Kit and method for HLA cross reaction group and HPA genotyping

Abstract

The invention discloses a kit and a method for HLA cross reaction group and HPA genotyping. The system for HLA cross reaction group and HPA genotyping detection is obtained through respective combination design of two different primers on the basis of common specific SNP loci, and rapid, simple, low-cost and high-throughput HLA epitope and HPA genotyping detection can be realized, so that platelet infusion ineffectiveness and immune platelet infusion ineffectiveness are more effectively prevented.

Description

Technical Field

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

[0002] Currently, there are 35 HPA antigens, designated HPA-1 to HPA-35 according to the time of their discovery, and they are genetically characterized as codominant and biallelic. HLA is a non-specific platelet antigen present on the platelet surface that is shared with other cells or tissues. HLA-I antigens are the predominant HLA antigens expressed on the platelet surface. Among HLA-I antigens, transfusions of platelets with matched HLA-A and HLA-B genotypes can significantly improve platelet transfusion refractoriness. However, due to the high polymorphism of HLA, the probability of finding a fully matched HLA allele for a patient is only 1 in 100,000. Therefore, establishing a large platelet donor pool is difficult both financially and in terms of donor availability. Studies have shown that recipients develop anti-donor HLA antibodies only against certain epitopes of their HLA antigens, rather than against the entire donor's HLA antigens. Further research has confirmed that matching the HLA epitopes between the donor and recipient can prevent the development of donor-specific antibodies. Clinical studies have shown that for patients who have been sensitized by HLA alloimmunization and those who are refractory to platelet transfusions, transfusions of HLA epitope-matched platelets based on HLA antigen cross-reactive groups (CREGs) are as effective as transfusions of HLA antigen-matched platelets, providing direct evidence that HLA epitope matching can replace HLA antigen-matched transfusions. Therefore, targeted testing of the HPA gene and HLA-AB epitopes in donors and recipients, and transfusion of compatible platelets with HPA gene and HLA epitope matching, are of great clinical significance.

[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 chip method, sequencing-based typing (SBT) and next-generation sequencing (NGS).

[0004] PCR-SSP is a typing method based on PCR technology. It uses the highly specific amplification effect of PCR and DNA sequence-specific hybridization to type the target DNA. However, PCR-SSP must be analyzed by gel electrophoresis, which is complicated to operate and prone to laboratory contamination.

[0005] The principle of PCR-SSO is to artificially synthesize complementary oligonucleotides, or SSO probes, based on the base sequences of various HLA alleles. These oligonucleotides are then hybridized with the HLA in the genomic DNA of the test specimen, and the HLA type of the test specimen is determined by observing whether hybridization occurs. Although this method is suitable for typing large quantities of samples, its disadvantages are that it requires high equipment and a complex operation process, making it difficult to complete the typing of all HLA alleles in a short period of time.

[0006] PCR-RFLP typing is far more accurate than serological methods, but it involves numerous steps and can be complex to interpret, especially in heterozygous cases. Furthermore, it requires digestion with multiple restriction endonucleases, which is time-consuming and expensive.

[0007] The DNA chip method is based on the technical design of anchored oligonucleotides. A series of oligonucleotide probes are introduced on the chip. The probes can bind to highly specific DNA sequences of different variant types on the HLA / HPA gene. When the probes successfully hybridize with the target DNA, the probes at specific positions on the chip will display a fluorescent signal, indicating that the DNA in the sample is a perfect match with this probe. However, this method is technically complex, expensive, and requires professional experimental techniques and data analysis capabilities, making it difficult to be widely used in clinical practice.

[0008] Sanger sequencing and NGS are currently the gold standards for clinical application. They use PCR to amplify HLA / HPA gene-specific sequences, and then perform Sanger sequencing on the amplified products, or extract the sample's whole-genome DNA and sequence and type the HLA genes using high-throughput sequencing technology. However, Sanger sequencing throughput is relatively low, and a single experiment can only sequence dozens to hundreds of target sequences. In addition, the target sequences need to be cloned individually, and multiple HLA alleles cannot be identified simultaneously. NGS requires a high level of operational technology, a large amount of data, special instruments and equipment, and professional data analysis technology. 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 One Lambda's HLA typing kit based on the PCR-SSP and PCR-SSO principles, and Debi's HLA nucleic acid typing kit based on the SBT principle. One Lambda's kit requires specialized testing equipment, high technical skills, and professional data analysis techniques, making it expensive and difficult to widely use in clinical practice. Debi'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 HLA testing in clinical practice.

[0010] In addition, existing literature also discloses technologies 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 fluorescent PCR melting curve method. The kit contains 262 primers for amplification in a 96-well optical reaction plate. The double-stranded DNA produced by PCR amplification binds to SYBR Green I, and the different melting curves generated by different products are used to comprehensively determine the low-resolution genotyping of HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1. However, the use of SYBR Green I to bind to the double-stranded DNA products is non-specific, resulting in poor specificity and accuracy. Multiple reactions are difficult to perform in a single-well PCR tube, resulting in extremely low detection throughput.

[0011] Chinese patent document CN101845520A uses specific MGB probes to type the HPA1-5, 15 alleles. The allele genotype is determined based on the distribution of two different fluorescence curves and scatter plots after amplification with common primers. The MGB probes used in this method are expensive and costly, making it unsuitable for widespread application. In addition, only one allele is detected per well, resulting in low efficiency.

[0012] 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

[0013] In order to solve at least some of the technical problems existing in the prior art, the present invention groups HLA genes based on SNP sites according to the HPA / HLA gene sequences published in the IPD-IMGT / HLA and Versiti databases, and obtains multiple HLA subgroups. At the same time, the present invention also groups HLA genes based on SNP sites, especially HPA genes with a high low frequency in the population, i.e., HPA Specific primers and probes are designed for the a and b subtypes of genes 1-6, 9, 12, 13, 15, 21, 27, 30, and 31. The probes are labeled with fluorescent dyes such as FAM / VIC / ROX / CY7 / ATTO425 for detecting HLA and HPA-specific targets. Simultaneously, a pair of internal standard primers and probes, labeled with CY5, are added to each well to monitor for false-negative results caused by instrument failure, reagent factors, polymerase activity, or inhibitors in the sample. A negative control well is also provided to eliminate false-positive results caused by sample contamination. This method enables rapid, simple, low-cost, and high-throughput HLA epitope and HPA genotyping detection. This method can be widely applied to platelet bank establishment and the detection and treatment of patients who fail platelet transfusions. Specifically, the present invention includes the following.

[0014] In a first aspect, the present invention provides a method for constructing a detection system for HLA cross-reactive panels and HPA genes, comprising the following steps:

[0015] (1) analyzing the epitope gene sequence of each HLA gene in the cross-reactive group, screening for shared specific SNP sites, and grouping the HLA genes according to the shared specific SNP sites to obtain multiple HLA subgroups, wherein at least some of the HLA subgroups contain multiple HLA genes;

[0016] (2) Analyze the gene sequence of each HPA allele and screen for specific SNP sites of low-frequency and high-frequency HPA genes;

[0017] (3) designing primers and probes for the common specific SNP site and the frequency-specific SNP site, respectively, wherein when designing primers, a mutation retardation amplification system is preferably used to design primers for a single specific SNP site, and a U-shaped anchor primer is preferably used when a linked mutation base exists near the specific SNP site, wherein the probes are labeled with different fluorescent groups; and

[0018] (4) Assign responsibilities based on the type of fluorescent group in the probe and the position of the primer.

[0019] In certain embodiments, according to the method for constructing a detection system for an HLA cross-reactive panel and HPA genes according to the first aspect of the present invention, the HLA genes include HLA-A and HLA-B.

[0020] In certain embodiments, according to the method for constructing a detection system for HLA cross-reaction groups and HPA genes according to the first aspect of the present invention, the multiple HLA subgroups obtained after grouping in step (1) include:

[0021] A1C1 subgroup containing A01, A11, A36, and A80;

[0022] A1C2 subgroup containing A03 and A30;

[0023] A1C3 subgroup containing A29;

[0024] A1C4 subgroup containing A30 and A31;

[0025] A2C-A subgroup containing A02, A23, A24, A68, and A69;

[0026] A10C1 subgroup containing A32 and A74;

[0027] A10C2 subgroup containing A25, A26, A34, A66, and A43;

[0028] A10C3 subgroup containing A33;

[0029] B2C subgroup containing B57 and B58;

[0030] the B5C1 subgroup containing B35, B51, B52, B53, B58, and B78;

[0031] the B5C2 subgroup containing B15 and B46;

[0032] B5C3 subgroup containing B18;

[0033] B5C4 subgroup containing B49;

[0034] the B5C5 subgroup containing B50;

[0035] the B5C7 subgroup containing B73;

[0036] the B7C1 subgroup containing B7, B42, B54, B55, B56, B67, B81, and B82;

[0037] the B7C2 subgroup containing B54, B55, B56, and B59;

[0038] the B7C3 subgroup containing B8, B41, and B42;

[0039] the B7C4 subgroup, which contains B7, B13, B27, B40, B47, B48, and B81;

[0040] the B8C1 subgroup, which contains B8, B14, B38, B39, and B67;

[0041] B12C1 subgroup containing B13, B40, B41, B44, B45, B47, B49, B50; and

[0042] The B12C2 subgroup contains B37.

[0043] In certain embodiments, according to the method for constructing a detection system for HLA cross-reactive panels and HPA genes according to the first aspect of the present invention, when designing primers using a mutation retardation amplification system, introducing mismatched bases at the 3' end of the primers comprises:

[0044] a. First, a single-base mismatch is introduced, and the specificity of the amplified product is determined based on the melting curve of real-time fluorescent PCR, i.e., whether a specific peak exists;

[0045] b. When the melting curve shows no specific peak, further introduce additional base mismatches and again determine whether a specific peak exists based on the melting curve of real-time fluorescent PCR;

[0046] Optionally, the method further comprises repeating step b until a specific peak is obtained.

[0047] In certain embodiments, according to the method for constructing a detection system for the HLA cross-reactive panel and the HPA gene according to the first aspect of the present invention, the U-shaped anchor primer comprises a 5'-end binding region, a 3'-end binding region, and a vacuolar structure region located therebetween, and designing the U-shaped anchor primer comprises the following steps:

[0048] a. Adjust the number of binding bases at the 3' end of the primer and the vacuolar structure of the linker, and select a primer with good specificity as the initial primer based on the results of the SYBR dye method;

[0049] b. further optimizing the number of bases in the linker of the preliminary primer to obtain an optimized primer;

[0050] c. Adjust the length of the 5' binding region of the U-shaped anchor primer according to the Taqman probe method and real-time fluorescence PCR method.

[0051] The second aspect of the present invention provides a detection system for HLA cross-reaction groups and HPA genes, which is obtained by the construction method described in the first aspect of the present invention.

[0052] In certain embodiments, the detection system for the HLA cross-reactive panel and HPA gene according to the second aspect of the present invention comprises primers having sequences as shown in SEQ ID Nos. 1-100; and probes having sequences as shown in SEQ ID Nos. 101-150, wherein the probes further comprise a fluorescent group, and the detection system comprises a plurality of independent subsystems, each of which comprises primers and probes for a specific SNP site. Information on the primers and probes in the subsystems is shown in Table 3.

[0053] In a third aspect, the present invention provides a detection kit for the HLA cross-reaction panel and the HPA gene, comprising reagents and apparatus for constructing the detection system described in the second aspect of the present invention, wherein the apparatus comprises a space capable of accommodating multiple independent subsystems; optionally, the apparatus further comprises internal standard primers and internal standard probes.

[0054] A fourth aspect of the present invention provides a method for typing or detecting HLA cross-reactive panels and HPA genes, comprising the steps of performing multiplex PCR using the detection system described in the second aspect; and

[0055] The steps of interpretation based on different colors of fluorescent signals.

[0056] The detection system of the present invention can effectively identify HLA epitopes and HPA antigen-specific SNP sites, and is combined with TaqMan probes to establish a rapid, simple, low-cost and high-throughput multiplex real-time fluorescence PCR detection method, thereby providing a convenient and effective detection method for preventing platelet transfusion ineffectiveness and treating patients with immune platelet transfusion ineffectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] 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 specific detection sites in CREG grouping.

[0058] Figure 2 Schematic diagram of the specific locations of HLA-B-related specific detection sites in the CREG grouping.

[0059] Figure 3 Examples of ARMS and UAP primer optimization are shown. The figure shows the primer optimization process for the 1C1 target sequence within the HLA-A locus. The blue curve represents the melting curve when using the SYBR real-time PCR method to screen primers; the orange curve represents the amplification curve when using the selected specific primers with the TaqMan real-time PCR method.

[0060] Figure 4 Negative quality control system detection.

[0061] Figure 5 Schematic diagram of amplification curves for HLA-A and B epitope and HPA allele typing in clinical samples. AL: Corresponds to test results for tubes 1-12, respectively. DETAILED DESCRIPTION

[0062] 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.

[0063] 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.

[0064] 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.

[0065] As used herein, the term "shared specific SNP site" refers to a SNP site that is shared among multiple HLA genes in a specific HLA gene group, particularly within a cross-reacting group (CREG), but not present in any HLA gene within other cross-reacting groups (CREGs). A shared specific SNP site is typically a single base, but sometimes also includes a combination of two or more bases. The distance between two or more bases is not limited, and 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 two or more bases are adjacent.

[0066] Herein, the term "binding region" refers to a partial region within an oligonucleotide that can specifically bind to a portion of a continuous sequence of a target gene, and is a sequence consisting of a plurality of continuous nucleotides.

[0067] As used herein, the term "specific binding," also referred to as "specific hybridization," includes the following meanings: (1) there is only one target gene or target DNA region to which the oligonucleotide hybridizes in a given template, and each base in the oligonucleotide pairs with the corresponding base in the target gene or target DNA. That is, the oligonucleotide and the target DNA are completely matched; and (2) under conditions suitable for a PCR reaction, only one detectable fragment of 25-350 bp in length is obtained after hybridization of the first oligonucleotide and the second oligonucleotide to the same template.

[0068] In this article, the term "high-frequency HPA gene" or "type a HPA" refers to a SNP locus of HPA with a frequency greater than 50% in a specific population. Similarly, "low-frequency HPA gene" or "type b HPA" refers to a SNP locus of HPA with a frequency less than 50% in a specific population. Among them, the low-frequency genes of HPA-1, 2, 3, 4, 5, 6, 9, 12, 13, 15, 21, 27, 30, and 31 have a frequency higher than

[0069] 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.

[0070] In the present invention, the reaction system includes: 10-50mM (preferably 20-40mM) Tris, pH 8.0-9.0), 50-500mM (preferably 100-200mM) KCl, 1-50mM (preferably 5-20mM) MgCl2, 0.01-10mM (preferably 0.1-2mM) dNTP, 0.01-10mM (preferably 0.1-2mM) 50% trehalose, and 1-10U / Test (preferably 1-5U / Test) Taq hot start enzyme.

[0071] Primers and probes for HLA-A and HLA-B epitopes, HPA alleles, and random sequences are placed in a 12-tube PCR system along with the above-described system and the internal reference gene HLA-B primer and probe according to Table 9. The final concentration of each primer in each well is 50-500 nM, preferably 100-200 mM, and the final concentration of the probe is 50-500 nM, preferably 50-150 mM.

[0072] 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; the fifth reaction system includes primers shown in SEQ ID No. The primers shown in SEQ ID Nos. 23-26, 43-44, 91-92 and the probes shown in SEQ ID Nos. 112-113, 122 and 127 were used as the reaction system; the sixth reaction system included the primers shown in SEQ ID Nos. 17-18, 33-34, 39-40, 77-78, 82-83 and the probes shown in SEQ ID Nos. 109-110, 120, 139 and 141; the seventh reaction system included the primers shown in SEQ ID Nos. 29-30, 35-38, 79-81 and 85 and the probes shown in SEQ ID Nos. 115, 118-119, 139 and 141; the eighth reaction system included the primers shown in SEQ ID Nos. 45-46, 57-58, 65-66 and 71-74 and the probes shown in SEQ ID Nos. 123, 129, 133, 135 and 137; the ninth reaction system included the primers shown in SEQ ID Nos. The primers shown in No.47-48, 59-60, 67-70, 75-76 and the probes shown in SEQ ID No.123, 129, 133, 135 and 137 were used; the tenth reaction system included the primers shown in SEQ ID No.49-50, 61-62, 85-86, 93-94, 97-98 and the probes shown in SEQ ID No.125, 131, 143, 147 and 149; the eleventh reaction system included the primers shown in SEQ ID No.52-53, 63-64, 87-88, 95-96 and 99-100 and the probes shown in SEQ ID No.125, 131, 143, 147 and 149; the twelfth reaction system included a negative control.

[0073] In the present invention, the above system contains, in addition to the above-mentioned probes and primers, further comprising an internal reference primer and probe, wherein the internal reference primer and probe have the sequences shown in SEQ ID No. 151, 152 and 153, respectively.

[0074] 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.

[0075] The present invention provides a method for typing or detecting HLA cross-reactive panels and HPA genes, sometimes referred to herein as the "typing or detection method of the present invention." The method includes commercial experimental tests for non-diagnostic purposes as well as tests for diagnostic purposes. Test methods for diagnostic purposes can be understood as methods for diagnostic use.

[0076] Example

[0077] 1. HLA and HPA sequence analysis and plasmid standard construction

[0078] HLA-A and HLA-B sequences are polymorphic, so the complete sequences of HLA-A and HLA-B were downloaded from the AFND and GenBank databases, and compared using Mega software. Primers that can amplify the full-length genomes of HLA-A and HLA-B were designed in the conserved regions. The sequences of HPA are relatively conserved compared to HLA. According to the HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 genes in the Versiti and GenBank databases (the frequency of low-frequency antigen genes in the population is higher than that of HLA genes in the human population), the HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 genes in the human population are relatively conserved. ) sequence to design primers capable of amplifying the corresponding gene fragments; human gDNA with known HLA-A and B gene subtypes was used as a template, and the designed primers were used to amplify the various HLA-A, HLA-B, and HPA gene fragments by PCR. For samples with extremely low gene frequencies or no corresponding gene subtypes, site-directed mutagenesis was used to construct the fragments, or the fragments were synthesized by a genetic biology company. Successfully amplified products were subjected to agarose gel electrophoresis, and bands with expected fragment lengths were excised and recovered. They were then ligated with T4 DNA to a T vector or B vector, and subsequently transformed into DH5α cells. Plasmids were extracted and digested for enzyme identification. Positive plasmids screened were sent to Shanghai Bioengineering for sequencing analysis. Plasmids with correct sequencing results were retained as templates for testing during the establishment of the methodology. Table 1 shows the information for HLA-A, HLA-B, and HPA plasmids with correct sequencing results.

[0079] Table 1. Genotyping results of Sanger sequencing of constructed standard plasmids

[0080]

[0081]

[0082] 2. HLA-A, HLA-B, HPA sequence analysis, specific primer design and optimization plan

[0083] MUSCLE iterative sequence analysis was performed based on all HLA-A and HLA-B sequences in the IPD-IMGT / HLA database. At the same time, group analysis was performed based on the sequences of HLA subtypes with the same epitope in the CREG table. The groups in the CREG table can be further grouped according to the epitope-specific SNP sites. HLA-A can be divided into 8 groups and HLA-B can be divided into 14 groups. Specific UAP and / or ARMS primers were designed based on the SNP sites in each group. The specific groups are shown in Tables 2 and 3. Figure 1 As shown;

[0084] Table 2 Examples of CREGs grouping, corresponding group antigens and SNP sequences

[0085]

[0086]

[0087] For HPA, different HPA loci are located on different chromosomes or distant gene segments on the same chromosome. The typing of different HPA subtypes is mainly based on the difference of single SNPs. Therefore, UAP and / or ARMS primers are designed based on specific SNP sites in HPA-1-6, 9, 12, 13, 15, 21, 27, 30, and 31 genes.

[0088] The primer can only be extended when the base at the 3' end of the ordinary primer is completely complementary to the template. When the base at the 3' end of the primer is mismatched with the template, the primer extension is inhibited or even completely terminated. However, similar sequences will produce non-specific amplification. Therefore, the design structure of the ARMS primer based on the mutation retardation amplification system can eliminate this type of non-specific amplification, that is, artificially introduce mismatched bases at the 3' end of the primer. The type of base mismatch and the number of mismatches introduced will affect the amplification effect of the primer. Taking the design and optimization of primers for the 1C1 target-specific sequence in the HLA-A locus as an example, the design and optimization scheme of the ARMS primer is as follows: Figure 1As shown in the upper half: In the first round, a single base mismatch was used, and the specificity of the amplified product was judged based on the melting curve of the SYBR dye real-time fluorescence PCR. The Tm value of the highest peak of the melting curve was determined by the X-axis result. The increase in temperature caused the melting of the DNA double helix. When the temperature reached a specific value, all double-stranded DNAs melted into single-stranded DNA, and the SYBR Green dye dissociated from the melted DNA double helix, and the fluorescence signal reached a peak. The peak of the melting curve corresponded to the Tm value of the specific amplified product. The Y-axis result was used to judge the fluorescence signal intensity of the fluorescent dye bound to the DNA chain. The more products, the stronger the fluorescence intensity. The difference in product between the specific peak and the non-specific peak can be determined based on the fluorescence intensity of the melting curve peak. Usually, the melting curve peak greater than 80°C is the specific peak. The figure shows that the ARMS primer with a single base mismatch will produce a non-specific peak greater than 80°C after amplification. Therefore, the primer structure needs to be further optimized. In the second round of optimization, the second and third mismatched bases were artificially introduced. Although the introduction of multiple mismatches affects The amplification efficiency of the primers is improved, but 2-3 base mismatches can increase the complexity of the primers and inhibit nonspecific amplification. As shown in the figure, the nonspecific melting curve peaks of the AC and TC base mismatch primers are weaker than those of the single-base C mismatch, proving that double-base mismatches can effectively inhibit the amplification of nonspecific products. Furthermore, by interpreting the melting curves, primers with better specificity were screened for TaqMan probe real-time fluorescence PCR detection, and the specificity of the designed primers was confirmed by directly reading the △CT value between the specific and nonspecific amplification curves. In addition, among the screened ARMS primers, the 5' end primer length was optimized and fine-tuned to further screen primers with better specificity.

[0089] Similarly, because the sequences between HLA-A and B locus subtypes are highly polymorphic and in linkage disequilibrium, that is, the differences in some gene segments are not single bases, when designing specific primers for SNPs, the linked mutation bases before and after the SNP will make ARMS primer design difficult. For this reason, UAP primers are also designed to screen specific primers. Because UAP primers have two relatively independent primer fragments and form a bridge structure through a deoxyadenine nucleotide linker, they can effectively avoid other base mutations adjacent to the SNP. Since UAP primers have two binding regions, the primer structure and binding sequence at the 5' end and 3' end can be adjusted separately. Taking the design and optimization of primers for the 1C1 target-specific sequence in the HLA-A locus as an example, the specific optimization scheme for UAP primers is as follows. Figure 2As shown in the lower panel, in the first round of UAP primer optimization, the number of binding bases at the 3' end of the primer and the type of linker (vacuolar bridge and replacement bridge) were adjusted. Primers with better specificity were selected for further optimization based on the results of the SYBR dye method. In the second round of optimization, the number of linker bases (3-9 deoxyadenine nucleotides) was optimized. Finally, the primers with better specificity were tested by TaqMan probe real-time fluorescence PCR. The length of the 5' end of the UAP primer was further adjusted, and finally, UAP primers with better specificity were selected.

[0090] For each SNP site, ARMS and UAP primers were designed and optimized, and finally the primers with the best specificity were comprehensively selected for multiplex PCR.

[0091] Table 3 shows the primer and probe sequences for the specific SNP sites of different groups in different CREG tables and the HPA specific SNP sites after screening and optimization in the present invention.

[0092] Table 3 CREGs and HPA primer probe sequences

[0093]

[0094]

[0095]

[0096]

[0097] 3. Optimization of reaction conditions

[0098] To balance the specificity of the ARMS and UAP primer systems, the present invention optimized the reaction procedure and annealing parameters. By comparing the two-step and three-step methods and optimizing the annealing temperature and annealing time using a gradient method, the most suitable reaction conditions for ARMS and UAP primers were selected, resulting in the highest specificity for ARMS and UAP primers. The results are shown in Table 4. The experimental results showed that under the same annealing conditions, the two-step method exhibited better specificity than the three-step method, with the three-step method achieving a nonspecific amplification Ct value of less than 35. Gradient optimization of the annealing temperature revealed that lower annealing temperatures (56 and 58°C) resulted in significant nonspecific amplification, while higher annealing temperatures (62 and 64°C) inhibited the reaction. Optimization of the annealing time revealed that longer annealing times resulted in primer binding to nonspecific templates, with nonspecific amplification Ct values ​​of less than 35 when the annealing time was ≥45 seconds. However, shorter annealing times resulted in incomplete target amplification, with specific amplification Ct values ​​exceeding 30. Therefore, a two-step amplification program was adopted with an annealing condition of 60°C for 30 seconds. Under these conditions, the system has both high specificity and stable amplification efficiency, which was used as the reaction condition for subsequent primer screening and multiplex real-time fluorescence PCR.

[0099] Table 4: Effects of different reaction conditions on specific amplification of ARMS and UAP primer systems

[0100]

[0101] 3. Screening of primers and probes for internal reference genes

[0102] The present invention designed 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. A BLAST comparison was performed against the NCBI database to verify the absence of homologous interference from other genes in the amplicon, confirming that this region is not conserved in non-human primates and other species, thus avoiding false positives from contaminated samples. Because the internal reference primer sequence is conserved, amplification efficiency is higher than that of other targets. This consumes dNTPs and Taq DNA enzyme activity in earlier amplification cycles, leading to delayed or inhibited amplification of other targets in multiplex real-time fluorescent PCR. Therefore, 1-2 base mismatches were introduced into the 3' end of each bidirectional primer to adjust primer amplification efficiency. The designed primers are shown in Table 5. The designed primers were paired with normal upstream and downstream primers and tested on gDNA samples. The results, as shown in Tables 6 and 7, show that 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 downstream primers), the amplification curves for gDNA samples were S-shaped, with Ct values ​​ranging from 28 to 30. The two primer pairs were replicated on 10 gDNA samples. When HLA-B-2427-2449-F-TT was used as the upstream primer, the intra- 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 standard primer HLA-B-2568-2592-R were selected as internal standard primers for establishing a multiplex real-time fluorescence PCR system. The final internal standard primer and probe sequences are shown in Table 8.

[0103] Table 5: Internal standard primer design sequences

[0104]

[0105] a The underlined bases are mismatched bases designed for the internal standard primer sequence

[0106] Table 6: Results of gDNA sample detection using internal standard primers

[0107]

[0108]

[0109] Table 7: Results of repeated experiments for screening internal standard primers

[0110]

[0111] Table 8 Screened reference gene primer probe sequences

[0112]

[0113] 4. Establishment of negative quality control system

[0114] The present invention prepares an adenovirus solution coated with an irrelevant sequence as a negative control. The solution is used as a negative control for extraction. The negative control well contains primers and probes for both the internal reference gene and the irrelevant sequence, which are used to control false positives during the sample extraction process and the detection process. In the negative control well, the internal reference gene is not amplified, but the irrelevant sequence detection Ct value is <35, then the detection result of the entire detection system is valid. When the primer probe of the irrelevant sequence and the primer probe of the internal reference gene are double-detected, the Ct values ​​of each other are not affected ( Figure 3 ).

[0115] 5. Multi-tube multiplex PCR system for detection of HLA-A, B epitopes and HPA alleles

[0116] (1) The magnetic bead-based 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 was 1.7-2.0.

[0117] (2) 2×Eagle Premix (PCR) reaction solution (Brand: Beijing Jinnuomei, Product No.: SJ-PCR002) was used. This premix contains all the components required for the reaction except primers, Taqman probes, and templates: 35 mM Tris (pH = 8.5), 150 mM KCl, 14 mM MgCl2, 0.6 mM dNTPs, 0.25 mM 50% trehalose, and 2 U / Test Taq hot start enzyme. Primers and probes for HLA-A and HLA-B epitopes, HPA alleles, and random sequences were placed in a 12-tube PCR system along with the premix and the internal reference gene HLA-B primer probe 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. The volume was finally made up to 40 μl with ddH2O.

[0118] 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

[0119]

[0120]

[0121]

[0122] (3) Amplification was performed using an 8-channel PCR amplifier (Hongshi 96H, China) with the following reaction conditions:

[0123]

[0124] (4) Interpretation criteria

[0125] A.Quality Control

[0126] ① If the internal reference gene curve in the negative control well (well 12) does not rise or the Ct value is ≥32, and the FAM amplification curve of the unrelated sequence is normal and the Ct value is <32, then this experiment is valid and there is no false positive;

[0127] 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, the amplification results of the wells are valid;

[0128] The test results that meet the above conditions are valid. For reactions that do not meet the above conditions, repeat the experiment or re-extract and amplify according to the specific situation. The specific situations and solutions are shown in Table 10 below.

[0129] Table 10 Multiplex real-time fluorescence PCR detection problems and solutions

[0130]

[0131] B. Interpretation of results

[0132] The experimental results that met the quality control standards were analyzed for HLA-A and B epitopes and HPA genotyping. All target detection results used the same interpretation criteria, that is, if the amplification curve of the probe of any marker of different targets was normal and the Ct value was ≤35, it was judged as a positive result; if no amplification curve appeared or the Ct value was greater than 35, it was judged as a negative reaction.

[0133] 6. Limit of Detection (LOD)

[0134] Based on the gDNA sample test results, LOD analysis was performed on the target with the worst amplification effect in each tube to represent the minimum detection limit of each tube. The extracted gDNA samples were serially diluted and the diluted gDNA samples were repeatedly measured using multiplex PCR, 7 times per day for 3 consecutive days. The test results were analyzed using the probit probability method to determine the minimum detection limit. The specific results are shown in Table 11. The results show that the LODs for tubes 1-11 were 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 was 0.838 ng / μL.

[0135] Table 11 Detection limit (LOD) results of gDNA samples detected by 12-tube multiplex PCR system

[0136]

[0137]

[0138]

[0139] 7. Reagent Precision Verification

[0140] The precision of the 11-well multiplex PCR system was verified using gDNA samples at low and high concentrations. All samples were tested for three consecutive days, with seven replicates per day. Inter- and intra-assay precision was calculated. The results are shown in Table 12. Both intra- and inter-assay precision for each target gene were less than 5%, demonstrating the excellent precision of the multiplex PCR system.

[0141] Table 12 Precision results of multiplex PCR system for gDNA samples

[0142]

[0143] 8. Clinical Sample Verification

[0144] The Tiangen kit was used to extract genomic DNA from 211 clinical samples with known typing. The 211 samples were tested and analyzed according to the method and judgment rules provided by the present invention. All samples were consistent with the known typing, with a consistency rate of 100%. Figure 4This is the CREGs grouping and HPA typing amplification curve of one of the samples. Through interpretation of the amplification curve results, the CREGs grouping result of this sample was 2C-A (HLA-A), 5C1 (HLA-B), 5C2 (HLA-B), and the HPA typing result was 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, HPA-31a31a.

[0145] Technical advantages: This method designs specific ARMS and UAP primers based on specific SNP sites in different HLA-AB epitopes and different HPA subtypes. A large number of primers are screened and optimized, and ultimately the primers with the best amplification effect are selected and combined with TaqMan probes. Furthermore, the combination of multiplex PCR has been optimized multiple times to ensure that the primers and probes amplified in each tube are independent of each other, ultimately enabling the detection of HLA-AB epitopes and HPA genes.

[0146] 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, there is currently no kit for detecting HLA epitopes in clinical practice. All tests are performed on HLA antigen genes, resulting in a waste of reagents. In addition, the operation process is complicated, the detection equipment is expensive, and the technical requirements are high. It is difficult to popularize clinical application. The present invention not only provides a method for HLA epitope detection, but also provides a method for HPA detection. A single person's sample can be tested in 12 tubes. The invention will greatly simplify the platelet HLA / HPA matching process and greatly reduce its testing costs, allowing more small and medium-sized laboratories to use conventional testing instruments to carry out HLA epitope and HPA antigen testing of platelet transfusion donors and recipients, thereby improving the effectiveness of platelet transfusions; in addition, the HLA epitope test results can not only be applied to patients for whom platelet transfusion is ineffective, but also, since the concept and principle of HLA epitope matching are also used in the selection of HLA-matched donors for organ transplantation, it can also be applied to the HLA epitope matching of some organ transplant patients and donors, and therefore has broad application prospects.

[0147] 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 and HPA genes, characterized in that: The following steps are involved: (1) analyzing the epitope gene sequence of each HLA gene in the cross-reactive group, screening for shared specific SNP sites, and grouping the HLA genes according to the shared specific SNP sites to obtain multiple HLA subgroups, wherein at least some of the HLA subgroups contain multiple HLA genes; (2) Analyze the gene sequence of each HPA allele and screen for specific SNP sites of low-frequency and high-frequency genes of different HPA subtypes; (3) designing primers and probes for the common specific SNP site and the frequency-specific SNP site, respectively, wherein, when designing primers, a mutation retardation amplification system is preferably used to design primers for a single specific SNP site, and a U-shaped anchor primer is preferably used when there is a linked mutation base near the specific SNP site, wherein the probes are labeled with different fluorescent groups; and (4) Assign responsibilities based on the type of fluorescent group in the probe and the position of the primer.

2. The method for constructing a detection system for HLA cross-reaction panels and HPA genes according to claim 1, characterized in that: The HLA genes include HLA-A and HLA-B.

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

4. The method for constructing a detection system for HLA cross-reaction panels and HPA genes according to claim 1, characterized in that: When designing primers using the mutation retardation amplification system, mismatches are introduced at the 3' end of the primers, including: a. First, a single-base mismatch is introduced, and the specificity of the amplified product is determined based on the melting curve of real-time fluorescent PCR, i.e., whether a specific peak exists; b. When the melting curve shows no specific peak, further introduce additional base mismatches and again judge whether a specific peak exists based on the melting curve of real-time fluorescent PCR; Optionally, the method further comprises repeating step b until a specific peak is obtained.

5. The method for constructing a detection system for HLA cross-reaction panels and HPA genes according to claim 1, characterized in that: The U-shaped anchor primer includes a 5' end binding region, a 3' end binding region and a vacuolar structure region located therebetween, wherein designing the U-shaped anchor primer includes the following steps: a. Adjust the number of binding bases at the 3' end of the primer and the vacuolar structure of the linker, and select a primer with good specificity as the initial primer based on the results of the SYBR dye method; b. further optimizing the number of bases in the linker of the preliminary primer to obtain an optimized primer; c. Adjust the length of the 5' binding region of the U-shaped anchor primer according to the Taqman probe method and real-time fluorescence PCR method.

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

7. The detection system for HLA cross-reaction panel and HPA gene according to claim 6, characterized in that: It includes primers whose sequences are shown as SEQ ID No. 1-100; and probes with sequences as shown in SEQ ID No.101-150, and the probes further comprise a fluorescent group, and the detection system comprises a plurality of independent subsystems, each of which comprises primers and probes for a specific SNP site, and the information of the primers and probes in the subsystems is shown in Table 3.

8. A detection kit for HLA cross-reaction panel and HPA gene, characterized in that: The invention comprises reagents and devices for constructing the detection system according to claim 6, wherein the device comprises a space capable of accommodating multiple independent subsystems.

9. The kit according to claim 8, characterized in that It further optionally comprises an internal standard primer and an internal standard probe.

10. A method for typing or detecting HLA cross-reactive panels and HPA genes, characterized in that: comprising the steps of performing multiplex PCR using the detection system according to claim 6 or 7; and The steps of interpretation based on fluorescent signals of different colors.

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