Reagent, detection system and method for detecting HPA genotyping of Chinese population

Abstract

The invention discloses a reagent, a detection system and a method for HPA genotyping detection of Chinese population. 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 a relatively high practical value.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to a reagent, a detection system and a method for HPA genotyping detection in the Chinese population. Background Art

[0002] Platelets are produced by megakaryocytes in the bone marrow and released into the bloodstream, playing a crucial role in hemostasis and thrombosis. A complex system of antigens is present on the platelet membrane, which can be divided into two categories based on their distribution characteristics. The first category includes carbohydrate antigens (such as ABO, H, and Lewis) and human leukocyte antigens (HLA-I), which are referred to as platelet-nonspecific or platelet-associated antigens. The second category is the relatively specific platelet alloantigen system (HPA), which is composed of antigenic determinants unique to platelets and is considered platelet-specific. The HPA system exhibits significant genetic polymorphism and is expressed on both platelets and their precursor cells, megakaryocytes. Genetic polymorphism in HPA is primarily due to single nucleotide polymorphisms (SNPs) in the structural genes of platelet membrane glycoproteins. These polymorphisms result in changes in single amino acids at specific positions, resulting in distinct antigenic phenotypes. HPA is a double dominant co-allele, and its naming follows a unified principle: "HPA" is used as the prefix, followed by a number for identification, and the 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 vital importance in clinical and transfusion medicine, as well as in genetics and anthropology. Previously, HPA antigen typing relied primarily on serological methods, including the mixed passive hemagglutination assay (MPHA), the platelet antigen monoclonal antibody immobilization assay (MAIPA), the enzyme-linked immunosorbent assay (ELISA), and the simplified sensitized red blood cell and platelet serological assay (SEPSA). However, due to limited antiserum sources and difficulties in obtaining platelets from patients, the application of serological methods has been significantly limited, hindering widespread adoption. With the continuous advancement of molecular biology techniques, HPA genotyping has become increasingly popular, becoming 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 often based on polymerase chain reaction (PCR). Among the numerous HPA genotyping techniques, PCR-SSP is the most common. However, it requires electrophoresis after PCR amplification, which is cumbersome and time-consuming. Results cannot be automatically obtained, are subjective, and require improved accuracy, thus limiting its application in large sample sizes. Regarding PCR-RFLP, incomplete enzymatic digestion during the procedure can also lead to erroneous typing results. Furthermore, not every HPA allele has suitable restriction sites. Direct sequencing-based genotyping offers high accuracy and is the gold standard for HPA genotyping. However, the testing cost and instrumentation requirements are high, and the procedure is cumbersome. Furthermore, other genotyping methods also present various cost and technical challenges.

[0005] 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

[0006] To address at least some of the technical issues existing in the prior art, the present invention, through rational grouping and combined with fluorescent probe melting curve analysis, enables efficient genotyping of 14 HPA antigen systems in Chinese individuals using only three systems. Specifically, the present invention encompasses the following:

[0007] In a first aspect, the present invention provides a reagent for HPA genotyping in the Chinese population, comprising the following three components:

[0008] (1) A first component comprising a primer set 1 having sequences as shown in SEQ ID Nos. 1, 2, 5-8, 11, 12, 19, and 20 and a probe set 1 having sequences as shown in SEQ ID Nos. 23, 25, 26, 28, and 36;

[0009] (2) The second component includes a primer set 2 having sequences as shown in SEQ ID Nos. 3-6, 9, 10, 15-18 and a probe set 2 having sequences as shown in SEQ ID Nos. 24, 27, 30, 32, and 35;

[0010] (3) The third component: It includes a primer set 3 whose sequences are shown as SEQ ID No. 5, 6, 11-14, 21, and 22, and a probe set 3 whose sequences are shown as SEQ ID No. 29, 31, 33, 34, and 37.

[0011] In certain embodiments, according to the reagent for HPA genotyping detection in the Chinese population according to the present invention, the first component, the second component and the third component are each used to form three different reaction systems.

[0012] In certain embodiments, according to the reagent for HPA genotyping detection in the Chinese population of the present invention, each probe in probe group 1, probe group 2, and probe group 3 has a different fluorescent group.

[0013] In certain embodiments, according to the reagent for HPA genotyping detection in the Chinese population according to the present invention, the HPA genotyping includes HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 gene loci.

[0014] In certain embodiments, according to the reagent for HPA genotyping detection in the Chinese population according to the present invention, the probes of probe set 1, probe set 2, and probe set 3 are as follows:

[0015]

[0016]

[0017] In certain embodiments, the reagent for HPA genotyping detection in the Chinese population according to the present invention further comprises an internal standard gene detection reagent.

[0018] A second aspect of the present invention provides a detection system (or reaction solution) for HPA genotyping detection in the Chinese population, comprising:

[0019] a. A first reaction system comprising a primer set 1 having a sequence as shown in SEQ ID No.1, 2, 5-8, 11, 12, 19, 20 and a probe set 1 having a sequence as shown in SEQ ID No.23, 25, 26, 28, 36;

[0020] b. A second reaction system comprising a primer set 2 having sequences such as SEQ ID No.3-6, 9, 10, 15-18 and a probe set 2 having sequences such as SEQ ID No.24, 27, 30, 32, 35;

[0021] c. The third reaction system comprises a primer set 3 whose sequences are shown as SEQ ID No. 5, 6, 11-14, 21, 22 and a probe set 3 whose sequences are shown as SEQ ID No. 29, 31, 33, 34, 37.

[0022] In certain embodiments, the detection system for HPA genotyping in Chinese population according to the present invention comprises:

[0023] The first reaction system comprises:

[0024]

[0025]

[0026] or

[0027] The second reaction system comprises:

[0028]

[0029] or

[0030] The third reaction system comprises:

[0031]

[0032]

[0033] The third aspect of the present invention provides a method for HPA genotyping in the Chinese population, comprising the step of using the reagent described in the first aspect.

[0034] In certain embodiments, the method for HPA genotyping in the Chinese population according to the present invention further comprises an interpretation step, wherein the interpretation rule is:

[0035]

[0036]

[0037] The present invention requires only three independent reaction systems to effectively detect a total of 14 HPA types unique to Chinese people, namely 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. Regarding result analysis, genotyping results for different loci can be obtained by simply interpreting the Tm values ​​of the melting curves corresponding to the corresponding fluorescent probes in each HPA system. When the locus is homozygous, a single melting curve corresponding to the Tm value of either HPA-a or HPA-b will appear; when the locus is heterozygous, Tm values ​​corresponding to both HPA-a and HPA-b will appear, and the melting curve will appear as a double peak, or the software can interpret the two Tm values ​​as a "steamed bun" peak.

[0038] The present invention also applied this detection system to samples of unknown HPA genotypes, comparing the results with those obtained from a commercial HPA gene typing kit using fluorescent probe qPCR. For samples with inconsistent typing results between the two methods, Sanger sequencing was used for further confirmation. The results demonstrated that the detection system of the present invention demonstrated higher reliability in terms of accuracy than the commercial kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Melting curves of primers and probes for preliminary screening of available HPA systems. The black lines in the figure indicate the melting curves when detecting the corresponding heterozygous templates of each HPA system.

[0040] Figure 2 Preliminary screening of the melting curves and amplification curves of the primers and probes for the HPA system to be optimized. The black lines in the figure indicate the melting curves / amplification curves when detecting the corresponding heterozygous templates of each HPA system.

[0041] Figure 3 The results of detecting the corresponding plasmids after optimization of HPA-6, 15, 27, 12, and 31 probes. The black lines in the figure indicate the melting curves / amplification curves when detecting the corresponding heterozygous templates of each HPA system. Figure 4 Screening of GAPDH primers and probes.

[0042] Figure 5 Three-tube multiplex PCR reaction system for detecting HPA genotyping (preliminary).

[0043] Figure 6 Preliminary validation of the HPA multiplex system on samples (without internal standard).

[0044] Figure 7 Optimization of the multiplex system of HPA tube 1 (i.e., the first reaction system).

[0045] Figure 8 Optimization of the multiplex system of HPA tube 2 (i.e., the second reaction system). The black line in the figure indicates the melting curve when detecting the HPA-6 system corresponding to the heterozygous template.

[0046] Figure 9 Screening of DMSO concentration in multiple systems of HPA tube 3 (i.e., the third reaction system).

[0047] Figure 10 Melting curve peak diagram and sequencing results of HPA-6 abnormal samples.

[0048] Figure 11 HPA-6 joint interpretation example.

[0049] Figure 12 Melting curves and genotyping results for each tube when HPA assays were performed on an exemplary sample.

[0050] Figure 13 Each channel of HPA tube 1 (i.e., the first reaction system) corresponds to the melting curve of the HPA system.

[0051] Figure 14 Each channel of HPA tube 2 (i.e., the second reaction system) corresponds to the HPA system melting curve.

[0052] Figure 15 Each channel of HPA tube 3 (i.e., the third reaction system) corresponds to the melting curve of the HPA system.

[0053] Figure 16 A three-tube multiplex PCR reaction system for HPA genotyping.

[0054] Figure 17 The typing confirmed by Sanger sequencing did not match the true genotyping of the sample. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The present invention provides an HPA detection reagent and method based on melting curve analysis. This method utilizes the physical property of different nucleotide melting temperatures due to different base sequences after PCR. By monitoring the nucleotide melting process, a characteristic melting curve can be obtained, enabling genotyping. In this method, an excess of reverse primers can be used in asymmetric PCR to amplify a large number of single-stranded positive strand products. In this case, a fluorescently labeled probe designed around the SNP site perfectly matches the antisense strand and exhibits a higher Tm value, while an incompletely matching fluorescently labeled probe exhibits a lower Tm value. In one specific embodiment, multiplex PCR technology based on fluorescent probe melting curves is used to permutate 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. This detection system is not only simple and rapid, but also accurate and efficient, and has high practical value.

[0059] In practical applications, for example, incompatible platelet antigens during transfusion between individuals can trigger platelet alloimmunization and the production of platelet alloantibodies, leading to alloimmune thrombocytopenia and various clinical conditions, including thrombocytopenia ineffectiveness, neonatal alloimmune thrombocytopenia, and post-transfusion purpura. In clinical transplantation, transplant rejection can also occur. In particular, the distribution of HPA gene frequencies varies among different ethnic groups. Therefore, rapid and accurate typing of the HPA antigen system is of vital importance in clinical and transfusion medicine, as well as in genetics and anthropology research. The detection system (including primers and probes) of the present invention is specifically designed for the Chinese population. The targeted HPA antigen system includes HPA gene loci with high-frequency HPA antigen a and low-frequency HPA antigen b. High frequency refers to the frequency of antigen a being no less than 50%, and low frequency refers to the frequency of antigen b being greater than 0.05%. Ultimately, the present invention performs genotyping for a total of 14 HPA antigen systems, including HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31.

[0060] Detection system or reaction solution

[0061] After the specific primers and probes are developed, the present application further finds that, on one hand, the single asymmetric PCR system is not directly applicable to multiplex detection, and on the other hand, the detection primers and probes need to be grouped and further optimized to realize accurate detection of HPA genotyping of Chinese population. For the probes, the present application finds that, for some antigen sites (e.g. HPA-27), increasing the length of the probes can enhance the resolution thereof, while for some antigen sites (e.g. HPA-6, HPA-15), shortening the length of the probes can enhance the resolution thereof. In addition, the present application also optimizes different fluorescent labels and probe types (Taqman or MGB probe). For the primers, in order to ensure the amplification efficiency, the present application optimizes the GC content of the primers, and simultaneously optimizes the additional components in the reaction system.

[0062] After the detection system or reaction solution is constructed, the present application further performs a large number of optimizations applicable to the detection method of the present application, wherein the grouping of the primers and probes takes into account the physical positions of the SNP sites of each antigen system, the fluorescence in each detection system is different and needs to be reasonably combined according to the strength of the fluorescence signal, and the concentrations of the additional components (e.g. dNTP, Mg 2+ , concentration of forward primers and reverse primers, concentration of probes, DMSO, etc.) need to be controlled within a reasonable concentration range.

[0063] In a preferred embodiment, the detection system comprises or consists of a first reaction system, a second reaction system and a third reaction system, wherein the first reaction system comprises:

[0064]

[0065]

[0066] The second reaction system comprises:

[0067]

[0068] The third reaction system comprises:

[0069]

[0070]

[0071] In the present application, the final concentration of MgCl2 in the reaction system is 0.1-10 mM, preferably 1-5 mM, more preferably 2-4 mM, and further preferably 2.5-3.5 mM.

[0072] In the present invention, the final concentration of dNTP in the reaction system is 100-500 μM, preferably 200-450 μM, further preferably 300-450 μM, and further preferably 350-450 μM.

[0073] In the present invention, the concentration of DMSO added to the third reaction system is not less than 4%, for example, it can be 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0074] In the present invention, the final concentration of the probe in the reaction system is 0.1-5 μM, preferably 0.2-1 μM, further preferably 0.4-0.8 μM, and further preferably 0.5-0.7 μM.

[0075] In the present invention, the concentration of the reverse primer is greater than that of the forward primer. The final concentration of the forward primer in the reaction system is 0.01-1 μM, preferably 0.01-0.5 μM, more preferably 0.01-0.1 μM, and further preferably 0.01-0.08 μM, for example 0.02-0.08 μM, 0.02-0.06 μM, or 0.04-0.06 μM. The final concentration of the reverse primer in the reaction system is 0.1-5 μM, preferably 0.2-1 μM, more preferably 0.2-0.8 μM, and further preferably 0.4-0.6 μM.

[0076] In the present invention, the melting curve analysis reaction solution can use a commercially available reaction solution for melting curve analysis, such as the GNM multiple asymmetric amplification and multi-color melting curve PCR reaction solution with the product number RXD03GS.

[0077] 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.

[0078] Method for HPA genotyping of Chinese population

[0079] The present invention provides a method for HPA genotyping in the Chinese population, sometimes referred to herein as the "detection method of the present invention," which includes commercial experimental detection for non-diagnostic purposes as well as detection for diagnostic purposes. Detection methods for diagnostic purposes can be understood as diagnostic uses.

[0080] The detection method of the present invention is not particularly limited to specific processes or steps as long as the detection system or kit of the present invention is used. In an exemplary method, the method of the present invention includes a PCR amplification step, particularly an asymmetric PCR amplification step.

[0081] 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.

[0082] Example

[0083] 1. Selection of HPA genotyping sites and construction of HPA plasmids

[0084] According to the frequencies of high-frequency antigen a and low-frequency antigen b at each HPA locus in the population as shown in Table 1, the present invention selected 14 systems, including HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, for genotyping detection, among which the frequency of HPA low-frequency antigen b was greater than 0.05%.

[0085] Table 1 Frequencies of HPA high-frequency antigen a and low-frequency antigen b

[0086]

[0087]

[0088] For each HPA system to be genotyped, the present invention constructed plasmids for the high-frequency antigen a and low-frequency antigen b of HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, respectively, to serve as detection templates for the established methodology. For the plasmids for the high-frequency HPA antigen a, the present invention used human genomic DNA (gDNA) of known HPA genotypes as a template, amplified the nucleic acid sequence of each HPA using standard PCR methods, and then ligated the amplified sequence into a T or B vector to obtain plasmids for the high-frequency antigen a at each HPA locus. Plasmids for the low-frequency HPA antigen b were generated by point mutation using the constructed plasmid for the high-frequency HPA antigen a. Sanger sequencing results for all constructed HPA plasmids were confirmed to be correct.

[0089] 2. Preliminary design and verification of primers and probes

[0090] Based on the nucleic acid sequence information and corresponding SNPs for HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, the present invention designed specific primers and fluorescently labeled probes. The primer design principles are as follows: 1) The amplification product includes the SNP polymorphisms for HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31, and the length is controlled between 100 and 500 base pairs to facilitate amplification; 2) Primer design avoids HPA SNP polymorphisms; 3) HPA polymorphisms within the same gene with close SNPs are systematically designed within a single primer pair (for HPA-3, -9, -27, and -30, only one specific primer pair, HPA-F3 / R3, is designed). The probe design principles are as follows: 1) Probe length should be 18-25 bp; 2) Probes should encompass the HPA SNP polymorphism, with the SNP located in the center of the probe; 3) A probe should be designed for each HPA locus, with the probe sequence fully matching the gene sequence of the high-frequency antigen a at that HPA locus. The sequences of the preliminary designed primers and probes are shown in Tables 2 and 3.

[0091] Table 2 Preliminary designed primers

[0092]

[0093] Table 3 Preliminary designed probes

[0094]

[0095]

[0096] Note: The bolded ones in the table are the SNP sites corresponding to each HPA system.

[0097] To verify the feasibility of the preliminarily designed primers and probes, the present invention first amplified plasmids of known HPA types using a single-plex asymmetric PCR system: plasmids of either HPA high-frequency antigen a or low-frequency antigen b were used to simulate HPA homozygous templates, and a mixture of high-frequency antigen a and low-frequency antigen b in equal proportions was used to simulate HPA heterozygous templates.

[0098] The experiment adopted a two-step reaction method with the following reaction conditions: 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, fluorescence signal acquisition at 50-70°C, denaturation at 90-98°C for 0.5-3 min, hybridization at 40-55°C for 1-10 min, and gradual heating from 45-90°C.

[0099] The results of single-plex asymmetric PCR on homozygous and heterozygous templates of different HPA systems are shown in Figure 2. Figure 1 and Figure 2 shown. Figure 2The results show that the primers and probes designed for HPA-1-5, 9, 13, 21, and 30 are available: when detecting homozygous templates, the melting curves of HPA-a or HPA-b show a single peak, and the Tm values of HPA-a and HPA-b also have certain differences; when detecting heterozygous templates, the melting curves show double peaks or "steamed bun peaks" that can be judged by software, and the software can simultaneously judge the Tm values of HPA-a and HPA-b, thereby distinguishing the heterozygous templates.

[0100] However, some primers and probes need to be optimized, such as Figure 3 As shown in the figure: the primers and probes for HPA-6 and HPA-15 are normal when detecting homozygous templates and heterozygous templates, the melting curves show a single peak when detecting homozygous templates, and the Tm values of HPA-a and HPA-b also have certain differences; but when detecting heterozygous templates, the melting curves only show a single peak, and the software can only judge one Tm value, which is between the Tm values of HPA-a and HPA-b, so it cannot effectively distinguish HPA-a and HPA-b when detecting heterozygous templates. For HPA-27, when detecting homozygous template HPA-27a, the amplification curve and melting curve are normal, but when detecting homozygous template HPA-27b, the amplification curve and melting curve are both absent, and when detecting heterozygous templates, only the Tm value of HPA-27a can be judged. For HPA-12 and HPA-31, whether it is a homozygous template or a heterozygous template, there is almost no amplification curve and melting curve.

[0101] 3. Optimization of primers and probes

[0102] For the primers and probes mentioned above that need to be optimized, the present application optimizes them by a variety of different strategies.

[0103] Firstly, for HPA-6, in order to solve the problem that it cannot distinguish HPA-6a and HPA-6b when detecting heterozygous templates, the present application has carried out a large number of optimizations, and found that shortening the length of the probe HPA Probe-6 can enhance its discrimination ability, and replacing the fluorescent label of HPA Probe-6 with Atto425 at the same time can greatly improve the amplification curve and melting curve that use CY7 to label HPA Probe-6 initially. The detection results of the optimized HPA-6 on the corresponding plasmid simulating homozygous templates and heterozygous templates are shown in the figure Figure 4 As shown in the figure, the optimized results show that the new probe can effectively distinguish HPA-6a and HPA-6b in the heterozygous template.

[0104] Secondly, for HPA-15, similar to HPA-6, the present invention shortened its probe length and optimized the Taqman probe to an MGB probe. The results showed that HPA-15a and HPA-15b in the hybrid template could be effectively separated. In addition, because HPA Probe-15 exhibited strong fluorescence signal intensity in a single-plex asymmetric PCR system when fluorescently labeled with ROX, considering the subsequent need for multiplex system combination, its fluorescent label was replaced with the slightly weaker fluorescent CY5.5 to ensure balanced fluorescence signal intensity in the multiplex system. The detection results after optimization are as follows: Figure 4 shown.

[0105] The main problem with HPA-27 is that the probe doesn't effectively bind when amplifying HPA-27b, resulting in neither amplification nor melting curves. Because the SNP site of HPA-27 is very close to that of HPA-9, the HPA-27 probe was initially designed to be shorter than other probes, at only 18 bp. This may be the reason for the probe's inability to effectively bind when amplifying HPA-27b. Therefore, the present invention appropriately extends the length of HPA-Probe27 to encompass the HPA-9 SNP site, ultimately effectively resolving the HPA-27b amplification and probe issues while also maintaining detection of the HPA-9 site. The optimized detection results are shown in Figure 2. Figure 4 shown.

[0106] As for HPA-12 and HPA-31, the common problem for both is that no amplification curve or melting curve is found when amplifying HPA-a or HPA-b. Analysis of the gene sequences of HPA-12 and HPA-31 found that the GC content of the HPA-12 and HPA-31 templates is relatively high (both >70%), which is not conducive to the PCR reaction. Therefore, the present invention redesigned primers with lower GC content, and at the same time changed the original CY5.5-labeled probes to ROX and Atto425, which have relatively stronger fluorescent signal intensities, and added DMSO to the system separately to promote the melting of high GC content templates and improve PCR amplification efficiency. The detection results of homozygous templates and heterozygous templates after optimization are as follows: Figure 4 shown.

[0107] 4. Design, screening and optimization of internal standard gene primers and probes

[0108] The present invention uses human whole blood as a sample, extracts human genomic DNA, and then performs testing. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is selected as the internal standard gene to participate in the amplification of the multiplex system. Only when the melting curve of the fluorescence channel corresponding to the internal standard in each well reaction system appears and the Tm value is within the correct range, the result of the well is considered to be valid; otherwise, it is considered to be invalid.

[0109] First, multiple pairs of GAPDH primers and corresponding probes were designed. Since the internal standard gene GAPDH is strongly expressed, in order to avoid the subsequent introduction of internal standard gene primers and probes affecting the detection of other targets of the HPA system in the multiplex system, FAM with relatively weak fluorescence signal intensity was selected to label the internal standard specific probe.

[0110] Then, the extracted human gDNA samples were amplified using the aforementioned single-plex asymmetric PCR method to screen for the most suitable specific primers and probes for amplifying GAPDH. Figure 5 As shown in the figure, the GAPDH-F / R primer pair and its corresponding probe, GAPDH-Probe, were selected based on the Ct values ​​of the amplification curves and the peak shapes of the melting curves. This combination resulted in a single, smooth melting curve peak. The Ct values ​​of the amplification curves were also relatively delayed, indicating that they would not excessively inhibit the amplification of other HPA targets in the multiplex system. Therefore, the GAPDH-F / R primers and GAPDH-Probe probe were selected as the internal standard gene primers and probes for GAPDH detection, ultimately added to the multiplex reaction system to monitor the effectiveness of the reaction system. The specific sequences of the GAPDH primers and probe are shown in Table 4.

[0111] Table 4 GAPDH primer and probe sequences

[0112]

[0113]

[0114] 5. Preliminary combination and verification of multiple systems

[0115] After designing, verifying and optimizing the primers and probes for each HPA system and the internal standard gene, a preliminary multiplex system combination was performed using the optimized primers and probes, resulting in a total of three tubes of multiplex asymmetric PCR systems. The primers and probes for each HPA system detected in each tube of the multiplex system were as follows: Figure 6 As shown in the figure, HPA tube 1 detects the HPA system including HPA-1, 4, 6, 21, and 3; HPA tube 2 detects the HPA system including HPA-2, 9, 5, 13, and 15; and HPA tube 3 detects the HPA system including HPA-27, 30, 12, and 31. Since HPA-3 includes HPA-12 and HPA-31, DMSO was added to the multiplex system, similar to the singleplex system, to promote the amplification of the high GC content templates of HPA-12 and HPA-31.

[0116] To evaluate the feasibility of simultaneous amplification of various HPA systems within a multiplex system, we first combined specific primers and probes for the corresponding HPA systems in each reaction tube without adding internal standard specific primers and probes. We then conducted a preliminary validation using human gDNA samples with known HPA genotypes. The experimental results are shown in Figure 2. Figure 6 shown.

[0117] The results showed that, overall, the peak Rm values ​​of the melting curves for each probe in the multiplex system of HPA tube 1 were slightly weaker than their respective Rm values ​​in the singleplex PCR system. Furthermore, when combining different fluorescently labeled probes in the multiplex system, the peak Rm values ​​of their melting curves also varied. The peak Rm value of the melting curve of VIC-labeled HPA Probe-4 was weaker than that of the other probes. Furthermore, after combining HPA-Probe 6 into the multiplex system and amplifying human gDNA samples, a new nonspecific peak appeared in the melting curve. The Tm value corresponding to this nonspecific peak was similar to the Tm value of this probe for detecting HPA-6b, potentially affecting the interpretation of HPA-6 genotyping results.

[0118] Similar to HPA tube 1, the multiplex system for HPA tube 2 showed a decrease in the peak Rm of the melting curves for each probe after combining different primers and probes into the multiplex system. Furthermore, the presence of a large pit in the melting curve for HPA-13 labeled with CY7 also detracted from the overall aesthetics.

[0119] HPA Tube 3 showed no significant issues with the overall amplification of the multiplex system, and the melting curve peak Rm was relatively uniform. Although a new nonspecific peak appeared when amplifying human gDNA samples with CY5-labeled HPA Probe-27, the Tm value of this nonspecific peak did not affect the interpretation of the final results.

[0120] In summary, except for the newly appeared hybrid peak of HPA Probe-6 in HPA tube 1, which affected the final result interpretation, the other multiplex system combinations of primers and probes correctly typed all known HPA genotyped samples, proving that the combination of the preliminary multiplex system is feasible.

[0121] 6. Optimization of multiple systems

[0122] 6.1 Optimization of HPA Tube 1

[0123] For HPA tube 1, we first took a lot of optimization measures to address the non-specific peaks that appeared in HPA Probe-6 and affected the interpretation of the final results. We found that the new probe redesigned for the HPA-6 SNP site can improve the interpretation of the results. The experimental results after optimization are as follows: Figure 7 As shown in A.

[0124] Secondly, since the peak Rm of the melting curve of each probe is slightly weaker than the Rm value in the single-plex PCR system, in order to improve the signal of the melting curve peak, the concentration of dNTP in the first HPA tube was optimized, and different Mg 2+ Concentration gradient to screen the optimal dNTP concentration and its corresponding Mg 2+ Finally, the gDNA samples with known HPA genotyping were tested for the concentration of three dNTPs and Mg 2+ The concentration combination was screened and the experimental results after optimization were as follows Figure 7 As shown in B. It can be found that the results of HPA genotyping using these three combinations are all correct, and the peak Rm of the second group of melting curves is relatively high, so the second group of dNTPs and Mg 2+ The concentration combination was used as the final reaction condition of HPA-1.

[0125] Finally, to address the weak signal intensity of VIC-labeled HPA Probe-4, in order to improve the signal intensity of the VIC channel and make the overall signal intensity more uniform, the forward primer and reverse primer corresponding to the HPA-4 system were increased to a certain extent; accordingly, the concentration of the HPA Probe-4 probe was also increased. The experimental results after optimization are as follows: Figure 7 As shown in C. It can be found that increasing the concentration of specific primers and probes corresponding to the HPA-4 system effectively increases the fluorescence signal intensity of HPA-4.

[0126] 6.2 Optimization of HPA Tube 2

[0127] As for HPA tube 2, we first addressed the "big pit" in the melting curve of CY7-labeled HPA-13 by changing its fluorescent label to Atto425, which to some extent improved the aesthetics of the melting curve finally displayed by the multiplex system. Figure 8 As shown in A.

[0128] Secondly, similar to HPA tube 1, in order to improve the signal intensity of each melting curve peak, the concentration of dNTP in HPA tube 2 was also optimized, and different Mg 2+ Finally, the gDNA samples with known HPA genotyping were tested for the three dNTPs and Mg 2+ The concentration combinations of Figure 8 As shown in B. It can be found that the results of genotyping using these three combinations are all correct, and for HPA tube 2, the peak value Rm of the second group of melting curves is relatively high. 2+The concentration combination of DMSO as the final reaction condition of HPA tube 2.

[0129] 6.3 Optimization of HPA tube 3

[0130] For HPA tube 3, due to the presence of two high GC content templates HPA-12 and HPA-31, the initial combination of multiple systems directly analogizes the addition of a certain concentration of DMSO in the single system to the multiple system. In order to explore a more suitable concentration of DMSO for the multiple system, five different DMSO concentration gradients: 1%, 2%, 3%, 4%, 5% were further set, and the known HPA typing human gDNA samples were detected, and the results are shown in Figure 9 The results show that when the DMSO concentration is <4%, HPA-12 and HPA-31 have poor amplification effect due to high GC content of the template. Only when the DMSO concentration is >4%, HPA-12 and HPA-31 can be well amplified. Therefore, not less than 4% concentration of DMSO is finally added to the system.

[0131] 7. Detection and further optimization of multiple detection system applied to clinical samples

[0132] After the above optimization of each aspect of the three-tube multiplex system of HPA tube 1, tube 2 and tube 3, a three-tube multiplex PCR reaction system was initially established. Then 100 clinical samples were collected, and gDNA was extracted from whole blood using the kit, and then detected using gDNA as the template. Most samples were detected, and the melting curve of each tube was normal, and the result interpretation of each HPA site to be typed was also normal. However, there were 9 samples in which the melting curve of HPA tube 1 HPA-Probe6 only showed a non-specific peak, and no specific peaks of a or b appeared (as shown in Figure 10 A-C of FIG. 6). After rechecking and verifying that the reason was not an operation problem and a reagent problem, Sanger sequencing was performed on the 9 samples after amplification by ordinary PCR. The Sanger sequencing results showed that the 9 samples were all HPA-6a homozygous samples. Then the sequence obtained by sequencing was compared with the sequence used for designing the primer probe by importing MEGA7, and the comparison results are shown in Figure 10 D of FIG. 6. It can be found that the commonality of the 9 samples is that compared with the sequence used for designing the primer probe, a G>A mutation occurs at the right adjacent base of the SNP site of HPA-6, and a A>G mutation occurs at the left adjacent 10 bases of the SNP site of HPA-6. The probe used in the multiple detection system contains both sites, and it is speculated that the 9 samples are due to too many mismatching sites between the template sequence and the probe, which causes the probe to fail to stably bind to the template sequence, so that the melting curve cannot be formed.

[0133] To optimize the HPA-Probe6 probe, the length was first shortened to avoid the A>G mutation within the 10 bases to the left of the HPA-6 SNP. Since the A>G mutation within the bases to the right of the HPA-6 SNP cannot be avoided, a base mismatch strategy was employed, with the probe design replacing the base at the altered site with a C. This ensured that the Tm value of the probe's melting curve remained consistent regardless of whether the base was an A or a G, ensuring detection of all HPA-6a variants. However, due to the shortened probe length, the probe designed with this mismatch strategy performed poorly for HPA-6b. A comparison of the performance of the pre-optimized probe (which detected HPA-6a without mutations adjacent to the SNP and all HPA-6b variants) with the probe designed with the mismatch strategy (which detected all HPA-6a variants but not HPA-6b variants) revealed that the combined use of these two probes could detect all possible HPA-6 variants. Therefore, the system was further optimized: the pre-optimized probe was named HPAProbe6-1, still labeled with Atto425, and placed in HPA tube 1. The mismatch strategy probe was named HPA Probe6-2, also labeled with Atto425, and placed in tube 3. Meanwhile, the original Atto425-labeled probe HPA Probe30 in tube 3 was replaced with CY5.5. When interpreting HPA-6 results, for HPA-6a, the peak of HPA Probe6-1 was first observed. If HPA Probe6-1 showed a peak specific to HPA-6a, the interpretation was normal. If HPA Probe6-1 did not show a peak specific to HPA-6a, the peak of HPA Probe6-2 was observed. If HPA Probe6-2 showed a peak specific to HPA-6a, it was HPA-6a with a mutation to the right of the HPA-6 SNP site. As for the interpretation of HPA-6b, it is only necessary to observe whether the corresponding b-specific peak appears in HPA Probe6-1 (such as Figure 11 shown).

[0134] 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 system for the three-tube multiplex PCR is shown in Tables 7-9. The experimental reaction conditions were the same as those for the singleplex asymmetric PCR described above.

[0135] Table 5 Primers used for HPA genotyping

[0136]

[0137]

[0138] Table 6 Probes used for HPA genotyping

[0139]

[0140] Table 7 HPA Tube 1 Multiple Reaction Detection System (1 person)

[0141]

[0142]

[0143] Table 8 HPA Tube 2 Multiple Reaction Detection System (1 person)

[0144]

[0145]

[0146] Table 9 HPA Tube 3 Multiple Reaction Detection System (1 person)

[0147]

[0148]

[0149] The optimized system was used to retest the nine samples that had previously tested abnormal in HPA-6, and another 100 clinical samples were collected. After extracting gDNA from whole blood, the samples were tested again using gDNA as a template. Figure 12 The figure shows the melting curve of each tube when testing one of the samples. Figure 13-15 It shows the detection of homozygotes and heterozygotes of the corresponding HPA system under different fluorescence channels in each reaction tube. Figure 16 Shown is the three-tube multiplex PCR reaction system ultimately used to detect HPA genotyping.

[0150] 8. Interpretation Rules

[0151] Due to HPA-b gene frequency and sample size limitations, some HPA systems currently only have HPA-a test data. Therefore, in Table 10, which lists reference Tm values ​​for HPA locus genotyping for different fluorescence channels on each HPA tube, the corresponding Tm values ​​for HPA-b without sample test data are based on previous plasmid testing results.

[0152] Table 10 Reference Tm values ​​of HPA locus genotyping corresponding to different fluorescence channels of each HPA tube

[0153]

[0154]

[0155] Note: The bold values ​​in the table are the corresponding Tm values ​​given based on the previous plasmid test results.

[0156] In order to verify the accuracy of the detection system, the test results of all 200 clinical samples were compared with the test results of the commercial HPA genotyping kit (item number: WY05E00101064), which covers 9 HPA antigen systems, HPA-1-6, 10, 15, and 21. The results showed that for the HPA systems 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 of 1, 7, and 9 samples, respectively, were inconsistent. For these abnormal samples, confirmation experiments were further carried out using Sanger sequencing, and the results showed that the sequencing results were consistent with the system detection results of this embodiment, and the commercial kit had misjudgments in many cases (such as Figure 17 Overall, the detection system of the present invention exhibits higher accuracy than the commercial kit.

[0157] 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 reagent for HPA genotyping detection in the Chinese population, characterized in that: It includes the following three components: (1) A first component comprising a primer set 1 having sequences as shown in SEQ ID Nos. 1, 2, 5-8, 11, 12, 19, and 20 and a probe set 1 having sequences as shown in SEQ ID Nos. 23, 25, 26, 28, and 36; (2) The second component includes a primer set 2 having sequences as shown in SEQ ID Nos. 3-6, 9, 10, 15-18 and a probe set 2 having sequences as shown in SEQ ID Nos. 24, 27, 30, 32, and 35; (3) The third component: It includes a primer set 3 whose sequences are shown as SEQ ID No. 5, 6, 11-14, 21, and 22, and a probe set 3 whose sequences are shown as SEQ ID No. 29, 31, 33, 34, and 37.

2. The reagent for HPA genotyping detection in Chinese population according to claim 1, characterized in that: The first component, the second component and the third component are respectively used to form three different reaction systems.

3. The reagent for HPA genotyping detection in Chinese population according to claim 1, characterized in that: Each probe in probe set 1, probe set 2, and probe set 3 has a different fluorescent group.

4. The reagent for HPA genotyping detection in Chinese population according to claim 1, characterized in that: The HPA genotyping includes HPA 1-6, 9, 12, 13, 15, 21, 27, 30, and 31 gene loci.

5. The reagent for HPA genotyping detection in Chinese population according to claim 1, characterized in that: The probes of probe group 1, probe group 2 and probe group 3 are as follows:

6. The reagent for HPA genotyping detection in Chinese population according to claim 1, characterized in that: Further includes an internal standard gene detection reagent.

7. A detection system or reaction solution for HPA genotyping detection in Chinese population, characterized in that: include: a. A first reaction system comprising a primer set 1 having a sequence as shown in SEQ ID No.1, 2, 5-8, 11, 12, 19, 20 and a probe set 1 having a sequence as shown in SEQ ID No.23, 25, 26, 28, 36; b. A second reaction system comprising a primer set 2 having sequences such as SEQ ID No.3-6, 9, 10, 15-18 and a probe set 2 having sequences such as SEQ ID No.24, 27, 30, 32, 35; c. The third reaction system comprises a primer set 3 whose sequences are shown as SEQ ID No. 5, 6, 11-14, 21, 22 and a probe set 3 whose sequences are shown as SEQ ID No. 29, 31, 33, 34, 37.

8. The detection system for HPA genotyping in the Chinese population according to claim 7, characterized in that: The first reaction system comprises: or The second reaction system comprises: or The third reaction system comprises:

9. A method for HPA genotyping in Chinese population, characterized in that: The method comprises the step of using the reagent according to any one of claims 1 to 6.

10. The method for HPA genotyping in Chinese population according to claim 9, characterized in that: The method further comprises a judgment step, wherein the judgment rule is:

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