A method for direct sequencing and typing of ABO blood group genes without nucleic acid extraction and its application

Abstract

This invention provides a simple, rapid, low-cost, high-throughput, and accurate integrated solution that eliminates the need for nucleic acid extraction and purification. It enables targeted capture, amplification, and first-generation sequencing analysis of all key regions of the ABO gene directly from various clinical samples, including whole blood, saliva, oral swabs, and dried blood spots. The operation is simple and low-cost, thus achieving accurate ABO blood typing, subtype analysis, and the discovery of new variants. The method described in this invention can be applied to the identification of difficult blood types, and can discover new ABO alleles, blood group gene chimeras, and rare blood types such as subtype-related intron mutations.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals and biotechnology, specifically to a method for direct sequencing and typing of ABO blood group genes without nucleic acid extraction and its application. Background Technology

[0002] The ABO blood group system is the most important blood group system for humans. ABO blood type incompatibility can trigger fatal acute hemolytic transfusion reactions, playing a crucial role in neonatal hemolysis and organ transplantation. Therefore, accurate ABO blood typing is an indispensable first step in ensuring clinical transfusion safety. However, conventional serological methods relying on antigen-antibody reactions are easily affected by various factors such as disease state, subtype, autoantibodies, irregular antibodies, and false or weak agglutination, which can lead to difficulties in typing or misinterpretation of results, failing to consistently meet the absolute accuracy requirements of clinical practice.

[0003] To overcome the limitations of serological methods, and with the development of genomics and precision medicine, DNA sequence-based blood typing technology has emerged. This technology analyzes the genetic nature of ABO blood types at the gene level, providing a more direct and reliable tool for typing, and has irreplaceable advantages in solving difficult clinical blood type identification and ensuring transfusion safety. The ABO gene is located on chromosome 9 (9q34.1-34.2) and contains 7 exons ranging in length from 28-688 bp and 6 introns of approximately 19514 bp, with a total length of about 18-20 kb. Its gene encoding product is glycosyltransferase, with most of the coding sequences located on exons 6 and 7, responsible for encoding the catalytic region of glycosyltransferase. These transferases control the biosynthesis of ABO blood group antigens, thereby determining blood type.

[0004] Currently, sequence-specific primer PCR (PCR-SSP) and fluorescence quantitative PCR are commonly used ABO genotyping methods in clinical practice. However, these methods are essentially targeted detection methods for "known" mutations. The design of these methods relies on pre-set known sequences and cannot perform comprehensive sequence scanning of key regions of the ABO gene. Therefore, it is difficult to discover new and unknown gene variations, and they have inherent defects in resolving complex genotypes and rare subtypes.

[0005] Sequencing technology can directly obtain the complete nucleotide sequence of the target gene fragment, identify unknown mutations, and is regarded as the gold standard for gene testing. This has enabled blood type analysis to move from serological phenotypic identification to a new stage of precise genotypic decoding, making it possible to identify rare subtypes and discover new mutations.

[0006] While first-generation sequencing (Sanger sequencing) offers high accuracy and simple, direct data analysis, current methods require the independent amplification and sequencing of each exon of the ABO gene, resulting in a cumbersome process. Second-generation sequencing, though boasting high throughput, relies on complex library construction and bioinformatics analysis, leading to long testing cycles and low cost-effectiveness. Third-generation single-molecule sequencing offers the advantage of long reads, but suffers from a high initial error rate and extremely high equipment and data management costs. These technical challenges collectively create a contradiction between high accuracy, high throughput, speed, convenience, and low cost, severely restricting the widespread deployment of ABO gene sequencing technology in clinical transfusion safety and other scenarios requiring rapid and universal application.

[0007] Regardless of the ABO genotyping technology mentioned above, a common bottleneck lies in sample pretreatment, with nucleic acid extraction being an indispensable step. Existing sequencing-based methods almost universally rely on cumbersome and time-consuming genomic DNA extraction and purification procedures. This not only increases operational time, cost, and the risk of human error, but also makes the technology difficult to promote in resource-constrained scenarios (such as on-site emergency care and primary blood banks) or for applications involving trace amounts or degraded samples (such as dried blood spots).

[0008] Existing ABO genotyping methods typically require cumbersome and time-consuming nucleic acid extraction and purification steps. This not only increases operation time, cost, and the risk of human error, but also limits their application in rapid on-site testing and large-scale screening.

[0009] Existing methods have poor compatibility with complex samples. For samples containing PCR inhibitors, such as saliva and oral swabs, or dried blood spots that have been stored for a long time and whose DNA may have degraded, conventional methods are prone to typing failure or decreased accuracy because they heavily rely on high-quality DNA templates. Moreover, traditional genotyping techniques are based on DNA extraction methods and have strict requirements on sample quality and type. For non-standard or trace samples such as saliva, oral swabs, and dried blood spots, typing failure or decreased accuracy often occurs due to poor DNA quality, the presence of inhibitors, or insufficient sample volume.

[0010] Conventional methods such as PCR-SSP or PCR-RFLP can only detect known and limited specific SNP sites, and cannot achieve complete sequencing of key regions of ABO genes (such as all 7 exons and important regulatory regions). Therefore, it is difficult to accurately identify rare subtypes, let alone discover and identify new and unknown gene variations.

[0011] Although second-generation sequencing and third-generation sequencing can perform complete sequencing, they usually require complex steps such as library construction and cluster amplification, generating massive amounts of data. Data analysis is complex and time-consuming, and the sequencing cost is relatively high, resulting in a low cost-effectiveness ratio for detection.

[0012] In the prior art, the "ABO genotyping method based on Sanger sequencing" is considered to be the most similar implementation to the present invention. This method typically involves designing specific primers to perform PCR amplification of the seven exons and key regulatory regions of the ABO gene one by one and independently. Subsequently, each amplification product is sequenced separately to obtain an accurate nucleotide sequence, thereby completing genotyping and having the ability to identify unknown mutations.

[0013] However, this technical approach has certain limitations: (1) Sequencing-based methods rely on cumbersome and time-consuming genomic DNA extraction and purification steps, which not only increases the operation time, cost and risk of human error, but also makes it easy to cause typing failure or decreased accuracy when detecting saliva, oral swabs or dried blood spots that have been stored for a long time, due to heavy reliance on high-quality DNA templates.

[0014] (2) Since the target fragments of the ABO gene are dispersed, they are usually split into several independent PCR and sequencing reactions in the design, which leads to complicated operation procedures and high costs. If multiple specific primers that do not affect each other can be designed for multiple amplification, and all 7 exons and two regulatory regions of the ABO gene can be amplified in a balanced manner at one time, the detection speed and cost can be greatly improved.

[0015] (3) It is limited to exon sequencing analysis of ABO genes and fails to analyze gene regulatory regions such as promoters and introns. The detection range is relatively limited and the ABO sequencing information provided is limited.

[0016] Existing ABO blood group genotyping technologies, due to inherent methodological limitations, struggle to balance throughput, process simplification, cost-effectiveness, and the ability to detect new variants, creating significant technical barriers. In particular, these technologies typically cannot eliminate the cumbersome nucleic acid extraction step and lack compatibility with complex samples such as saliva and dried blood spots. These inherent defects in existing technologies severely restrict the widespread application of accurate ABO genotyping in clinical transfusion safety, thus creating an urgent technical need and clear innovation space for the proposed invention—a nucleic acid extraction-free direct sequencing and genotyping method for ABO blood group genes.

[0017] In summary, this invention aims to provide an integrated solution that is simple to operate, fast, low-cost, and accurate. It eliminates the need for nucleic acid extraction and purification, and can directly perform targeted capture, amplification, and first-generation sequencing analysis of all key regions of the ABO gene from various clinical samples (whole blood, saliva, oral swabs, dried blood spots). The operation is simple and low-cost, thereby achieving accurate ABO blood typing, subtype analysis, and discovery of new variants. Summary of the Invention

[0018] To address the shortcomings of existing technologies, this invention aims to provide a simple, rapid, low-cost, high-throughput, and accurate integrated solution. It eliminates the need for nucleic acid extraction and purification, enabling targeted capture, amplification, and first-generation sequencing analysis of all key regions of the ABO gene directly from various clinical samples (whole blood, saliva, oral swabs, dried blood spots). The method is simple to operate and low-cost, thus achieving accurate ABO blood typing, subtype analysis, and the discovery of new variants. Applied to the identification of difficult blood types, this method has led to the discovery of new ABO alleles, blood group gene chimeras, and rare blood types such as subtype-related intron mutations.

[0019] To achieve this objective, the present invention adopts the following technical solution: This invention provides a method for direct sequencing and typing of ABO blood group genes without nucleic acid extraction. The method includes the following steps: (1) probe and primer design, (2) pre-coating of universal probes into PCR well plates, (3) capture of ABO genes, (4) PCR amplification, (5) purification of PCR products, (6) cyclic sequencing reaction, (7) purification of sequencing PCR products, and (8) placing the purified samples on a sequencing analyzer for high-throughput capillary electrophoresis sequencing. Specifically, step (3) capture of ABO genes involves directly immobilizing and capturing the target region of ABO genes from whole blood, saliva, oral swabs, and dried blood spot sample lysates through complementary hybridization of specific capture probes and universal probes. Specifically, step (4) PCR amplification involves multiplex PCR amplification using primers covering the promoter, 7 exons, and 1 key intron.

[0020] On the one hand, the specific capture probe and universal probe sequences designed in this invention are shown in SEQ ID No. 1-24. Part of the capture probe binds complementary to the specific region of the ABO gene, and the other part binds to the universal probe coated on the PCR plate, thereby achieving direct capture of the ABO gene in the sample to be tested.

[0021] Furthermore, the cyclic sequencing reaction specifically comprises a sequencing PCR reaction performed by PCR sequencing primers, the sequences of which are shown in SEQ ID No. 41-49; and a PCR amplification performed by multiplex PCR amplification primers, the sequences of which are shown in SEQ ID No. 25-40.

[0022] Furthermore, the probe primers are capture probe primers and universal probe primers, and the sequences of the capture probe primers and universal probe primers are shown in SEQ ID No. 1-24.

[0023] Furthermore, the amplification primer sequences are shown in SEQ ID No. 25-40.

[0024] Furthermore, the sequences of the sequencing primers are shown in SEQ ID No. 41-49.

[0025] This invention also provides a kit for a direct sequencing and typing method of ABO blood group genes without nucleic acid extraction, characterized in that it contains the aforementioned probe primers, amplification primers, and sequencing primers.

[0026] The present invention also provides an application of the aforementioned method in identifying difficult blood types, discovering new alleles, and accurately detecting ABO blood group chimeras.

[0027] The beneficial effects of this invention are as follows: 1. A revolutionary "nucleic acid extraction-free" direct detection method with wide sample applicability (whole blood, saliva, oral swabs, dried blood spots) completely eliminates the cumbersome and time-consuming nucleic acid extraction and purification steps required in traditional molecular detection, greatly simplifying the operation, shortening the detection time, and laying the foundation for achieving high throughput.

[0028] 2. Integrated "pre-packaged probe capture" system A universal probe is pre-coated onto a PCR plate, and a specific capture probe is used to hybridize with and capture the target ABO gene fragment in the sample, achieving purification and enrichment of the target ABO gene region. Capture and subsequent PCR amplification can be performed in the same reaction well, reducing the need for opening and transferring the plate, thus improving efficiency and preventing contamination.

[0029] 3. Innovative Detection Scope: Covering key regulatory and coding regions of the ABO gene, the primer set is designed not only for a few common exons, but also systematically covers the promoter region, all seven exons, and key intron regions. This comprehensive design enables it not only to detect coding mutations in exons, but also to discover mutations in promoter and intron regulatory regions. This is the molecular basis for discovering novel subtypes and interpreting complex serological phenotypes. Attached Figure Description

[0030] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Four cases of ABO blood type chimerism were identified. Samples 1, 2, and 3 showed a low bimodal distribution at the O01 allele, while sample 4 showed a low bimodal distribution at the B allele, suggesting possible chimerism. Detailed Implementation

[0031] 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.

[0032] This invention discloses a method for direct sequencing and typing of ABO blood group genes without nucleic acid extraction and its applications. The core of this method lies in pre-coating a universal probe onto a PCR plate. Through complementary hybridization between a specific capture probe and the universal probe, the target region of the ABO gene from whole blood, saliva, oral swabs, and dried blood spot lysates is directly immobilized and captured. Multiplex PCR amplification is then performed using primers covering the promoter, seven exons, and one key intron. Finally, gene sequence analysis is achieved through sequencing PCR and capillary electrophoresis sequencing. This method overcomes the bottleneck of cumbersome and time-consuming traditional nucleic acid extraction steps, achieving high-throughput, rapid, and accurate typing. Its significant application value lies in successfully solving the problem of difficult blood group identification. In practice, in addition to identifying 20 ABO blood group subtypes, it also identified 4 chimeras, 5 new subtype mutations not reported by the International Society of Blood Transfusion (ISBT), and 3 subtypes caused by intron mutations. This demonstrates its superior ability to discover new alleles and resolve complex gene structures, which is of great significance for improving transfusion safety and enriching the blood group gene database.

[0033] Example 1: A direct sequencing and typing method for ABO blood group genes without nucleic acid extraction (1) Probe and primer design Based on the ABO gene sequence (reference sequence: NG_006669.2) published in the National Center for Biotechnology Information (NCBI) database, specific capture probes and amplification primers were designed targeting the promoter, seven exons, and regulatory elements in intron 1 of the ABO gene. Among them, some of the eight pairs of capture probes bind complementary to specific regions of the ABO gene, while others bind to universal probes coated on PCR plates, thereby achieving direct capture of the ABO gene in the sample to be tested.

[0034] The designed capture probe and universal probe sequence are shown in SEQ ID No. 1-24, wherein: SEQ ID No. 1-3 are capture probes designed for the ABO gene promoter and exon 1 region, which can specifically capture the ABO gene promoter and exon 1 to the PCR plate and serve as templates for subsequent PCR amplification; SEQ ID No. 4-6 are capture probes designed for the exon 2 region of the ABO gene, which can specifically capture the second exon of the ABO gene to the PCR plate and serve as a template for subsequent PCR amplification; SEQ ID No. 7-9 are capture probes designed for the exon 3 region of the ABO gene, which can specifically capture the third exon of the ABO gene to the PCR plate and serve as a template for subsequent PCR amplification; SEQ ID No. 10-11 are capture probes designed for the exon 4 region of the ABO gene, which can specifically capture the fourth exon of the ABO gene to the PCR plate and serve as a template for subsequent PCR amplification; SEQ ID No. 12-13 are capture probes designed for the exon 5 region of the ABO gene, which can specifically capture the 5th exon of the ABO gene to the PCR plate and serve as a template for subsequent PCR amplification; SEQ ID No. 14-16 are capture probes designed for the exon 6 region of the ABO gene, which can specifically capture the 6th exon of the ABO gene to the PCR plate and serve as a template for subsequent PCR amplification; SEQ ID No. 17-20 are capture probes designed for the exon 7 region of the ABO gene, which can specifically capture the 7th exon of the ABO gene to a PCR plate and serve as a template for subsequent PCR amplification; SEQ ID No. 21-23 are capture probes designed for the regulatory region of ABO gene intron 1. They can specifically capture the regulatory region of ABO gene intron 1 (GATA1 regulatory protein binding region) to the PCR plate and serve as a template for subsequent PCR amplification. SEQ ID No. 24 is a universal probe pre-coated on a PCR plate. Its 5' end is labeled with an amino group (Amino), which reacts with the active group on the plate surface, and its 3' end is labeled with C12. It is reverse complementary to the tail sequence (bolded portion) at the 3' end of all capture probes, capturing ABO gene-specific sequences onto the PCR plate through complementary binding to the capture probe.

[0035] Table 1. Capture probes and universal probe sequences (2) Universal probes were pre-coated onto PCR well plates PCR plates pre-coated with universal probes can be stored at 4°C for 2 years, so multiple PCR plates can be pre-coated without the need for a coating step before each test.

[0036] The specific coating steps are as follows: Dissolve the poly-L-lysine-phenylalanine copolymer powder in 1x phosphate buffer (1x PBS) to obtain a 0.1 mg / ml copolymer mixture (pH=6.0). Add 100 μL of the copolymer mixture to each well and incubate at 37°C for >1 hour. After washing twice with PBS, add 100 μL / well of pre-coating buffer: containing 138 ml of PBS (0.012 M, pH 6.0), 150 μM universal probe, and 5.25 mg of sulfosuccinimide octanoate (BS3). After adding, incubate at 4°C for >2 hours. Prepare an alkaline SDS washing buffer (containing 0.1875 M NaOH and 0.13% SDS). Wash the plate three times with this buffer, then add 120 μL of blocking buffer (50 mg sulfosuccinimide octanoate (BS3) dissolved in 150 ml of 1 x PBS) to each well. Incubate at 4°C for >2 hours, then wash twice with 1 x PBS. After drying, seal and store at 4°C.

[0037] (3) Capture of ABO genes The samples to be tested can be whole blood, saliva, oral swabs, and dried blood spots. The capture systems for different samples are as follows: The whole blood capture system (150 μL) consists of: 50 μL of 3 x lysis buffer, 7 μL of proteinase K, 8 μL of DMSO, 30 μL of whole blood sample, and 1 μL of 10 μM capture probe. Add deionized water to a final volume of 150 μL and mix well.

[0038] The saliva capture system (150 μL) consists of: 50 μL of 3 x lysis buffer, 7 μL of proteinase K, 8 μL of DMSO, 30 μL of saliva sample, and 1 μL of 10 μM capture probe. Deionized water is added to bring the volume to 150 μL and the mixture is stirred.

[0039] The oral swab capture system (150 μL) consists of: 50 μL of 3 x lysis buffer, 7 μL of proteinase K, 8 μL of DMSO, 1 μL of 10 μM capture probe, and deionized water to a final volume of 150 μL. The collected oral swab is then added and thoroughly mixed.

[0040] The dried blood spot capture system (150 μL) consists of: 50 μL of 3 x lysis buffer, 7 μL of proteinase K, 8 μL of DMSO, 1 μL of 10 μM capture probe, and deionized water to a final volume of 150 μL. Two dried blood spot samples with a diameter of 3 mm are then added and thoroughly mixed.

[0041] After vortexing, incubate at 56°C and 800 rpm for 10 minutes to mix thoroughly. Then, add 100 μL of the capture system to the PCR plate pre-coated with the universal probe and incubate at 55°C and 1000 rpm for 30 minutes. (4) PCR amplification PCR amplification was performed using 16 multiplex PCR primers for amplifying the promoter, exons 1-7, and intron regulatory regions of the ABO gene in the human blood group system; the sequences of the multiplex PCR primers are shown in SEQ ID No. 25-40, respectively. Table 2 Primer sequences for multiplex PCR amplification After capture, the plate was washed three times with washing buffer (0.1 × sodium citrate saline (SSC), 0.1% sodium dodecyl sulfate (SDS)). 25 μL of PCR amplification system was added, containing 1 x Rapid Taq PCR Master Mix and primers at a final concentration of 0.5 M. The PCR amplification program is shown in Table 3. Table 3 PCR amplification program (5) Purification of PCR products Take 20 μL of PCR product and add 5 U of exonuclease I and 2 U of shrimp alkaline phosphatase SAP to the purification reagent. Incubate at 37°C for 15 minutes, then incubate at 80°C for 15 minutes to remove any remaining primers, dNTPs, enzymes, and other impurities from the original PCR reaction system to prevent interference with subsequent sequencing reactions.

[0042] (6) Cyclic sequencing reaction The sequencing PCR reaction was performed by nine PCR sequencing primers, the sequences of which are shown in SEQ ID No. 41-49, respectively, enabling sequencing PCR reactions of the ABO gene promoter, exons 1-7 and intron regulatory regions.

[0043] Table 4 PCR Sequencing Primers Prepare the sequencing PCR reaction system according to the ABI BigDye terminator sequencing kit instructions. A 10 μL system contains 1 x sequencing buffer, 0.8 μL BigDye mix, 5 pmol sequencing primers, and 1 μL of purified PCR product. Using the purified multiplex PCR product as a template, perform nine separate sequencing PCR reactions. The reaction procedures are shown in Table 5. Table 5 PCR reaction procedure (7) Purification of sequencing PCR products Add 125 mM EDTA and 3 M sodium acetate to 10 μL of sequencing reaction product, mix well, then add 2.5 volumes of 100% ethanol and vortex vigorously. Incubate at room temperature or 4°C in the dark for 15 minutes. Centrifuge at 15000g for 20 minutes and discard the supernatant. Wash the precipitate with pre-cooled 75% ethanol, centrifuge at 15000g for 5 minutes, and carefully discard the supernatant. Air-dry the precipitate for 10 minutes. Dissolve the dried precipitate in 10 μL of Hi-Di formamide to prevent DNA denaturation and maintain single-stranded DNA.

[0044] Samples were placed on an ABI 3730xl sequencing analyzer for high-throughput capillary electrophoresis sequencing. The instrument automatically performed fragment separation, laser-induced fluorescence excitation, and data analysis. The resulting sequences were analyzed using Chromas 2.0 software and referenced the NCBI and ISBT databases.

[0045] Example 2: Performance Validation of ABO Blood Group Subtype Identification - Discovery of 20 ABO Subtypes In clinical blood typing, 20 samples presented a complex situation where serological typing yielded inconsistent results, making a definitive classification impossible. The nucleic acid extraction-free ABO blood group gene direct sequencing and typing method established in this invention was used to analyze these 20 samples. The results showed that all 20 problematic samples were caused by mutations in different regions of the ABO gene, resulting in ABO subtypes. This method successfully transformed the "uncertain" results of serological typing into definitive molecular diagnostic conclusions, providing a crucial tool for accurate clinical blood typing and safe transfusion.

[0046] Table 6. Serological and sequencing results of 20 samples. Example 3: Performance Validation of ABO Blood Group Subtype Identification - Detection of Novel ABO Gene Mutations and Intron Gene Regulatory Region Mutations To verify the effectiveness of this method, we selected 8 difficult-to-identify blood types that could not be determined by serological and PCR-SSP methods. We used the nucleic acid extraction-free ABO blood group gene direct sequencing and typing method established in this invention to perform sequencing analysis on these 8 samples. Five samples showed novel mutations not included in the International Society of Blood Transfusion (ISBT) database, highly suspected to be related to ABO subtypes. Additionally, three samples had mutations in the transcriptional regulatory region of intron 1 (GATA1 regulatory protein binding region), which may affect ABO gene transcription and thus ABO antigen expression.

[0047] Table 7. Serological and sequencing results of difficult samples whose blood types could not be determined by serological and PCR-SSP methods. Example 4: Performance Verification of ABO Blood Typing - Detection of ABO Blood Type Chimeras Facing the clinical challenge of ABO blood group chimerism, the method of this invention demonstrates excellent resolving power, successfully detecting 4 cases of ABO blood group chimerism (see...). Figure 1 Samples 1, 2, and 3 showed low bimodal peaks in the O01 allele, while sample 4 showed low bimodal peaks in the B allele, suggesting possible chimerism. The results were confirmed by third-generation sequencing and short tandem repeat (STR) analysis, which solved the bottleneck of serological identification and fully verified the unique performance advantages of this method in resolving complex blood types.

[0048] Table 8 Comparison of different methods for determining ABO chimeric blood types 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 method for direct sequencing and typing of ABO blood group genes without nucleic acid extraction, characterized in that, The method includes the following steps: (1) probe and primer design, (2) pre-coating universal probes into PCR well plates, (3) capture of ABO genes, (4) PCR amplification, (5) purification of PCR products, (6) cyclic sequencing reaction, (7) purification of sequencing PCR products, and (8) placing the purified samples on a sequencing analyzer for high-throughput capillary electrophoresis sequencing. Specifically, step (3) capture of ABO genes involves directly immobilizing and capturing the target region of ABO genes from whole blood, saliva, oral swabs, and dried blood spot lysates through complementary hybridization of specific capture probes and universal probes. Specifically, step (4) PCR amplification involves multiplex PCR amplification using primers covering the promoter, 7 exons, and 1 key intron.

2. The method according to claim 1, characterized in that, The designed specific capture probe and universal probe sequences are shown in SEQ ID No. 1-24. Part of the capture probe binds complementary to the specific region of the ABO gene, and the other part binds to the universal probe coated on the PCR plate, thereby achieving direct capture of the ABO gene in the sample to be tested.

3. The method according to claim 1, characterized in that, The cyclic sequencing reaction specifically refers to the sequencing PCR reaction performed by PCR sequencing primers, the sequences of which are shown in SEQ ID No. 41-49 respectively; the PCR amplification is performed by multiplex PCR amplification primers, the sequences of which are shown in SEQ ID No. 25-40 respectively.

4. A probe primer used in the method according to claim 1, characterized in that, The probe primers are capture probe primers and universal probe primers, and the sequences of the capture probe primers and universal probe primers are shown in SEQ ID No. 1-24.

5. An amplification primer used in PCR amplification according to claim 1, characterized in that, The amplification primer sequences are shown in SEQ ID No. 25-40.

6. A sequencing primer used in a cyclic sequencing reaction according to claim 1, characterized in that, The sequences of the sequencing primers are shown in SEQ ID No. 41-49.

7. A kit for a direct sequencing and typing method of ABO blood group genes without nucleic acid extraction, characterized in that, It contains the probe primers of claim 4, the amplification primers of claim 5, and the sequencing primers of claim 6.

8. The application of the method described in claim 1 in identifying difficult blood types, discovering new alleles, and accurately detecting ABO blood group chimeras.

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