HPA genotyping detection reagent based on melting curve analysis method, detection method and application

Abstract

The invention relates to an HPA genotyping detection reagent based on a melting curve analysis method, a detection method and application, in particular to a detection reagent for HPA genotyping detection of Chinese population, a detection method and application. According to the invention, a multiplex PCR technology based on a fluorescent probe melting curve is adopted, specific primers of different HPA systems and fluorescent labeled probes are arranged and combined, and finally, only three reaction tubes are used for simultaneously carrying out genotyping on 14 HPA antigen systems of Chinese population at a time. In addition, the detection system disclosed by the invention is simple, convenient, rapid, accurate and efficient, and has relatively high practical value and clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the field of gene detection, specifically to an HPA genotyping detection reagent, detection method, and application based on melting curve analysis, and particularly to a reagent, detection system, detection method, kit, and application for HPA genotyping detection in the Chinese population. Background Technology

[0002] Platelets are produced by megakaryocytes in the bone marrow and released into the bloodstream, playing a crucial role in hemostasis and thrombosis. The platelet membrane surface possesses a complex antigen system, which can be divided into two categories based on their distribution characteristics: The first category includes carbohydrate antigens (such as ABO, H, and Lewis antigens) and human leukocyte antigens (HLA-I antigens), known as platelet non-specific antigens or platelet-associated antigens. The second category is the relatively specific platelet alloantigen system (HPA), composed of platelet-specific antigenic determinants, belonging to platelet-specific antigens. The HPA system exhibits significant genetic polymorphism and is expressed on both platelets and their precursor cells, megakaryocytes. HPA gene polymorphism mainly originates from single nucleotide polymorphisms (SNPs) in platelet membrane glycoprotein structural genes. This polymorphism leads to changes in a single amino acid at a specific location, resulting in different antigenic phenotypes. HPA is a double dominant coalele, and its naming follows a unified principle: it is prefixed with "HPA" followed by a number for identification, and alleles are distinguished by the letters "a" and "b", where "a" indicates an allele with a gene frequency greater than 50%, and "b" indicates an allele with a gene frequency less than 50%.

[0003] Rapid and accurate typing of the HPA antigen system is of great significance in clinical and transfusion medicine, as well as in genetic and anthropological research. Previously, HPA antigen typing primarily relied on serological methods, including the Mixed Passive Hemagglutination Assay (MPHA), the Monoclonal Antibody Immobilization Assay (MAIPA), the Enzyme-Linked Immunosorbent Assay (ELISA), and the Simplified Sensitized Red Blood Cell Platelet Serological Assay (SEPSA). However, due to limited antiserum sources and difficulties in obtaining platelets from patients, the application of serological methods was significantly restricted and difficult to widely promote. With the continuous advancement of molecular biology techniques, HPA genotyping has gradually become more widespread, emerging as a more efficient and reliable typing method. It has been applied to population surveys, genotyping of clinical cases, and the establishment of platelet donor banks with known HPA types.

[0004] HPA genotyping is primarily based on polymerase chain reaction (PCR). Among the various HPA genotyping techniques, PCR-SSP is the most common. However, it requires electrophoresis identification after PCR amplification, making the process cumbersome and time-consuming. Results cannot be automatically obtained, are subjective, and their accuracy needs improvement, thus limiting its application in large-scale sample collections. As for PCR-RFLP, incomplete enzyme digestion during the process can also lead to erroneous genotyping results, and not every HPA allele has suitable restriction enzyme sites. Direct sequencing genotyping is highly accurate and is the gold standard for HPA genotyping, but it is costly, requires advanced equipment, and involves complex procedures. Furthermore, other genotyping methods also have varying degrees of cost or technical limitations.

[0005] 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

[0006] Addressing at least some of the technical problems existing in the prior art, this invention, through reasonable grouping and combination with fluorescent probe melting curve analysis technology, enables efficient genotyping detection of 14 HPA antigen systems in Chinese individuals in a single, simultaneous operation using only 3 systems. Specifically, this invention includes the following:

[0007] In a first aspect, the present invention provides an HPA genotyping detection reagent based on melting curve analysis, particularly a reagent for HPA genotyping detection in the Chinese population, comprising the following three components: (1) The first component includes primer set 1 with sequences as shown in SEQ ID No. 1, 2, 5-8, 11, 12, 19, 20 and probe set 1 with sequences as shown in SEQ ID No. 23, 25, 26, 28, 36; (2) Second component: It includes primer set 2 with sequences as shown in SEQ ID No. 3-6, 9, 10, 15-18 and probe set 2 with sequences as shown in SEQ ID No. 24, 27, 30, 32, 35; (3) The third component includes primer set 3 with sequences as shown in SEQ ID No. 5, 6, 11-14, 21, 22 and probe set 3 with sequences as shown in SEQ ID No. 29, 31, 33, 34, 37.

[0008] In some embodiments, the reagent for HPA genotyping detection in the Chinese population according to the present invention is wherein the first component, the second component, and the third component are each used to form three different reaction systems.

[0009] In some embodiments, the reagent for HPA genotyping detection in the Chinese population according to the present invention includes probes in probe group 1, probe group 2 and probe group 3, each probe having a different fluorescent group.

[0010] In some embodiments, the reagent for HPA genotyping detection in the Chinese population according to the present invention includes HPA genotyping of HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 gene loci.

[0011] In some embodiments, the reagent for HPA genotyping detection in the Chinese population according to the present invention includes probes for probe set 1, probe set 2, and probe set 3 as follows:

[0012] In some embodiments, the reagent for HPA genotyping detection in the Chinese population according to the present invention further includes an internal standard gene detection reagent.

[0013] A second aspect of the present invention provides an HPA genotyping reaction solution based on melting curve analysis, and particularly provides a detection system (or reaction solution) for HPA genotyping detection in the Chinese population, comprising: a. A first reaction system comprising primer set 1 with sequences as shown in SEQ ID No. 1, 2, 5-8, 11, 12, 19, 20 and probe set 1 with sequences as shown in SEQ ID No. 23, 25, 26, 28, 36; b. A second reaction system comprising primer set 2 with sequences as shown in SEQ ID No. 3-6, 9, 10, 15-18 and probe set 2 with sequences as shown in SEQ ID No. 24, 27, 30, 32, 35; c. A third reaction system comprising primer set 3 with sequences as shown in SEQ ID No. 5, 6, 11-14, 21, 22 and probe set 3 with sequences as shown in SEQ ID No. 29, 31, 33, 34, 37.

[0014] In some embodiments, according to the detection system for HPA genotyping in the Chinese population described in this invention, wherein: The first reaction system includes: ;or The second reaction system includes: ; or The third reaction system includes:

[0015] A third aspect of the present invention provides a method for HPA genotyping in a Chinese population, comprising the step of using the reagents described in the first aspect.

[0016] In some embodiments, the method for HPA genotyping in the Chinese population according to the present invention further includes an interpretation step, wherein the interpretation rule is:

[0017] In a fourth aspect, the present invention provides an HPA genotyping kit based on melting curve analysis, comprising the above-described detection reagents, reaction system or reaction solution.

[0018] In some embodiments, according to the kit of the present invention, the kit further includes instructions for executing interpretation rules.

[0019] In a fifth aspect, the present invention provides the application of the above-described detection reagents, reaction systems, or reaction solutions in the preparation of HPA genotyping kits based on melting curve analysis.

[0020] This invention requires only three independent reaction systems to effectively detect 14 HPA genotypes unique to Chinese individuals, namely genotypes 1-6, 9, 12, 13, 15, 21, 27, 30, and 31. The entire detection process takes only 100 minutes, making it not only simple and rapid but also accurate and efficient, with high practical value. In terms of result analysis, the genotyping results for different loci can be obtained simply by interpreting the Tm values ​​of the melting curves of the corresponding fluorescent probes for each HPA system: when the locus is homozygous, a single melting curve corresponding to the Tm value of HPA-a or HPA-b will appear; while when the locus is heterozygous, both HPA-a and HPA-b Tm values ​​will appear simultaneously, with the melting curve showing a double peak or a "bun peak" where the software can interpret the two Tm values.

[0021] Furthermore, this invention also applied the detection system to detect unknown HPA genotype samples, and compared the results with those obtained from commercial HPA genotyping kits based on fluorescent probe qPCR. For samples with inconsistent genotyping results from the two methods, Sanger sequencing was used for confirmation. The final results demonstrate that the detection system of this invention exhibits higher reliability in terms of accuracy compared to commercial kits. Attached Figure Description

[0022] Figure 1Melting curves of primers and probes for the initial screening of available HPA systems are shown in the figure. The black lines in the figure indicate the melting curves when detecting the corresponding heterozygous templates of each HPA system.

[0023] Figure 2 The melting and amplification curves of primers and probes for the HPA system to be optimized were initially screened. The black lines in the figure indicate the melting / amplification curves when detecting the corresponding heterozygous templates of each HPA system.

[0024] Figure 3 The results of detecting the corresponding plasmids after optimizing HPA-6, 15, 27, 12, and 31 probes are shown in the figure. The black lines in the figure indicate the melting curves / amplification curves when detecting the corresponding heterozygous templates of each HPA system.

[0025] Figure 4 Screening of GAPDH primers and probes.

[0026] Figure 5 Preliminary three-tube multiplex PCR reaction system for HPA genotyping.

[0027] Figure 6 HPA multiplex system for preliminary validation of the sample (without internal standard).

[0028] Figure 7 Optimization of the HPA tube 1 (i.e., the first reaction system) multi-system.

[0029] Figure 8 The optimization of the HPA tube 2 (i.e., the second reaction system) multi-system is shown in the figure. The black line in the figure marks the melting curve when detecting the corresponding hybrid template of the HPA-6 system.

[0030] Figure 9 Screening of DMSO concentration in multiple systems using HPA tube 3 (i.e., the third reaction system).

[0031] Figure 10 HPA-6 abnormal sample melting curve peak diagram and sequencing results.

[0032] Figure 11 HPA-6 joint interpretation example.

[0033] Figure 12 The melting curves and genotyping results of each tube when using HPA to test exemplary samples.

[0034] Figure 13 The melting curves of each channel in HPA tube 1 (i.e., the first reaction system) correspond to the HPA system melting curves.

[0035] Figure 14 The melting curves of each channel in HPA tube 2 (i.e., the second reaction system) correspond to the HPA system melting curves.

[0036] Figure 15 The melting curves of each channel in HPA tube 3 (i.e., the third reaction system) correspond to the HPA system melting curves.

[0037] Figure 16 A three-tube multiplex PCR reaction system for detecting HPA genotyping.

[0038] Figure 17 Sanger sequencing confirmed that the genotyping did not match the true genotyping of the sample. Detailed Implementation

[0039] 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.

[0040] 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.

[0041] 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.

[0042] This invention provides an HPA detection reagent and method based on melting curve analysis. It utilizes the physical property that different base sequences cause different nucleotide melting temperatures after PCR. By monitoring the nucleotide melting process, characteristic melting curves can be obtained, leading to genotyping. In this invention, using an excess of reverse primers in asymmetric PCR amplifies a large number of single-stranded positive-strand products. At this point, fluorescently labeled probes designed around SNP sites perfectly match the antisense strand, exhibiting a high Tm value, while partially mismatched fluorescently labeled probes exhibit a low Tm value. In one specific embodiment, multiplex PCR technology based on fluorescent probe melting curves is used to arrange and combine specific primers and fluorescently labeled probes for different HPA systems, ultimately enabling simultaneous genotyping of all 14 HPA antigen systems in the Chinese population using only three reaction tubes in a single test. This detection system is not only simple and rapid but also accurate and efficient, possessing high practical value.

[0043] In practical applications, for example, incompatible transfusions of platelet antigens between individuals can trigger platelet alloimmune reactions, producing platelet alloantibodies, leading to alloimmune thrombocytopenia and causing various clinical conditions such as ineffective platelet transfusion, neonatal alloimmune thrombocytopenia, and post-transfusion purpura. In clinical transplantation, transplant rejection may also occur. Furthermore, the distribution of HPA gene frequencies varies across different ethnic groups. Therefore, rapid and accurate typing of the HPA antigen system is of great significance in clinical and transfusion medicine, as well as in genetic and anthropological research. The detection system of this invention (including primers and probes) is specifically designed for the Chinese population. The HPA antigen system it targets includes HPA gene loci with high-frequency antigen a and low-frequency antigen b. High frequency means that the frequency of antigen a is not less than 50%, and low frequency means that the frequency of antigen b is greater than 0.05%. Ultimately, this invention performs genotyping on 14 HPA antigen systems, including HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31.

[0044] Detection system or reaction solution 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 HPA genotyping detection in the Chinese population. Regarding probes, this invention found that for certain antigenic sites (e.g., HPA-27), increasing probe length enhances resolution, while for other antigenic sites (e.g., HPA-6, HPA-15), shortening probe length enhances resolution. Furthermore, this invention optimized different fluorescent labels and probe types (Taqman or MGB probes). For primers, to ensure amplification efficiency, this invention optimized the GC content of the primers and also optimized the additional components added to the reaction system.

[0045] After constructing the detection system or reaction solution, this invention further optimized the detection method to suit the present invention. The grouping of primers and probes took into account the physical location of SNP sites in each antigen system. Different detection systems exhibit different fluorescence levels, requiring a reasonable combination of fluorescence signal intensities. Simultaneously, the concentrations of added components (e.g., dNTPs, Mg) were adjusted. 2+ The concentrations of forward and reverse primers, probe concentrations, DMSO, etc., need to be controlled within a reasonable range.

[0046] In a preferred embodiment, the detection system includes or comprises a first reaction system, a second reaction system, and a third reaction system, wherein the first reaction system includes: ; The second reaction system includes: ; The third reaction system includes:

[0047] 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.

[0048] In this invention, the final concentration of dNTP in the reaction system is 100-500 µM, preferably 200-450 µM, even more preferably 300-450 µM, and even more preferably 350-450 µM.

[0049] 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%.

[0050] In this invention, the final concentration of the probe in the reaction system is 0.1-5 µM, preferably 0.2-1 µM, even more preferably 0.4-0.8 µM, and even more preferably 0.5-0.7 µM.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Methods for HPA Genotyping in the Chinese Population This invention provides a method for HPA genotyping in the Chinese population, sometimes referred to herein as "the detection method of this invention," which includes commercial experimental tests for non-diagnostic purposes as well as tests for diagnostic purposes. Detection methods for diagnostic purposes can be understood as diagnostic applications.

[0055] 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.

[0056] 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.

[0057] Example 1. Selection of HPA genotyping sites and construction of HPA plasmids Based on the frequency of high-frequency antigen a and low-frequency antigen b at each HPA locus shown in Table 1, this invention selects 14 HPA systems (1-6, 9, 12, 13, 15, 21, 27, 30, and 31) with a frequency of HPA low-frequency antigen b greater than 0.05% for genotyping detection.

[0058] Table 1. Frequency of HPA high-frequency antigen a and low-frequency antigen b For each HPA system to be genotyped, this invention constructed plasmids for high-frequency antigen a and low-frequency antigen b of HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, respectively, as detection templates for methodological establishment. For the high-frequency antigen a plasmid of HPA, this invention used human genomic DNA (gDNA) of known HPA genotypes as templates, amplified the nucleic acid sequences of each HPA using conventional PCR, and then ligated the amplified sequences into T or B vectors to obtain plasmids for high-frequency antigen a at each HPA locus. For the low-frequency antigen b plasmid of HPA, point mutations were constructed using the constructed high-frequency antigen a plasmid of HPA. All constructed HPA plasmids were correctly sequenced using Sanger sequencing.

[0059] 2. Preliminary design and validation of primers and probes Based on the nucleic acid sequence information and corresponding SNP sites of HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, this invention designs specific primers and fluorescently labeled probes. The primer design principles are: 1) The amplification product contains SNP polymorphic sites of HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, and the length is controlled between 100-500 bp to facilitate amplification; 2) The primer design avoids HPA SNP polymorphic sites; 3) HPA systems located in the same gene and with close SNP polymorphic sites are designed in one pair of primers (only one pair of specific primers, HPA-F3 / R3, is designed for HPA-3, -9, -27, and -30). The probe design principles are as follows: 1) The probe length is 18-25 bp; 2) The probe design includes SNP polymorphic sites of HPA, and the SNP sites are located in the center of the probe; 3) One probe is designed for each HPA site, and the probe sequence completely matches the gene sequence of the high-frequency antigen a of that HPA site. The preliminary primer and probe sequences are shown in Tables 2 and 3.

[0060] Table 2 Preliminary primer design Table 3 Preliminary probe design Note: The bolded entries in the table represent the SNP sites corresponding to each HPA system.

[0061] To verify the feasibility of the initially designed primers and probes, this invention first amplifies plasmids of known HPA types using a single asymmetric PCR system: plasmids containing either high-frequency HPA antigen a or low-frequency antigen b are used to simulate HPA homozygous templates, while plasmids containing high-frequency antigen a and low-frequency antigen b in equal proportions are used to simulate HPA heterozygous templates.

[0062] The experiment employed a two-step reaction method. The reaction conditions were as follows: 90-98°C for 1-5 min, 90-98°C for 5-30 s, 50-70°C for 0.5-3 min, 45-55 cycles, with fluorescence signal acquisition at 50-70°C. Denaturation occurred at 90-98°C for 0.5-3 min, hybridization at 40-55°C for 1-10 min, and the temperature was gradually increased from 45-90°C.

[0063] The results of singleton asymmetric PCR performed on homozygous and heterozygous templates from different HPA systems are as follows: Figure 1 and Figure 2 As shown. Figure 2 The results showed that the primers and probes designed for the HPA systems HPA-1-5, 9, 13, 21, and 30 were usable: when detecting homozygous templates, the melting curves for HPA-a or HPA-b showed a single peak, and the Tm values ​​of HPA-a and HPA-b also showed some differences; when detecting heterozygous templates, the melting curves showed a double peak or a "bun peak" that could be interpreted by the software, and the software could simultaneously interpret the Tm values ​​of HPA-a and HPA-b, thus distinguishing the heterozygous template.

[0064] However, some primers and probes need to be optimized, such as... Figure 3 As shown: For HPA-6 and HPA-15, primers and probes produced normal amplification curves when detecting homozygous and heterozygous templates. The melting curve for homozygous templates showed a single peak, and the Tm values ​​for HPA-a and HPA-b also showed some difference. However, when detecting heterozygous templates, the melting curve only showed a single peak, and the software could only interpret one Tm value, which was between the Tm values ​​of HPA-a and HPA-b. Therefore, it was impossible to effectively distinguish between HPA-a and HPA-b when detecting heterozygous templates. For HPA-27, when detecting homozygous template HPA-27a, both amplification and melting curves were normal. However, when detecting homozygous template HPA-27b, neither amplification nor melting curves were observed, and only the Tm value of HPA-27a could be interpreted when detecting heterozygous templates. For HPA-12 and HPA-31, almost no amplification or melting curves were observed for either homozygous or heterozygous templates.

[0065] 3. Optimization of primers and probes For the primers and probes mentioned above that require optimization, this invention has optimized them using a variety of different strategies.

[0066] Firstly, regarding HPA-6, to address the issue of its inability to distinguish between HPA-6a and HPA-6b when detecting heterozygous templates, this invention underwent extensive optimization. It was found that shortening the length of the HPA Probe-6 probe enhances its resolving power. Furthermore, replacing the fluorescent label of HPA Probe-6 with Atto425 significantly improved the smoothness of the initial amplification and melting curves using CY7 for HPA Probe-6 fluorescent labeling. The optimized detection results for simulated homozygous and heterozygous templates of the corresponding HPA-6 plasmid are as follows: Figure 4 As shown, the optimized results indicate that the new probe can effectively distinguish between HPA-6a and HPA-6b in the hybrid template.

[0067] Secondly, similar to HPA-6, this invention shortens the probe length for HPA-15 and optimizes the Taqman probe to an MGB probe. Results show that HPA-15a and HPA-15b can be effectively separated in the heterozygous template. Furthermore, because HPA Probe-15 exhibits strong fluorescence signal intensity in singlet asymmetric PCR systems when fluorescently labeled with ROX, considering the need for subsequent multiplex PCR combinations, and to ensure uniform fluorescence signal intensity across multiplex systems, the fluorescent label was replaced with a slightly weaker fluorescent CY5.5. The optimized detection results are as follows. Figure 4 As shown.

[0068] The main problem with HPA-27 is that the probe cannot effectively bind during HPA-27b amplification, resulting in the inability to generate amplification and melting curves. Since the SNP sites of HPA-27 and HPA-9 are very close, the initial probe length for HPA-27 was shorter than other probes, only 18 bp. This may be the reason why the probe cannot effectively bind during HPA-27b amplification. Therefore, this invention appropriately extends the length of HPA-Probe27 to include the SNP site of HPA-9, effectively solving the amplification and probe problem of HPA-27b without affecting the detection of the HPA-9 site. The optimized detection results are as follows: Figure 4 As shown.

[0069] Regarding HPA-12 and HPA-31, a common problem is that neither amplification nor melting curves are observed for either HPA-a or HPA-b. Analysis of the gene sequences of HPA-12 and HPA-31 revealed high GC content (>70%) in both templates, which is detrimental to PCR. Therefore, this invention redesigned primers with lower GC content, and replaced the original CY5.5-labeled probes with ROX and Atto425, which have relatively stronger fluorescence signals, respectively. DMSO was also added separately to the system to promote the melting of high-GC-content templates and improve PCR amplification efficiency. The optimized detection results for homozygous and heterozygous templates are as follows: Figure 4 As shown.

[0070] 4. Design, screening, and optimization of internal standard gene primers and probes. The goal of this invention is to use human whole blood as a sample, extract human genomic DNA, and then perform detection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is selected as an internal standard gene to participate in the amplification of multiplex systems. Only when the fluorescence channel corresponding to the internal standard in each well shows a melting curve and the Tm value is within the correct range is the result of that well considered valid; otherwise, it is considered invalid.

[0071] First, multiple pairs of GAPDH primers and corresponding probes were designed. Since the expression of the internal standard gene GAPDH is strong, in order to avoid the introduction of the internal standard gene primers and probes affecting the detection of other targets in the HPA system in the multiplex system, FAM, which has a relatively weak fluorescence signal intensity, was selected to label the internal standard specific probe.

[0072] Then, the extracted human gDNA samples were amplified using the aforementioned singlet asymmetric PCR method to screen for the most suitable specific primers and probes for amplifying GAPDH. The screening results are as follows: Figure 5 As shown, based on the CT value of the amplification curve and the peak shape of the melting curve, the GAPDH-F / R primer pair and its corresponding probe GAPDH-Probe were selected: when this primer-probe pair is used, the melting curve has a single peak and a smooth peak shape; the Ct value of the amplification curve is also relatively delayed, and it will not excessively inhibit the amplification of other HPA targets in the multiplex system. Therefore, primers GAPDH-F / R and probe GAPDH-Probe were selected as the primers and probes for the internal control gene finally added to the multiplex reaction system for the detection of GAPDH, in order to monitor the effectiveness of the reaction system. The specific sequences of the corresponding primers and probes for GAPDH are shown in Table 4.

[0073] Table 4 GAPDH primer and probe sequences 5. Preliminary combination and verification of multiple systems After designing, validating, and optimizing primers and probes for each HPA system and internal standard gene, preliminary multiplex PCR systems were combined using the optimized primers and probes, resulting in three multiplex asymmetric PCR systems. The primers and probes for each HPA system detected in each multiplex system are as follows: Figure 6 As shown. The HPA system detected by HPA tube 1 includes HPA-1, 4, 6, 21, and 3; the HPA system detected by HPA tube 2 includes HPA-2, 9, 5, 13, and 15; the HPA system detected by HPA tube 3 includes HPA-27, 30, 12, and 31. Since HPA-3 contains HPA-12 and HPA-31, DMSO was added to the multiplex system, analogous to the singlet system, to promote the amplification of high GC content templates of HPA-12 and HPA-31.

[0074] To evaluate the feasibility of simultaneous amplification of various HPA systems within a multiplex system, specific primers and probes for each HPA system were combined in each reaction tube without the addition of internal standard specific primers and probes. Preliminary validation was performed using gDNA samples from individuals with known HPA genotypes. The experimental results are as follows: Figure 6 As shown.

[0075] The results showed that, overall, in the HPA tube 1 multiplex system, the peak Rm of the melting curve of each probe was slightly weaker than its Rm value in the singlet PCR system. Furthermore, the peak Rm of the melting curves of probes with different fluorescent labels varied in strength after being combined into the multiplex system; the peak Rm of the melting curve of VIC-labeled HPA Probe-4 was weaker compared to other probes. In addition, for HPA-Probe6, after being combined into the multiplex system and amplified on human gDNA samples, a new non-specific peak appeared in the melting curve, and the Tm value corresponding to this non-specific peak was similar to the Tm value when the probe detected HPA-6b, which may affect the interpretation of HPA-6 genotyping results.

[0076] Similar to HPA tube 1, for the multiplex system of HPA tube 2, the peak Rm of the melting curves for each probe decreased overall after combining different primers and probes into the multiplex system. Furthermore, the presence of large pits in the melting curve of HPA-13 labeled with CY7 also affected the overall aesthetics.

[0077] The multiplex amplification of HPA tube 3 showed no obvious problems, and the peak Rm of the melting curve was relatively uniform. Although new nonspecific peaks appeared when CY5-labeled HPA Probe-27 amplified human gDNA samples, the Tm values ​​of the impurity peaks did not affect the interpretation of the final results.

[0078] In summary, except for the newly appearing impurities in HPA Probe-6 in HPA tube 1 which affected the final result interpretation, the other multiplex combinations of primers and probes correctly genotyped all known HPA genotyping samples, proving that the initial multiplex combination is feasible.

[0079] 6. Optimization of multiple systems 6.1 Optimization of HPA tube 1 For HPA tube 1, firstly, extensive optimization was performed to address the impact of the non-specific peaks from HPA Probe-6 on the final result interpretation. The results showed that a newly designed probe targeting the HPA-6 SNP site improved the interpretation of the results. The optimized experimental results are as follows: Figure 7 As shown in Figure A.

[0080] Secondly, since the peak Rm values ​​of the melting curves of each probe were slightly weaker than their respective Rm values ​​in the singlet PCR system, the concentration of dNTPs in the first tube of the HPA assay was optimized to improve the signal of the melting curve peaks. Different Mg levels were also set. 2+ Concentration gradients were used to screen for the optimal concentrations of dNTPs and their corresponding Mg. 2+ The concentration of Mg was ultimately determined by detecting the gDNA samples with known HPA genotypes, comparing the three dNTPs with Mg. 2+ The concentration combinations were screened, and the optimized experimental results are as follows: Figure 7 As shown in Figure B, it can be observed that the HPA genotyping results using all three combinations are correct, and the second group has a relatively higher peak Rm in the melting curve. Therefore, the second group of dNTPs and Mg was ultimately chosen. 2+ The concentration combination was used as the final reaction condition for HPA-1.

[0081] Finally, to address the weak signal intensity of VIC-labeled HPA Probe-4, and to improve the signal intensity of the VIC channel and make the overall signal intensity more uniform, the number of both the forward and reverse primers for the HPA-4 system was increased to a certain extent; correspondingly, the concentration of the HPA Probe-4 probe was also increased. The optimized experimental results are as follows: Figure 7 As shown in Figure C, it can be observed that increasing the concentration of the specific primers and probes corresponding to the HPA-4 system effectively improved the fluorescence signal intensity of HPA-4.

[0082] 6.2 Optimization of HPA tube 2 Regarding HPA tube 2, firstly, addressing the significant issue with the melting curve of the CY7-labeled HPA-13, its fluorescent label was changed to Atto425, which improved the aesthetics of the final melting curve of the multi-system mixture to some extent. Optimized detection results are as follows: Figure 8 As shown in Figure A.

[0083] Secondly, similar to HPA tube 1, the concentration of dNTPs in HPA tube 2 was also optimized to improve the signal intensity of the peak values ​​of each melting curve, and different Mg concentrations were also set. 2+ Concentration gradient. Ultimately, the three dNTPs and Mg were detected in gDNA samples with known HPA genotypes. 2+ The concentration combinations were screened. The results are as follows: Figure 8 As shown in Figure B, it can be observed that the genotyping results using these three combinations are all correct, and for HPA tube 2, the peak Rm of the melting curve of the second group is relatively higher. Therefore, the second group of dNTPs and Mg was ultimately adopted. 2+ The concentration combination was used as the final reaction conditions for HPA tube 2.

[0084] 6.3 Optimization of HPA tube 3 For HPA tube 3, due to the presence of the two high-GC templates, HPA-12 and HPA-31, a certain concentration of DMSO was added to the multiplex system initially, similar to the singleton system. To explore a more suitable DMSO concentration for the multiplex system, five different DMSO concentration gradients were further established: 1%, 2%, 3%, 4%, and 5%. These were used to detect known HPA-typed human gDNA samples, and the results are as follows... Figure 9 As shown in the figure, the results indicate that when the DMSO concentration is <4%, HPA-12 and HPA-31 exhibit poor amplification due to the high GC content in the template. Only when the DMSO concentration is >4% can HPA-12 and HPA-31 be amplified effectively. Therefore, a concentration of at least 4% DMSO was ultimately added to the system.

[0085] 7. Application and further optimization of multiplex detection systems in clinical sample testing. After optimizing various aspects of the three-tube multiplex PCR system (HPA tubes 1, 2, and 3), a preliminary three-tube multiplex PCR reaction system was established. Next, 100 clinical samples were collected, and gDNA was extracted from whole blood using a kit. The gDNA was then used as a template for detection. In most samples, the melting curves of each tube were normal, and the interpretation of the results for each HPA locus to be genotyped was also normal. However, in 9 samples, when genotyping the HPA-6 locus, the melting curve of HPA-Probe6 in HPA tube 1 only showed a non-specific peak, without the specific peaks of a or b (e.g., ...). Figure 10(As shown in the AC diagram). After retesting and verifying that the cause was not an operational or reagent issue, the nine samples were amplified using standard PCR and then subjected to Sanger sequencing. The Sanger sequencing results showed that all nine samples were homozygous for HPA-6a. The sequenced sequences were then compared with the sequences used to design the primers and probes in a MEGA7 PCR machine. The comparison results are shown below. Figure 10 As shown in Figure D, these nine samples share a common feature: compared to the sequences used to design the primers and probes, a G>A mutation occurred at the base adjacent to the right of the SNP site in HPA-6, and an A>G mutation occurred 10 bases adjacent to the left of the SNP site in HPA-6. The probes used in the multiplex detection system contain both of these sites. It is speculated that the excessive number of mismatched sites between the template sequence and the probe in these nine samples prevented the probe from stably binding to the template sequence, thus failing to generate a melting curve.

[0086] To optimize the detection process, the probe length of HPA-Probe6 was first shortened to avoid the A>G mutation at the 10 bases adjacent to the left of the SNP site in HPA-6. Since the A>G mutation at the right adjacent base of the SNP site in HPA-6 could not be avoided, a base mismatch strategy was adopted. The probe was designed with a C base corresponding to this site, ensuring that the Tm value of the melting curve remained consistent regardless of whether the site was A or mutated to G, thus guaranteeing the detection of all HPA-6a. However, due to the shortened probe length, the probe designed with this mismatch strategy performed poorly in detecting HPA-6b. Comparing the detection performance of the probe before optimization (detecting HPA-6a without SNP mutations and all HPA-6b) with the probe after optimization using the mismatch strategy (detecting all HPA-6a but not HPA-6b), it was found that using both probes in combination for interpretation could detect all possibilities of the HPA-6 site. Therefore, the system was further optimized: the probe before optimization was named HPAProbe6-1, still labeled with Atto425, and placed in HPA tube 1 unchanged; the mismatch strategy probe was named HPA Probe6-2, also labeled with Atto425, and placed in tube 3. Simultaneously, the probe HPA Probe30 originally labeled with Atto425 in tube 3 was replaced with CY5.5. When interpreting the results for HPA-6, for HPA-6a, the peaks of HPA Probe6-1 were first observed. If HPA Probe6-1 showed a specific peak for HPA-6a, it could be interpreted normally. If HPA Probe6-1 did not show a specific peak for HPA-6a, the peaks of HPA Probe6-2 needed to be observed. If HPA Probe6-2 showed a specific peak for HPA-6a, it indicated that HPA-6a was a mutation at the site to the right of the SNP site in HPA-6. As for interpreting HPA-6b, it is only necessary to observe whether HPA Probe6-1 shows a specific peak for the corresponding b (e.g., Figure 11 (As shown).

[0087] After final system optimization, the primers and probes for each HPA system used for HPA genotyping are shown in Tables 5-6, and the specific reaction systems for triplet multiplex PCR are shown in Tables 7-9. The experimental reaction conditions are the same as those for the aforementioned singlet asymmetric PCR.

[0088] Table 5 Primers used for HPA genotyping detection Table 6 Probes used for HPA genotyping Table 7. HPA Tube 1 Multiple Reaction Assay System (1 person) Table 8. HPA tube 2 multiple reaction assay system (per person) Table 9. HPA tube 3 multiple reaction assay system (per person) Nine samples that previously showed abnormal HPA-6 results were retested using the optimized system, and another 100 clinical samples were collected. gDNA was extracted from whole blood and tested again using gDNA as a template. Figure 12 The melting curves of each tube as a whole are shown when testing one of the samples. Figure 13-15 This demonstrates the detection of homozygotes and heterozygotes in the corresponding HPA system under different fluorescence channels in each reaction tube. Figure 16 The system shown is a three-tube multiplex PCR reaction system ultimately used for HPA genotyping.

[0089] 8. Interpretation Rules Due to limitations in gene frequency and sample size for HPA-b, some HPA systems currently only have detection data for HPA-a. Therefore, in Table 10, which provides reference Tm values ​​for HPA loci genotyping for different fluorescence channels in each HPA tube, the corresponding Tm values ​​for HPA-b for which no sample detection data is available are given based on the results of previous plasmid detection.

[0090] Table 10 Reference Tm values ​​for HPA loci genotyping corresponding to different fluorescence channels in each HPA tube. Note: The bolded values ​​in the table are the corresponding Tm values ​​given with reference to previous plasmid detection results.

[0091] To verify the accuracy of the detection system, the test results of all 200 clinical samples were compared with those of a commercial HPA genotyping kit (catalog number: WY05E00101064), which covers nine HPA antigen systems: HPA-1-6, 10, 15, and 21. The results showed that for HPA-1, 4, 5, 6, 10, and 21, the genotyping results obtained by the two methods were consistent. However, for the HPA-2, HPA-3, and HPA-15 systems, the genotyping results were inconsistent in 1, 7, and 9 samples, respectively. For these abnormal samples, further confirmatory experiments were performed using Sanger sequencing. The results showed that the sequencing results were consistent with the detection results of the system in this embodiment, indicating that the commercial kit has several potential misclassification scenarios (e.g., Figure 17 (As shown). Overall, the detection system of this invention exhibits higher accuracy compared to the commercially available kit.

[0092] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for HPA genotyping in the Chinese population, characterized in that, This includes the steps of using a test reagent containing the following three components: (1) The first component includes primer set 1 with sequences as shown in SEQ ID No. 1, 2, 5-8, 11, 12, 19, 20 and probe set 1 with sequences as shown in SEQ ID No. 23, 25, 26, 28, 36; (2) Second component: It includes primer set 2 with sequences as shown in SEQ ID No. 3-6, 9, 10, 15-18 and probe set 2 with sequences as shown in SEQ ID No. 24, 27, 30, 32, 35; (3) The third component includes primer set 3 with sequences as shown in SEQ ID No. 5, 6, 11-14, 21, 22 and probe set 3 with sequences as shown in SEQ ID No. 29, 31, 33, 34, 37.

2. A reagent for HPA genotyping based on melting curve analysis, characterized in that, It comprises the following three components: (1) The first component includes primer set 1 with sequences as shown in SEQ ID No. 1, 2, 5-8, 11, 12, 19, 20 and probe set 1 with sequences as shown in SEQ ID No. 23, 25, 26, 28, 36; (2) Second component: It includes primer set 2 with sequences as shown in SEQ ID No. 3-6, 9, 10, 15-18 and probe set 2 with sequences as shown in SEQ ID No. 24, 27, 30, 32, 35; (3) The third component includes primer set 3 with sequences as shown in SEQ ID No. 5, 6, 11-14, 21, 22 and probe set 3 with sequences as shown in SEQ ID No. 29, 31, 33, 34, 37.

3. The HPA genotyping detection reagent based on melting curve analysis according to claim 2, characterized in that, The first component, the second component, and the third component are each used to form three different reaction systems.

4. The HPA genotyping detection reagent based on melting curve analysis according to claim 2, characterized in that, Each probe in probe group 1, probe group 2 and probe group 3 has a different fluorescent group; Preferably, the fluorescent group is selected from ROX, BHQ2, CY5, VIC, BHQ1, MGB, ATTO 425, and CY5.

5.

5. The HPA genotyping reagent based on melting curve analysis according to claim 2, characterized in that, The HPA genotyping includes HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 gene loci.

6. The HPA genotyping detection reagent based on melting curve analysis according to claim 2, characterized in that, Further, it includes internal standard gene detection reagents.

7. An HPA genotyping reaction system based on melting curve analysis, characterized in that, This includes the following independently configured first reaction system, second reaction system, and third reaction system: a. A first reaction system comprising primer set 1 with sequences as shown in SEQ ID No. 1, 2, 5-8, 11, 12, 19, 20 and probe set 1 with sequences as shown in SEQ ID No. 23, 25, 26, 28, 36; b. A second reaction system comprising primer set 2 with sequences as shown in SEQ ID No. 3-6, 9, 10, 15-18 and probe set 2 with sequences as shown in SEQ ID No. 24, 27, 30, 32, 35; c. A third reaction system comprising primer set 3 with sequences as shown in SEQ ID No. 5, 6, 11-14, 21, 22 and probe set 3 with sequences as shown in SEQ ID No. 29, 31, 33, 34, 37.

8. The HPA genotyping reaction system based on melting curve analysis according to claim 7, characterized in that: The first reaction system, the second reaction system, and the third reaction system each independently further include dNTPs and Mg. 2+ ; Preferably, it further includes DMSO.

9. An HPA genotyping kit based on melting curve analysis, characterized in that, Includes the detection reagent as described in any one of claims 2-6; Preferably, the kit further includes instructions for executing interpretation rules; Preferably, the interpretation rule is as follows: 。 10. The use of the detection reagent according to any one of claims 2-6 in the preparation of an HPA genotyping kit based on melting curve analysis; Preferably, the sample to be tested is a fluid sample; Preferably, the sample to be tested is a blood sample.

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