Method and kit for genotyping of multi-system red blood cell (RBC) blood group based on next-generation sequencing (NGS)

This patent application claims the benefit and priority of Chinese Patent Application No. 2024104330574, filed with the China National Intellectual Property Administration on Apr. 10, 2024, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.

A computer readable XML file entitled “Sequence Listing”, that was created on Dec. 26, 2024, with a file size of 364, 120 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety.

The present disclosure belongs to the technical field of blood group genotyping, and specifically relates to a method and a kit for genotyping of 25 coding genes in 17 red blood cell (RBC) blood group systems based on next-generation sequencing (NGS).

A human blood group system consists of red blood cell (RBC) surface antigens (proteins, glycoproteins, or glycolipids). The blood group system is inherited and controlled by a single gene or a gene cluster of two or three closely-linked homologous genes. As of July 2023, there are a total of 378 RBC blood group antigens recognized by the International Society of Blood Transfusion (ISBT), of which 360 belong to 45 blood group systems. Human RBCs selectively express antigens from different blood group systems, which constitute each person's unique blood group combination. In addition to ABO, there are 17 most clinically important RBC blood group systems in China, including H, RH (including RHD and RHCE), MNS, Kidd, Diego, Duffy, Lewis, Kell, P1Pk, GLOB, Lutheran, Yt, SC, DO, CO, LW, and JR.

To ensure the safety of clinical blood transfusion, a primary factor is that blood groups of the donor and the patient are consistent. There are two problems:

First, unconventional blood group systems cause adverse reactions to blood transfusions. Nowadays, ABO and RhD blood groups (conventional blood groups) are those that medical institutions conduct routine test and homotype transfusion; for blood group systems (non-conventional blood groups) other than the above two, routine test is omitted due to technical and cost limitations. Therefore, for patients with chronic blood transfusions, multiple transfusions of unconventional blood with an incompatible blood group may stimulate the immune system to produce blood group alloantibodies, triggering transfusion reactions that may even be life-threatening in severe cases. According to statistics from Serious Hazards of Transfusion (SHOT) in the UK, all hemolytic transfusion reactions in the past five years were caused by incompatible transfusions with unconventional blood group systems. A key to solving this problem lies in matching blood transfusions with unconventional blood group systems in patients with chronic blood transfusions.

Second, the supply of blood for rare blood groups should be guaranteed. A rare blood group does not refer to a specific blood group, but refers to a blood group whose frequency is less than one in a thousand among all people. For example, the above-mentioned 17 most clinically important blood group systems in China mainly include 31 key rare blood groups. Blood of rare blood groups is an extremely scarce resource. It is currently the only way to solve the problem of blood supply for clinical rare blood types by conducting large-scale screening among blood donors to find those with rare blood groups and freezing blood of the rare blood groups. However, the difficulty in screening rare blood groups is that there is no technology suitable for simultaneous detection of multiple blood group systems yet at home and abroad.

RBC blood group identification belongs to the field of in vitro diagnostics, and identification methods include serological test and genetic diagnosis. The serological test is the most traditional blood group identification method as well as a commonly used blood group identification method in clinical medical institutions. The RBC blood group is identified by specific reaction in vitro between serum (antibody reagent) containing known specific antibodies and the antigens on a RBC surface to be tested, showing the advantages of simple operation, rapidity, and easy interpretation. However, the serological test is limited by the availability of blood group antibody reagents, and most unconventional blood group systems do not have commercially available antibody reagents globally; a few commercially available reagents completely depend on imports, and are expensive and in short supply. This is a “bottleneck” faced by the current in vitro diagnostic industry of blood groups.

When the serological test was limited, RBC blood group genetic diagnosis based on PCR and sequencing came into being. There are 1,568 alleles listed in a blood group gene mutation database of the National Center for Biotechnology Information (NCBI). Blood type can be predicted by detecting the coding genes of blood group antigens and analyzing genetic polymorphism characteristics. The most significant advantage of genotyping to detect blood groups is to supplement and overcome the deficiencies of serology. For example, diseases, drugs, and treatments (especially recent blood transfusions) interfere with the serological test, making blood group results ambiguous; there are individuals with genetic mutations that result in reduced antigen expression or with unexpected antibodies. In addition, for rare blood groups for which serological test antibody reagents are not available, genotyping is the only way to predict the blood group.

The blood group genetic testing technologies currently widely used in clinical and blood collection and supply institutions in China are polymerase chain reaction-sequence specific primer (PCR-SSP) and first-generation sequencing (Sanger). A prerequisite for the PCR-SSP to identify blood group alleles is to clarify DNA sequences and single nucleotide polymorphisms (SNPs) in order to prepare corresponding primers or probes. Therefore, this technique is not suitable for detecting mutations of unknown sequences or blood group gene with high polymorphism. The Sanger is a gold standard for genetic testing, and can accurately obtain the nucleotide sequence of a target gene or a certain fragment and identify unknown mutations. However, this technique cannot directly distinguish between two haplotypes and may sometimes produce ambiguous typing results. Moreover, as low-to-medium-throughput genetic testing techniques, the PCR-SSP and Sanger are in a high cost and less efficient for screening rare blood groups. In this case, next-generation sequencing (NGS), also known as high-throughput sequencing, is more appropriate. The NGS determines a DNA sequence while synthesizing same by capturing newly synthesized end tags, and allows the detection of one single haplotype DNA sequence to avoid ambiguous typing results. In addition, the NGS has an extremely high detection throughput and can greatly increase the detection throughput, and is highly suitable for large-scale screening of rare blood groups.

A purpose of the present disclosure is to provide a method and a kit for genotyping of 25 coding genes in 17 red blood cell (RBC) blood group systems based on next-generation sequencing (NGS). In the present disclosure, the method based on NGS and multiplex PCR effectively improves both accuracy and detection efficiency for the genotyping of RBC blood group, and can save the manpower in genotyping of the RBC blood group to a certain extent.

The present disclosure provides a primer set for simultaneously detecting genotyping of 17 RBC blood group systems, including primers with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 336.

Preferably, a first primer set includes the primers with the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 108;

a second primer set includes the primers with the nucleotide sequences shown in SEQ ID NO: 109 to SEQ ID NO: 222; and a third primer set includes the primers with the nucleotide sequences shown in SEQ ID NO: 223 to SEQ ID NO: 336.

The present disclosure further provides a kit for simultaneously detecting genotyping of 17 RBC blood group systems, including the primer set and a PCR reaction solution.

Preferably, the first primer set, the second primer set, and the third primer set in the primer set are packaged independently.

The present disclosure further provides a method for simultaneously detecting genotyping of 17 RBC blood group systems, including the following steps: (1) subjecting three capture and amplification systems to first amplification separately to obtain three first amplification products, where the three capture and amplification systems are prepared by using a nucleic acid extracted from a sample as a template and a first primer set with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 108, a second primer set with nucleotide sequences shown in SEQ ID NO: 109 to SEQ ID NO: 222, and a third primer set with nucleotide sequences shown in SEQ ID NO: 223 to SEQ ID NO: 336, respectively;

(2) purifying the three first amplification products separately, and subjecting three resulting purified first amplification products to second amplification with a sequencing universal adapter carrying an index separately to obtain three second amplification products; and

(3) purifying and mixing the three second amplification products to allow sequencing to obtain the genotyping of the RBC blood group systems.

Preferably, the sample in step (1) is selected from the group consisting of a whole blood sample, a plasma sample, and a paraffin tissue sample.

Preferably, the three capture and amplification systems in step (1) each have a volume of 25 μL, and include 1 μL of a mixture of the first primer set or the second primer set or the third primer set, 3 μL of an enzyme mixture Pxp-1st-mix, 1 μL of the template, and nuclease-free water as a balance.

Preferably, a PCR reaction program of the first amplification in step (1) includes: initial denaturation at 98° C. for 20 min; 9 cycles of denaturation at 98° C. for 15 s, annealing at 62° C. for 20 min, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 5 min; and heat preservation at 10° C.

Preferably, a system of the second amplification in step (2) has a volume of 12.5 μL, and includes 6.5 μL of an enzyme mixture Pxp-2nd-mix, 4 μL for one of the three purified first amplification products, 1 μL of a universal adapter N5XX, and 1 μL of a universal adapter A7XX.

Preferably, a PCR reaction program of the second amplification in step (2) includes: initial denaturation at 98° C. for 2 min; 23 cycles of denaturation at 98° C. for 15 s, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 10 min; and heat preservation at 4° C.

Beneficial effects: the present disclosure provides a PCR primer set and a kit for genotyping and capturing 25 coding genes of 17 RBC blood group systems based on a MiSeq NGS platform of the Illumina platform. The primer and kit can simultaneously detect multiple samples and multiple genes, including hereditary mutations such as point mutations and short indels. The kit effectively improves detection efficiency and accuracy, while reducing costs, simplifying operating steps, and providing flexible packaging specifications.

In the present disclosure, a detection method for detecting 31 key rare blood groups of 17 RBC blood group systems is constructed based on the primer set or kit. High-throughput sequencing is conducted to simultaneously detect 17 RBC blood group system-related genes to improve detection efficiency and reduce costs. The high-throughput gene sequencing can break through the limitations of traditional detection technology and improve detection sensitivity. Meanwhile, variations including known mutation sites and unknown mutation sites can be detected to obtain comprehensive and accurate detection results. By sequencing 17 RBC blood group system-related genes and their mutation sites, specific gene sequence information can be obtained, thereby providing accurate and efficient detection for rare blood group detection.

FIG. 1 shows a flow chart for RBC blood group identification using the primer set or kit of the present disclosure;

FIG. 2 shows an electrophoresis pattern of primer specificity verification in Example 1;

FIG. 3 shows an electrophoresis pattern of primer detection sensitivity verification in Example 2; and

FIG. 4 shows results of the repeatability verification of primer detection in Example 3.

The present disclosure provides a primer set for simultaneously detecting genotyping of 17 RBC blood group systems, including primers with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 336.

In the present disclosure, the RBC blood group system and corresponding genetic information are shown in Table 1.

The present disclosure is based on 1,072 blood group system typing sites of 25 genes shown in Table 1: H (ISBT 018), Rh (ISBT 004), MNS (ISBT 002), Kidd (JK, ISBT 009), Diego (DI, ISBT 010), Duffy (FY, ISBT 008), Lewis (ISBT 007), Kell (ISBT 006), P1Pk (ISBT 003), Globoside (GLOB, ISBT 028), Lutheran (LU, ISBT 005), YT (ISBT 011), Scianna (SC, ISBT 013), Dombrock (DO, ISBT 014), Colton (CO, ISBT 015), Landsteiner-Wiener (LW, ISBT 016), and Junior (JR, ISBT 032). The present disclosure develops a multiplex PCR-based RBC blood type NGS typing primer set, kit and method adapted to the Illumina sequencing platform. The primer set is designed based on the target region of the above gene. The target region preferably includes the full length of the coding region sequences of the 25 antigen-coding genes of the above-mentioned 17 blood group systems and the splicing flanking region within 100 bp to 150 bp. In the present disclosure, the 26 genes are preferably selected from the University of California Santa Cruz (UCSC) database; the typing sites have been disclosed in databases and literature, most of which are collected in the ISBT database, and a small number are derived from literature.

In the present disclosure, the specific primers in the primer set are completely complementary or partially complementary to the above-mentioned target region. Preferably, each pair of specific primers for the target region contains two parts, labeled as 3′-end Tag1 and 5′-end Tag2. A fragment sequence of the 3′-end Tag1 is completely complementary or partially complementary to the target region, and is preferably a nucleic acid molecule with a specific sequence, with an unlimited number of bases, preferably 20 to 40. The 5′-end Tag2 is used to combine with the universal primer during the amplification of the target region, thus completing the specific labeling of the amplification products of the target regions of different samples to be tested. The 5′-end Tag2 has preferably 3 to 20, more preferably 4 to 10 bases.

In the present disclosure, a total of 168 pairs of primers are designed based on the above genes and design principles. Each pair of primers contains mutation sites in the gene. The size of a target region amplified by each pair of primers is 150 bp to 300 bp, with wide coverage and multiple detection sites; the primers have a relatively uniform Tm value (57° C. to 63° C.); the primers show desirable specificity, stability, and uniformity during multiplex amplification, and have non-specific amplification. The primers preferably further include specific modifications, and the modified primers are not easily degraded by nucleases; specifically, the primer-specific modifications include one or more of thio modification, deoxyuracil modification, 5′-reverse dT modification, and 5′-amino modification. A thio-modified primer is a derivative in which the double-bonded oxygen atom on the oligonucleotide chain is replaced by a sulfur atom, and can resist the degradation of nuclease and enhance the stability of the primer. Each deoxythymine in the oligonucleotide sequence of a deoxyuracil-modified primer is replaced by deoxyuracil, and can increase the double-stranded dissolution temperature by 1.7° C., thereby increasing the stability of the double-stranded. The 5′ end of the oligonucleotide sequence in a 5′-reverse dT-modified primer contains a reverse dT, thereby forming a cross-link, which can inhibit the degradation of exonucleases during the extension of DNA polymerase. The 5′-amino modification includes C6 amino modification and C12 amino modification. The former can be used to connect some compounds that do not affect its function even near the oligonucleotide, while the latter is used to ligate the affinity purification group and some fluorescent marks.

In the present disclosure, according to the nature of each primer, such as the amplification interval, Tm value, primer sequence, and actual expansion during the later test, the primer set is divided into 3 pools, which are specifically shown in Table 2. The number marked after the sequence in Table 2 is the number of SEQ ID NO. For example, (1) indicates that the nucleotide sequence of a sequence is serialized as SEQ ID NO: 1. The primer set 1 includes: RB_018, RB_023, RB_026, RB_029, RB_032, RB_034, RB_037, RB_039, RB_063, RB_016, RB_053, RB_055, RB_057, RB_048, RB_051, RB_070, RB_064, RB_066, RB_068, RB_118, RB_123, RB_106, RB_108, RB_110, RB_013, RB_003, RB_093, RB_097, RB_099, RB_101, RB_104, RB_042, RB_126, RB_077, RB_079, RB_082, RB_085, RB_087, RB_089, RB_091, RB_125, RB_130, RB_134, RB_137, RB_144, RB_146, RB_149, RB_153, RB_155, RB_157, RB_160, RB_167, RB_138, RB_140, and their nucleotide sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 108 in sequence.

The primer set 2 includes: RB_020, RB_021, RB_022, RB_025, RB_027, RB_031, RB_035, RB_058, RB_060, RB_062, RB_015, RB_054, RB_046, RB_049, RB_052, RB_071, RB_072, RB_074, RB_065, RB_067, RB_069, RB_117, RB_120, RB_122, RB_105, RB_109, RB_112, RB_114, RB_010, RB_012, RB_002, RB_005, RB_007, RB_095, RB_098, RB_102, RB_041, RB_043, RB_045, RB_127, RB_081, RB_084, RB_088, RB_075, RB_131, RB_133, RB_136, RB_143, RB_147, RB_150, RB_152, RB_156, RB_158, RB_161, RB_163, RB_165, RB_141, and their nucleotide sequences are shown in SEQ ID NO: 109 to SEQ ID NO: 222 in sequence.

The primer set 3 includes: RB_019, RB_024, RB_028, RB_030, RB_033, RB_036, RB_038, RB_059, RB_061, RB_017, RB_056, RB_047, RB_050, RB_073, RB_116, RB_119, RB_121, RB_124, RB_107, RB_111, RB_113, RB_115, RB_009, RB_011, RB_014, RB_001, RB_004, RB_006, RB_008, RB_092, RB_094, RB_096, RB_100, RB_103, RB_040, RB_044, RB_076, RB_078, RB_080, RB_083, RB_086, RB_090, RB_128, RB_129, RB_132, RB_135, RB_142, RB_145, RB_148, RB_151, RB_154, RB_159, RB_162, RB_164, RB_166, RB_168, RB_139, and their nucleotide sequences are shown in SEQ ID NO: 223 to SEQ ID NO: 336 in sequence.

The present disclosure further provides a kit for simultaneously detecting genotyping of 17 RBC blood group systems, including the primer set, and a PCR reaction solution and a quality control.

In the present disclosure, the first primer set, the second primer set, and the third primer set in the primer set are packaged independently. The PCR reaction solution is preferably PCR Buffer, high-fidelity DNA polymerase, and nuclease-free water. In the examples, enzyme mixtures Pxp-1st-mix and Pxp-2nd-mix that mix the PCR Buffer with the high-fidelity DNA polymerase are selected, and the enzyme mixtures Pxp-1st-mix and Pxp-2nd-mix each preferably include commercial ingredients, such as main components in the enzyme mixture: high-fidelity enzyme and 5×HF PCR Buffer: Thermo Fisher scientific's “Phusion Hot Start II DNA Polymerase (2 U/μL)”, F565L; dNTP (dATP, dTTP, dCTP, dGTP): Sangon (BBI) A620046, A670046, A640046, 660046.

In the present disclosure, the quality control is preferably a human normal genomic DNA.

The present disclosure further provides a method for detecting genotyping of 17 RBC blood group systems, including the following steps as shown in FIG. 1 : (1) subjecting three capture and amplification systems to first amplification separately to obtain three first amplification products, where the three capture and amplification systems are prepared by using a nucleic acid extracted from a sample as a template and a first primer set, a second primer set, and a third primer set, respectively;

(2) purifying the three first amplification products separately, and subjecting three resulting purified first amplification products to second amplification with a sequencing universal adapter carrying an index separately to obtain three second amplification products; and

(3) purifying and mixing the three second amplification products to allow sequencing to obtain the genotyping of the RBC blood group systems.

In the present disclosure, three capture and amplification systems are subjected to first amplification separately to obtain three first amplification products, where the three capture and amplification systems are prepared by using a nucleic acid extracted from a sample as a template and a first primer set, a second primer set, and a third primer set, respectively. Preferably, the sample is extracted using magnetic beads, the concentration and purity are measured, and the nucleic acid is quantitatively diluted. Since nano-scale superparamagnetic carboxyl magnetic beads can reversibly adsorb nucleic acid molecules under different salt concentrations and hydrophobic environments, the nucleic acid molecules can be specifically adsorbed based on this. High-purity nucleic acid samples can be obtained through subsequent washing and elution. Concentration and purity are measured using Qubit or NanoDrop. According to a measured nucleic acid sample concentration, the nucleic acid sample is diluted to 50-250 ng/μL with 10 mM Tris-HCl, preferably 100-200 ng/μL serves as a starting concentration for amplification and library construction.

In the examples of the present disclosure, a quality of the diluted nucleic acid is preferably further evaluated by 1.5% agarose gel electrophoresis at 150 V for 30 min. The quality of the extracted nucleic acid is determined based on the uniformity and integrity of the agarose gel electrophoresis results. High integrity or slight degradation of the nucleic acid does not affect the library construction. The extracted nucleic acids are grouped and labeled separately after passing the concentration measurement, purity measurement, and agarose gel electrophoresis test. The sample is selected from the group consisting of a whole blood sample, a plasma sample, and a paraffin tissue sample. Nucleic acids are extracted from the above samples and diluted to the starting concentration for amplification and library construction. 3 tubes of capture and amplification system are constructed according to the grouping of the pools, where a primer pair of each pool is regarded as 1 tube. The three capture and amplification systems each have a volume of 25 L, and include preferably 1 μL of a mixture of the first primer set or the second primer set or the third primer set, 3 μL of an enzyme mixture Pxp-1st-mix, 1 μL of the template, and nuclease-free water as a balance. In the mixture of each pool primer set, 26 μL of Tris-HCl and 124 μL of primer set are preferably used to form a pool 1 primer with a total volume of 150 μL and a final concentration of 200 nM; except that the primers GATA1_001, KLF1_004, KLF1_008, and LU_004 are all 2 μL, and the primer Rh_020 is 5 μL, the other primers are all 1 μL. 32 μL of Tris-HCl and 168 μL of primer set are used to form a pool 2 primer with a total volume of 200 μL and a final concentration of 150 nM; except that the primer A4GALT_007 is 6 μL, the primers ABCG2_019, ABCG2_023, FUT_007, KLF1_005, KLF1_007, LU_001, LU_005, LU_008, MNS_002, and Rh_018 are 2 μL, the primer YT_002 is 3 μL, and the primer Rh_010 is 11 μL, the other primers are all 1 μL. 8 μL of Tris-HCl and 142 μL of primer set are used to form a pool 3 primer with a total volume of 150 μL and a final concentration of 200 nM; except that the primers A4GALT_005, ABCG2_006, ABCG2_024, KLF1_003, KLF1_006, LU_007, MNS_001, Rh_011, Rh_013, and SLC35C1_003 are 2 μL, the primer Rh_021 is 5 μL, the other primers are all 1 μL. When constructing each pool primer solution, each primer in each pool primer set has a concentration of 30 μM.

In the present disclosure, the three capture and amplification systems are preferably subjected to first amplification to obtain three first amplification products (pre-library products). Preferably, a PCR reaction program of the first amplification includes: initial denaturation at 98° C. for 20 min; 9 cycles of denaturation at 98° C. for 15 s, annealing at 62° C. for 20 min, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 5 min; and heat preservation at 10° C.

In the present disclosure, the three first amplification products are purified separately, and three resulting purified pre-library products are subjected to second amplification with a sequencing universal adapter carrying an index separately to obtain three second amplification products (library construction products). The purification preferably includes purifying the PCR amplification products (pre-library products) of each sample using purification magnetic beads, removing the remaining primers, gDNA, and dNTPs in the PCR products and recovering the amplification products.

In the present disclosure, after the purification is completed, sequencing universal adapters (N5XX and A7XX) carrying an index are used to further PCR-amplify the purified products. A reaction system of the PCR amplification is calculated as 12.5 μL, and preferably includes: enzyme mixture Pxp-2nd-mix 6.5 μL; pre-library product 4 μL; universal adapter N5XX 1 μL, universal adapter A7XX 1 μL, with a total volume of 12.5 μL. The same sample includes 3 pre-library products, which are added to 3 tubes of PCR master mix containing the same universal adapter. The PCR reaction program is preferably: initial denaturation at 98° C. for 2 min; 23 cycles of denaturation at 98° C. for 15 s, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 10 min; and heat preservation at 4° C.

In the present disclosure, after the PCR products are obtained, the library construction products of each sample are preferably separately purified using purification magnetic beads. The remaining primers and dNTPs are removed from the PCR product and the amplification products are recovered; the concentration and purity are measured, and the sample quality is tested by 1.5% agarose gel electrophoresis. After passing the test, the sample is diluted and mixed at equal concentrations, and then sequenced uniformly. The RBC blood group is determined based on sequencing results (Table 3). The mixed sample refers to mixing the products of the same sample amplified and purified in the first step using three sets of primers, and the purified products amplified in the second step using adapters carrying the same index. The sample mixing requirements: different samples are amplified in the second step using adapters carrying different indexes; 3 pools of the same sample are amplified in the second step using adapters with the same index.

To further illustrate the present disclosure, the primer set, the kit, and the method for genotyping multi-RBC blood group system based on NGS provided by the present disclosure are described in detail below in connection with examples, but these examples should not be construed as limiting the claimed scope of the present disclosure.