before confirming their CREG grouping. HLA genotyping methods include a series 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 sites, respectively. These two techniques have good accuracy, but low throughput and complex operation, limiting their clinical application. PCR-SSP and PCR-SSO techniques use specific primers or oligonucleotide probes for typing, significantly improving detection sensitivity and specificity compared to the previous two methods, but still require electrophoresis or hybridization steps, making them less convenient to operate. PCR-SBT involves PCR amplification of DNA fragments followed by sequencing, providing accurate results and identifying novel alleles. However, the equipment is expensive, and the testing cycle is long. NGS utilizes high-throughput sequencing technology to sequence the entire HLA genome or specific regions, performing HLA genotyping by alignment with HLA reference sequences. It offers advantages such as high throughput and high resolution. However, it generates massive amounts of data, involves complex data processing and analysis, and incurs high equipment maintenance and reagent costs.

[0046] Most existing technologies cannot directly detect HLA CREGs. They require separate genotyping of HLA-A and HLA-B before confirming the CREG grouping. This two-step process not only increases complexity but, more importantly, introduces the high difficulty and cost of accurate HLA-A and HLA-B genotyping. Existing methods for direct CREG detection are based on PCR-SSP technology, which requires numerous reaction tubes, has low throughput, complex formulation systems, and necessitates electrophoresis identification after amplification, increasing workload, detection time, and the possibility of human error, hindering large-scale clinical application. This invention adopts a strategy of directly detecting HLA CREGs at the design level. By analyzing different HLA gene sequences within the same CREG group, common sites are identified, and primers and specific probes are designed accordingly. This allows a single probe to simultaneously detect multiple HLA genotypes, significantly reducing the number of probes required and avoiding the complexity introduced by separate genotyping of each HLA gene, thereby effectively improving detection efficiency and reducing costs. At the technical level, based on multicolor probe melting curve analysis, it is possible to directly detect 7 CREG groups with only 3 reaction tubes. The results can be interpreted by combining the fluorescence channel and melting curve Tm value, without the need for electrophoresis identification. It has significant advantages such as simple operation, high throughput and short time consumption, and has good clinical application value.

[0047] Construction method

[0048] This invention first provides a method for constructing a detection system for HLA cross-reactivity groups, comprising:

[0049] (1) The HLA antigens in the HLA cross-reactive group are grouped to obtain the HLA cross-reactive subgroups. The indicators used for grouping include the SNP coordinate position of the HLA antigen.

[0050] (2) Design and synthesize primers and specific probes based on the SNP sites in each cross-reactive subgroup after grouping;

[0051] (3) The primers and probes are separated into individual tubes to obtain the detection system.

[0052] In step (1), grouping HLA antigens according to the SNP coordinates includes analyzing different HLA gene sequences located within the same CREG group, and dividing HLA antigens with common sites into the same subgroup. Specifically, if the physical locations of adjacent SNPs do not exceed 5 bp, HLA antigens containing the above-mentioned SNP sites are divided into the same subgroup.

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

[0054] In step (3), the designed primers and probes are divided into tubes to obtain the detection system. The tube division takes into account multiple factors such as the signal intensity of the fluorescent label, melting curve, primer position, and amplification product size, so as to realize the accurate detection of 7 CREG groups and 17 subgroups directly by 3 reaction tubes.

[0055] Detection system or reaction solution

[0056] After developing specific primers and probes, this invention, through in-depth research, discovered that, on the one hand, singleton asymmetric PCR systems are not directly suitable for multiplex detection; on the other hand, detection primers and probes require grouping and further optimization to achieve accurate detection of HLA cross-reactivity groups. Regarding probes, this invention found that for certain subgroups (e.g., B8C2 and B12C1 subgroups), shortening the probe length can actually enhance their resolution. Furthermore, this invention optimized different fluorescent labels and their placement (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 optimized the detection method applicable to the present invention, including the concentration of each added component (e.g., dNTP, Mg). 2+ The concentrations of the forward and reverse primers, probes, DMSO concentration, etc., can be controlled within a reasonable range to achieve the detection of HLA cross-reactive groups.

[0058] In a preferred embodiment, the detection system includes or comprises 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 this invention, the final concentration of MgCl2 in the reaction system is 0.1-10 mM, preferably 1-5 mM, even more preferably 2-4 mM, and even more preferably 2.5-3.5 mM.

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

[0068] In this 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 this invention, the final concentration of the probe in the reaction system is 0.01-10 μM, preferably 0.01-5 μM, and even more preferably 0.05-5 μM.

[0070] In this invention, the concentration of the reverse primer is greater than the concentration 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, even 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, even more preferably 0.2-0.8 μM, and further preferably 0.4-0.6 μM.

[0071] In this invention, the melting curve analysis reaction solution can be a commercially available reaction solution for melting curve analysis, such as the GNM multiplex asymmetric amplification and multicolor melting curve PCR reaction solution with catalog number RXD03GS.

[0072] 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.

[0073] Methods for detecting HLA crossreactivity

[0074] This invention provides a method for detecting HLA cross-reactivity groups, sometimes referred to herein simply as "the 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 application.

[0075] The detection method of this invention does not particularly limit the specific process or steps, as long as the detection system or kit described in this invention is used. In an exemplary method, the method of this 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 instrument known in the art.

[0076] 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.

[0077] Example

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

[0079] HLA-A and HLA-B sequence information was collected from real-time HLA databases (http: / / ftp.ebi.ac.uk / pub / databases / ipd / imgt / hla / and http: / / hla.alleles.org / nomenclature / index.html). Sequences of 7712 HLA-A genes and 9640 HLA-B genes were collected, and 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 from each low-resolution genotype of HLA-A and HLA-B genes for primer and probe design. After design, these sequences were aligned with all subtype sequences in the corresponding low-resolution genotype to ensure that at least 99% of the sequences in that genotype could be detected.

[0080] The primer and probe design is as follows: Within each CREG group, specific probes are designed by selecting SNP sites that cover as many subtypes as possible, rather than designing probes for each subtype individually. This design approach significantly reduces the number of probes required, reduces system complexity, and effectively improves detection efficiency. For example, in the A1C group, a single probe can detect all subtypes except A31 and A29, which is named the A1C1 subgroup; similarly, detection in the A2C1, A10C, B7C1, B8C2, B12C1, and A10C groups can even be achieved with just one probe. Furthermore, besides using fewer probes to detect more subtypes, the primer design, by covering multiple probe binding sites in a single amplification product, greatly reduces the number of primer pairs required, further reducing system complexity. Only 6 primer pairs are needed to detect the 17 CREG subgroups. The HLA CREG grouping detection design and required primers and probes are shown in Tables 1 and 2.

[0081] Table 1. HLA CREG detection grouping design

[0082]

[0083]

[0084] *SNP sites are color-coded. Group A1C2 is based on the detection of two SNPs that are far apart; Group B7C1 uses three SNP sites that are very close together (one probe can cover them). The HLA antigens detected by SNP sites of different colors are marked with the corresponding colors.

[0085] 2. Initial screening of probes with satisfactory performance using singleton asymmetric PCR and probe optimization.

[0086] Melting curve analysis of the reaction solution was performed using the Beijing Jin Nuomei Company. Pre-designed primers and probes for detecting different CREG groups / subgroups were added to the reaction system. Singleton asymmetric PCR was performed using previously constructed HLA plasmid standards as templates to determine the Tm value of the melting curve for each probe detecting different templates. In addition, different plasmid templates with similar Tm values ​​were mixed in equal volumes (hybrid templates) to simulate HLA alleles in real samples. The reaction program was: 90-98℃ for 1-5 min, 90-98℃ for 5-30 s, 55-65℃ for 0.5-3 min, 45-55 cycles. Denaturation was performed at 90-98℃ for 0.5-3 min, hybridization at 40-55℃ for 1-10 min, and the temperature was gradually increased from 45-90℃. The results showed that, among the 18 designed probes, except for A-Probe 2, A-Probe 3, B-Probe H, and B-Probe I, the remaining probes exhibited good peak shapes and distinguishable Tm values ​​in the melting curves of the hybrid template, and could be used in the subsequent establishment of multiplex systems. Figure 1 ).

[0087] Further optimizations were made to the four probes, including A-Probe 2, that exhibited poor melting curve peak shapes or were difficult to distinguish when detecting heterozygous templates. Firstly, when A-Probe 2 probe amplified single templates from different plasmids, the melting curve peaks showed uneven baselines with a clear upward trend; the Rm values ​​of the melting peaks were generally low, and the differences in Rm between replicates were also significant; when detecting heterozygous templates, the Rm of the melting peaks further decreased, and in one replicate, the low signal even prevented the identification of two genotypes (…). Figure 2(Left). 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 on the sequence, the modification was performed on the backbone (the backbone can only label CY5). After the intermediate modification, the melting curves obtained from single templates of different plasmids showed more uniform peak shapes, flatter baselines, significantly increased melting peak heights, and better consistency in duplicate wells. When detecting hybrid templates, the difference in Tm values ​​between the two melting peaks was significantly increased compared to before, and Rm was also higher, which is more conducive to software interpretation and differentiation of different subtypes. Figure 2 right).

[0088] The A-Probe 3 probe was used to detect the A1C3 subgroup. When amplifying single templates with different plasmids, the melting curves showed significant dips, and apart from the perfectly matched A*29 plasmid, several other HLA plasmids did not show obvious melting peaks. When detecting heterozygous templates, only the A*29 plasmid showed a melting peak, making it impossible to distinguish between different genotypes. Further sequence analysis revealed that the GC content in the probe's binding region was higher than 80%, leaving little room for adjustment within the same region. Therefore, other sites with relatively low GC content were replaced, and the probe was redesigned. After optimization, both A*29 and other plasmids produced melting peaks. When detecting heterozygous templates, the difference in Tm between the two melting peaks was 5.37℃, which the software could interpret, allowing for the differentiation of the two genotypes (the peak heights were not completely identical, which was related to the incomplete equivalence of the two plasmid concentrations). Figure 3 ).

[0089] The B-Probe H probe, labeled with CY7, was used to detect the B8C2 subgroup. When amplifying single templates from different plasmids with the same probe coverage region, the heterogeneity of melting curves (Tm and Rm) was significant, with large differences in Tm and Rm between replicates, resulting in disordered peak shapes and the appearance of some non-specific peaks at the front. When detecting hybrid templates, a single peak appeared in one replicate, making it impossible to reliably distinguish between different genotypes. Replacing the probe's fluorophore with Atto 425 and shortening the probe length by three bases, and repeating the experiment, showed that after changing the fluorophore and shortening the length, the consistency in detecting single templates from different plasmids with the same probe coverage region was significantly improved, the melting curve peak shape was more regular, and the Rm value of the melting peak was significantly increased. When detecting hybrid templates, the difference in Tm between the two melting peaks significantly increased, and the software could reliably distinguish between the two genotypes. Figure 4 ).

[0090] The B-Probe I probe, labeled with CY7, was used to detect the B12C1 subgroup. Its optimization method was similar to that of B-Probe H. Before optimization, the Tm and Rm values ​​of the polymorphic melting peaks in the B-Probe I probe varied significantly when detecting single plasmid templates (the Tm difference reached up to 1℃); when detecting hybrid templates, no two peaks were produced, making it impossible to distinguish different genotypes. After replacing the fluorophore of the probe with Atto425 and shortening it by two bases, the consistency of Tm and Rm values ​​in the polymorphic melting curves was significantly improved when detecting single plasmid templates, and the peak shape of the melting curves became more uniform; when detecting hybrid templates, two melting peaks were produced, clearly distinguishing different genotypes. 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 control gene in 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 primer and probe design. Amplification was performed using extracted human gDNA templates. Primers and probes with a single melting peak with good peak shape and a relatively high cycle threshold (Ct value) were ultimately selected. 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 validation of plasmid template

[0100] Probes that met the performance requirements in the initial screening were combined (to coordinate the fluorescence channels, the fluorescent group of B-Probe L, which was in the same tube as the optimized B-Probe H, was replaced with CY7, and the fluorescent group of B-Probe K, which was in the same tube as the optimized B-Probe I, was replaced with CY7), placing them in as few PCR reaction tubes as possible, ultimately forming three multiplex reaction systems. The first reaction system was used to detect the A1C and A2C1 groups in CREG (involving the HLA-A gene), and the other two systems were used to detect the remaining groups in CREG (involving the HLA-B gene). Initial validation of each multiplex system was performed using HLA plasmids as templates. Taking the detection of heterozygous templates as an example, the peak shape, Tm, and Rm values ​​of the melting curves of each channel in the three multiplex systems were found to be basically consistent with those in single-channel detection, and no interference between channels was observed. These results preliminarily confirm the feasibility of the three multiplex systems. Figure 6 ).

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

[0102] Further validation of the three multiplex detection systems was conducted using samples. Human genomic DNA (gDNA) was extracted from whole blood samples using the magnetic bead-based blood genomic DNA extraction kit from Beijing Tiangen Pharmaceuticals Co., Ltd., following the kit's instructions.

[0103] First, 19 known HLA-A and HLA-B genotyping samples were amplified using a multiplex system identical to the amplified plasmid template to preliminarily validate and optimize the multiplex system. Optimization primarily involved adjusting the dNTP concentration and adding dimethyl sulfoxide (DMSO). Taking 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). Furthermore, some channels in the multiplex system exhibited low melting curve signals, non-specific peaks, or only single peaks instead of double peaks when detecting samples. Adding a certain concentration of DMSO to the multiplex system significantly improved the peak shape and height of the melting curves, significantly enhanced the specific peaks, and effectively distinguished two melting peaks with similar Tm values. Figure 7 (BD). Examples of melting curves for the final three multi-system tests are shown below. Figure 7 As shown in E, the final three multi-systems after optimization are shown in Table 4, and the reaction procedure is the same as before.

[0104] Table 4. Multiple reaction system for detecting HLA CREG

[0105]

[0106]

[0107]

[0108] (3) Result Interpretation

[0109] If any subgroup within a CREG group is determined to be positive, the CREG group is considered positive; a CREG group is considered negative only if all subgroups within it are determined to be negative. Table 5 shows the rules for determining CREG subgroups.

[0110] Table 5. Methods for Determining Test Results

[0111]

[0112] (4) Validation based on clinical samples

[0113] This embodiment collected 100 clinical samples with confirmed HLA-A and HLA-B genotypes through sequencing. gDNA was extracted from the samples using a magnetic bead method. Using the gDNA as a template, amplification was performed in three multiplex systems under the aforementioned reaction conditions. Results showed that the Tm values ​​of the internal standard in all three systems met the positive criteria for all samples, indicating valid samples. The CREG grouping results analyzed by multicolor probe melting curves were consistent with the sequencing results, with a 100% concordance rate for both positive and negative samples (Table 6). Examples of clinical sample testing results are shown below. Figure 8 As shown.

[0114] Table 6. Degree of agreement between the sample detection results and sequencing results of the method of the present invention.

[0115]

[0116]

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

[0118] At the technical level, the technology of this invention has significant advantages in detecting HLA CREG, namely speed, ease of operation, and high throughput. Only three PCR reaction tubes are needed to detect 7 HLA CREG groups and 17 subgroups, significantly improving the detection throughput. In terms of operation, the results are interpreted by combining the fluorescence channel and melting curve, eliminating the need for electrophoresis, simplifying the operation, and effectively shortening the experimental time and improving the detection efficiency.

[0119] At the application level, multiple studies have confirmed the reliability and significant advantages of CREG-based HLA epitope typing in the prevention and treatment of immune PTR and organ transplantation. PTR is extremely common in clinical practice. The transfusion effect of platelets based on CREG epitope matching is not significantly different from that of platelets matched with HLA antigens. It can not only improve PTR, but also greatly reduce the difficulty of finding donors. In kidney transplantation, CREG-based epitope typing can not only significantly improve the matching rate between donor and recipient, but also the matching effect is comparable to that of antigen matching. Therefore, the CREG detection method of this invention has wide clinical applications in the fields of immune PTR and kidney transplantation.

[0120] 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 detection system for HLA cross-reactive groups, characterized in that, The cross-reactive group includes: (a) Subgroup 1: including A1C1 containing HLA antigens A01, A03, A11, A30, and A36; A1C2 containing HLA antigen A31; and A1C3 containing HLA antigen A29. (b) Second subgroup: including A2C1 containing HLA antigens A02, A23, A24, A68, and A69; and A2C2 containing HLA antigens B57 and B58; (c) Third subgroup: including A10C containing HLA antigens of A25, A26, A32, A33, A34, A66, and A74; (d) Subgroup 4: including B5C1 containing HLA antigens of B35, B51, B52, B53, B58, and B78; B5C2 containing HLA antigens of B15, B45, B46, B49, and B50; B5C3 containing HLA antigen of B18; and B5C4 containing HLA antigen of B73. (e) Subgroup 5: including 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) The sixth subgroup includes B8C1 containing the HLA antigen B8; and B8C2 containing the HLA antigens B14, B18, B38, B39, and B67. (g) Subgroup 7: including B12C1 containing HLA antigens of B13, B40, B41, B44, B45, B47, B49, and B50; and B12C2 containing HLA antigen of B37; The detection system for detecting the cross-reactive group includes: The first detection system for detecting the HLA-A gene includes a primer set with the sequences shown in SEQ ID No. 1-6 and a probe set with the sequences shown in SEQ ID No. 13-18; The second detection system for detecting the HLA-B gene includes a primer set with the sequence shown in SEQ ID No. 7-12 and a probe set with the sequence shown in SEQ ID No. 19-21, 25-26, and 30; The third detection system for detecting the HLA-B gene includes a primer set with the sequences shown in SEQ ID No. 7-10 and a probe set with the sequences shown in SEQ ID No. 22-24 and 27-29.

2. The detection system for HLA cross-reactive groups according to claim 1, characterized in that, The first detection system includes: The second detection system includes: The third detection system includes:

3. A test kit for HLA cross-reactive groups, characterized in that, Includes the detection system described in claim 1 or 2.

4. The test kit for HLA cross-reactive groups according to claim 3, characterized by, It further includes internal standard primers and internal standard probes.

5. The test kit for HLA cross-reactive groups according to claim 4, characterized in that, The sequences of the internal standard primers are shown in SEQ ID No. 31 and 32.

6. The test kit for HLA cross-reactive group according to claim 4, characterized by, The sequence of the internal standard probe is shown in SEQ ID No.

33.

7. The test kit for HLA cross-reactive group according to claim 3, characterized by, The kit may further include instructions for performing the detection method and interpretation rules.

8. The test kit for HLA cross-reactive groups according to claim 7, characterized by, The detection method comprises a step of asymmetric PCR using the detection system according to claim 1 or 2.

9. The test kit for HLA cross-reactive groups according to claim 8, characterized in that, The detection method further comprises a step of combined interpretation according to fluorescence channel and melting curve.

10. Use of reagents for the preparation of a HLA cross-reactive group detection kit, characterized in that, The reagent comprises the detection system according to claim 1 or 2.

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