A probe, method and kit for discriminating ABO blood group genotyping and A2, O3 subtypes

Abstract

This invention provides a probe-based quantitative polymerase chain reaction (qPCR) method for precise typing of ABO blood group genes and some subtype genes. By designing multiple sets of allele-specific primers and corresponding double-labeled oligonucleotide probes, this method utilizes the primer-probe combination in the reaction system. During PCR amplification, the Taq enzyme hydrolyzes the probes, separating the reporter and quencher groups and generating a fluorescence signal proportional to the amplification amount of the target DNA. The kit developed in this invention can identify ABO blood types and detect common ABO subtype genes simultaneously, making it suitable for rapid testing of routine and complex blood typing samples in clinical settings.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biology, specifically to a probe, method, and kit for identifying ABO blood group genotyping and A2 and O3 subtypes. Background Technology

[0002] The ABO blood group system is the most important and fundamental blood group system in human transfusion medicine and organ transplantation. The ABO blood group system is characterized by high genetic polymorphism. The ABO gene encodes glycosyltransferases that synthesize specific antigenic determinants; therefore, key single nucleotide polymorphisms (SNPs) are directly related to the expression of blood group antigens. Accurate ABO blood typing is crucial for ensuring clinical transfusion safety, preventing hemolytic transfusion reactions, and improving transplant success rates. Traditional serological methods, which classify blood types by detecting red blood cell surface antigens, are simple to perform but prone to misdiagnosis in cases of blood type subtype, neonates, or patients with weakened antigens due to certain disease states, posing significant clinical safety risks.

[0003] With the maturation of molecular biology techniques, ABO blood typing has become a key method for solving difficult serological identification problems. This technology designs primers or probes targeting specific mutation sites of the ABO gene and its subtypes (such as c.467C>T, c.802G>A) to directly analyze blood type at the gene level, overcoming the limitations of serology in subtype identification. Currently, common molecular typing techniques include: PCR-Restriction Fragment Length Polymorphism (PCR-RFLP): cumbersome, time-consuming, and dependent on specific restriction enzyme sites, unable to cover all mutation types. PCR-Sequence Specific Primers (PCR-SSP): cumbersome, result analysis relies on open-top electrophoresis, greatly increasing the risk of aerosol contamination and potentially leading to false positives. Furthermore, the interpretation of gel electrophoresis results relies on human experience, and the identification of weak or non-specific bands may be subject to subjective errors, leading to misinterpretations. Sequencing methods (first-generation, second-generation, and third-generation sequencing): capable of detecting ABO gene and subtype mutations, but costly, complex data analysis, and time-consuming, making them unsuitable for rapid and large-scale clinical testing. Gene chip technology: high throughput, but the platform is expensive and requires a large initial investment, so it is not widely used in routine clinical laboratories.

[0004] Quantitative real-time PCR (qPCR) technology is widely used in pathogen detection and gene expression analysis due to its high sensitivity, closed-tube operation (preventing contamination), and rapid speed (1-2 hours). This technology is mainly divided into two categories: dye-based methods (such as SYBR Green I) and probe-based methods (such as TaqMan).

[0005] In publicly available technical solutions, ABO genotyping or ABO subtype genotyping largely relies on PCR-SSP or dye-based qPCR melting curve analysis. However, PCR-SSP requires electrophoresis analysis, which is complex and prone to contamination. Dye-based qPCR relies on melting curve analysis, has poor specificity, and is susceptible to interference from non-specific amplification. The dye binds to all double-stranded DNA in the reaction system (including primer dimers and any non-specific amplification products), generating heterogeneous peaks, leading to difficulties in result interpretation or even misinterpretation. Furthermore, when the Tm values ​​of amplification products corresponding to different SNP sites are very close, the melting curves cannot effectively distinguish them. In addition, sequencing technologies, including Sanger sequencing, second-generation sequencing, and third-generation sequencing, are also used as a solution for ABO genotyping or ABO subtype genotyping, but their inherent high cost, long cycle time, and complex data analysis limit their application in routine rapid clinical testing.

[0006] At the market level, several internationally renowned diagnostic companies have become major players in this field. For example, companies like OrthoClinical Diagnostics, Bio-Rad Laboratories, Grifols, and QuidelOrtho primarily offer solutions focused on serological testing or microplate agglutination assays. Meanwhile, companies like Illumina and Qiagen focus more on providing high-throughput, broad-spectrum molecular diagnostic platforms such as gene chips, which are more expensive and less widely used in routine clinical laboratories.

[0007] In existing technologies, the "ABO genotyping method based on SYBR Green I real-time quantitative PCR combined with melting curve analysis" is considered the closest to the present invention. This method designs primers targeting different alleles of the ABO gene, amplifies the gene using conventional qPCR, and then performs melting curve analysis. Because the amplification products of different alleles differ in sequence length and base composition, their melting temperatures (Tm values) also differ. The ABO genotype of the sample is inferred by analyzing the characteristic peaks of the Tm values ​​of the products. Although this method attempts to use qPCR for genotyping, avoiding the cumbersome process of traditional electrophoresis, its inherent limitations make it difficult to meet clinical needs for accurate ABO genotyping, especially subtype identification. The main problems are as follows: (1) Poor specificity and susceptible to interference from non-specific amplification: SYBR Green dye binds to all double-stranded DNA in the reaction system (including primer dimers and any non-specific amplification products) to generate fluorescence signals. These non-specific products can interfere with the shape of the melting curve, producing impurities and making it difficult to interpret the results or even misinterpret them, which seriously affects the specificity and accuracy of the detection.

[0008] (2) Insufficient resolution of key SNPs: The core of ABO subtyping lies in identifying several key SNP sites. However, when the Tm values ​​of the amplification products corresponding to different SNP sites are very close, the melting curves cannot effectively distinguish them. For example, when identifying the key SNP cluster of the A2 subtype, its melting peak may overlap with the peaks of wild-type A1 or other variants, making it impossible to clearly and unambiguously identify key alleles.

[0009] (3) The process is not completely simplified: Although the amplification process is closed, it still relies on the additional and time-consuming step of melting curve analysis. Data analysis is easily interfered with, and the interpretation experience of operators is highly required, which is not conducive to achieving fully automatic and standardized result output.

[0010] The probe-based real-time PCR technique is simple to operate, highly specific, and fast (less than 2 hours) to detect. Some literature has reported its application in ABO genotyping, but these studies focus on simple ABO blood typing and cannot detect complex ABO subtype alleles.

[0011] In summary, the existing closest technical solutions have significant shortcomings in terms of detection specificity, resolution of key SNPs, and process simplification due to the inherent limitations of their methodologies. This creates space and necessity for the proposal of this invention. Summary of the Invention

[0012] This invention is based on the principle of probe-based quantitative polymerase chain reaction (qPCR). By designing multiple sets of allele-specific primers and corresponding double-labeled oligonucleotide probes, it achieves precise typing of ABO blood group genes and some subtype genes. This method utilizes the primer-probe combination in the reaction system. During PCR amplification, the hydrolysis by Taq enzyme separates the reporter group and quencher group on the probe, generating a fluorescence signal proportional to the amplification amount of the target DNA.

[0013] To achieve this objective, the present invention employs the following technical solution: On the one hand, the present invention provides a specific primer and TaMan probe for identifying ABO blood group genotyping and A2 and O3 subtypes, characterized in that the sequences of the primer and probe are as shown in SEQ ID No. 1-28.

[0014] On the other hand, the present invention provides a kit for ABO blood group genotyping and identification of A2 and O3 subtypes, characterized in that it contains probe primers as shown in SEQ ID No. 1-28.

[0015] Furthermore, the kit also includes a reaction solution consisting of 2×qPCRMasterMix and an aqueous component.

[0016] On the other hand, the present invention also provides a method for ABO blood group genotyping and identification of A2 and O3 subtypes, characterized by comprising the following steps: (1) Collect 200 μL of EDTA-anticoagulated whole blood from the subjects to be tested and extract genomic DNA; (2) Remove the required amount of reagent from the freezer, thaw at room temperature, and vortex to mix. (3) Add 12.5 μL of 2×qPCRMasterMix, 1.25 μL of the corresponding forward and reverse primers, 0.3 μL of the corresponding FAM probe, 0.5 μL of the corresponding HEX probe, and 9.2 μL of deionized water to each AG detection well, and add 1 μL of the DNA to be tested. The total volume of each qPCR reaction system is 25 μL. The composition of the qPCR amplification system is shown in Table 2: (4) Add 12.5 μL of 2×qPCRMasterMix and 12.5 μL of deionized water to the H well (blank control well); (5) After adding the ingredients, seal the cap, centrifuge briefly, and then load the product onto the machine; (6) Place the sealed PCR plate into a real-time PCR instrument for PCR amplification; (7) Perform quality control and result interpretation on the experimental results according to the result classification table. A positive result is one with an amplification curve and Ct≤37, and a negative result is one without an amplification curve.

[0017] Furthermore, in step (1), the DNA concentration is 20-60 ng / μL, and the A260 / A280 value is 1.6-2.0.

[0018] Furthermore, the conditions for PCT amplification in step (6) are: pre-denaturation at 95°C for 10 minutes, followed by 38 cycles of reaction at 95°C for 10 seconds and 59°C for 40 seconds.

[0019] The beneficial effects of this invention are as follows: By monitoring the signal intensity of different fluorescence channels in real time, results can be directly interpreted based on whether each channel is amplified and the Ct value, without relying on melting curve analysis. The dual fluorescence detection channels in each detection system serve as controls, providing strict quality control for system effectiveness. This method uses closed-tube operation, effectively avoiding cross-contamination. Simultaneously, the high specificity of the probes accurately identifies key SNP sites, and finally, qualitative interpretation of ABO genotype and subtype is completed according to the preset positive / negative combination rules, fully demonstrating the technical advantages of simple operation, accurate results, and high throughput. The kit established in this invention can identify ABO blood types while also detecting common ABO subtype genes (such as A201, A205, O3, etc.), making it suitable for rapid detection of routine and difficult blood type samples in clinical practice. Detailed Implementation

[0020] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0021] Example 1: Primer and Probe Design Based on the ABO gene sequence (reference sequence: NG_006669.2) published in the National Center for Biotechnology Information (NCBI) database, specific primers and a dual-channel TaqMan-MGB probe (FAM channel for detecting wild-type sites, HEX channel for detecting mutant sites) were designed targeting the ABO gene sites. All sequences were validated for high specificity by BLAST. A total of 7 sets of primers and probes were developed, and the sequence listings of these 7 sets of specific primers and TaMan probes are shown in Table 1, SEQ ID Nos. 1-28. The first set of specific primers and TaMan probes, as shown in SEQ ID No. 1-4, were used to detect the genetic locus corresponding to the O01 / O02 gene in the ABO gene (mutation site at position 261 in the coding region). The second set of specific primers and TaMan probe primers, as shown in SEQ ID No. 5-8, are used to detect the genetic locus corresponding to the O02 gene in the ABO gene (mutation site at position 681 in the coding region). The third set of specific primers and TaMan probes, as shown in SEQ ID No. 9-12, detect the genetic locus corresponding to gene B in the ABO gene (mutation site at position 796 in the coding region). The fourth set of specific primers and TaMan probes, as shown in SEQ ID No. 13-16, were used to detect the genetic locus corresponding to gene B in the ABO gene (mutation site at position 703 in the coding region). The fifth set of specific primers and TaMan probes, as shown in SEQ ID No. 17-20, were used to detect the genetic locus corresponding to the A201 gene in the ABO gene (mutation site at position 1009 in the coding region). The sixth set of specific primers and TaMan probes, as shown in SEQ ID No. 21-24, were used to detect the genetic locus corresponding to the A205 gene in the ABO gene (the mutation site at position 1061 in the coding region). The seventh set of specific primers and TaMan probes, as shown in SEQ ID No. 25-28, were used to detect the genetic locus corresponding to the O03 gene in the ABO gene (mutation site at position 53 in the coding region). Table 1 Primer and probe set information Specific primers and TaMan probes targeting different ABO alleles are placed in separate reaction wells. The FAM and HEX labeled probes can detect ABO blood type information in the samples and also serve as control wells for quality control of the detection system. Furthermore, the detection results from each well can be interpreted collaboratively and cross-validated. Therefore, this invention achieves accurate ABO blood typing while also implementing built-in quality control of the detection process, ensuring the accuracy of the analysis.

[0022] Example 2: Composition of the kit and probe-based real-time PCR for ABO blood group genotyping and identification of A2 and O3 subtypes. These seven probe and primer sets were used to prepare a probe-based real-time PCR kit for ABO blood group genotyping and A2 / O3 subtype identification. The kit also includes a reaction solution consisting of 2×qPCRMasterMix and water. The kit is available in 8-well / person kits (well positions are shown in Table 2), in either single-person (8-well PCR) or 12-person (96-well PCR plate) specifications, and comes with the corresponding number of sealing films / caps. The usage steps of this kit are as follows: Table 2. Detection Hole Location Table and Result Analysis 1. Collect 200 μL of EDTA-anticoagulated whole blood from the subject to be tested, and extract genomic DNA. The required DNA concentration is 20-60 ng / μL, and the A260 / A280 value is 1.6-2.0. 2. Remove the required amount of reagent from the freezer, thaw at room temperature, and vortex to mix. 3. Add 12.5 μL of 2×qPCRMasterMix, 1.25 μL of the corresponding forward and reverse primers, 0.3 μL of the corresponding FAM probe, 0.5 μL of the corresponding HEX probe, and 9.2 μL of deionized water to each AG detection well. Add 1 μL of the DNA to be tested. The total volume of each qPCR reaction system is 25 μL. The composition of the qPCR amplification system is shown in Table 2. 4. Add 12.5 μL of 2×qPCRMasterMix and 12.5 μL of deionized water to the H well (blank control well); 5. After adding the ingredients, seal the cap, centrifuge briefly, and then transfer to the machine; 6. Place the sealed PCR plate into a real-time PCR instrument for PCR amplification. Perform pre-denaturation at 95℃ for 10 minutes, followed by 38 cycles of reaction at 95℃ for 10 seconds and 59℃ for 40 seconds. 7. Perform quality control and result interpretation based on the result classification table (Tables 2 and 3). A positive result is indicated by an amplification curve and Ct ≤ 37, while a negative result is indicated by the absence of an amplification curve. The blank control well should not show an amplification curve. At least one channel of the FAM and HEX channels in the AG detection well should show a positive result; otherwise, the detection well is considered invalid and the detection fails.

[0023] Table 3 Interpretation Table of Detection Hole Results Example 3: Performance evaluation of probe-based real-time PCR detection: The invented probe-based real-time PCR kit was evaluated by comparing the results of 100 samples with those of a control reagent (Sanger sequencing). All sample results were identical to the sequencing genotyping results, showing 100% consistency (Table 4). Table 4: Performance of the probe-based real-time PCR kit and Sanger sequencing for ABO blood type and subtype detection. The invented probe-based real-time PCR kit was used to test 10 samples with inconsistent serological typing, making it difficult to determine blood type. It was able to provide accurate ABO typing results for the 10 samples and distinguish between A2 and O3 subtypes (Table 5).

[0024] Table 5. Serological results and analysis results of probe-based quantitative PCR kit for 10 cases with discrepancies between forward and reverse typing. Serological results are indicated by the intensity of agglutination of the sample with the corresponding reagent antibody or indicator cell, w: weak agglutination.

[0025] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A specific primer and TaMan probe for identifying ABO blood group genotyping and A2, O3 subtypes, characterized by, The sequences of the specific primers and TaMan probes are shown in SEQ ID No. 1-28.

2. A kit for ABO blood group genotyping and A2, O3 subtyping, characterized in that, The kit contains the probe primers as described in claim 1.

3. The kit of claim 2, wherein The kit also contains a reaction solution consisting of 2×qPCRMasterMix and an aqueous component.

4. A method for ABO blood group genotyping and A2, O3 subtype discrimination, characterized by, The method includes the following steps: (1) Collect 200 μL of EDTA-anticoagulated whole blood from the subjects to be tested and extract genomic DNA; (2) Remove the required amount of reagent from the freezer according to claim 2, thaw at room temperature, and vortex to mix. (3) Add the corresponding forward and reverse specific primers, the corresponding FAM probe, the corresponding HEX probe, and deionized water to the AG detection wells respectively, and then add the DNA to be tested for amplification: (4) Add 12.5 μL of 2×qPCRMasterMix and 12.5 μL of deionized water to the H well (blank control well); (5) After adding the ingredients, seal the cap, centrifuge briefly, and then load the product onto the machine; (6) Place the sealed PCR plate into a real-time PCR instrument for PCR amplification; (7) Perform quality control and result interpretation on the experimental results according to the result classification table. A positive result is one with an amplification curve and Ct≤37, and a negative result is one without an amplification curve.

5. The method according to claim 4, characterized in that, The DNA concentration in step (1) is 20-60 ng / μL, and the A260 / A280 value is 1.6-2.

0.

6. The method according to claim 4, characterized in that, The PCR amplification conditions in step (6) are: pre-denaturation at 95℃ for 10 minutes, followed by 38 cycles of reaction at 95℃ for 10 seconds and 59℃ for 40 seconds.

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