Quality control product for blood type gene detection and preparation method thereof

Abstract

The invention relates to the technical field of blood type gene detection, and provides a quality control product for blood type gene detection and a preparation method thereof. The preparation method of the quality control product comprises the following steps: preparing AB type and O type suspensions, preparing A205 DNA, and uniformly mixing the A205 DNA with the AB type and O type suspensions to form the quality control product. Carrying out detection operation on the quality control product and a sample to be detected according to the same mode, wherein the detection operation comprises extraction, amplification, analysis and sequencing; in the detection result, if the quality control product is out of control, the detection result of the to-be-detected sample is not acquired. The quality control product capable of being used for blood group gene detection is successfully constructed, and the quality control product is high in effectiveness and stability in quality control and good in accuracy and can be suitable for various different blood group gene detection technologies and detection items.

Description

Technical Field

[0001] The present invention belongs to the technical field of blood type gene detection, and in particular relates to a quality control product for blood type gene detection and a preparation and use method thereof. Background Art

[0002] Blood typing has applications in blood transfusion, anthropology, genetics, forensic medicine, transplant immunity, and disease resistance (or susceptibility). In clinical medicine, in addition to blood transfusion and transplant immunity, testing for specific antibodies to hemolytic disease of the newborn and autoimmune hemolytic anemia also requires knowledge of blood typing and related techniques.

[0003] The rapid development of genomics has impacted all areas of medicine. In transfusion medicine, the rapid development of genotyping technology has made it a strong support and auxiliary technology for serological blood type testing. In actual clinical work, blood type gene testing technology is an important means of accurately identifying blood type for patients with positive direct antiglobulin test results, multiple or recent blood transfusions, autoimmune hemolytic anemia, multiple autoantibodies, and when fetal or newborn blood type identification is difficult, especially when testing for rare blood type antigens for which commercial serological reagents are not available.

[0004] Recent studies have revealed that hemolytic transfusion reactions are a major cause of transfusion mortality, with immune hemolysis caused by blood type incompatibility being a major factor. This is because, while hundreds of blood type antigens have been identified on the surface of red blood cells, each person selectively expresses only a subset. In other words, the blood type antigens expressed by each person's red blood cells vary. If a person's red blood cell surface antigens occur at a rate less than one in a thousand in the general population, or if their red blood cells lack the antigens expressed by the majority of the population, their blood type is considered rare. For patients with rare blood types, once antibodies are developed, subsequent transfusions of incompatible blood can easily lead to transfusion reactions or even death. Often, when patients urgently need blood, compatible rare blood types are difficult to find quickly. Therefore, screening for rare blood types and establishing rare blood type banks are crucial for transfusion safety. Serology is the classic method for detecting blood type antigens. However, due to the scarcity and high cost of monoclonal antibodies for most rare blood type antigens, large-scale screening of donors for rare blood types using serology is impractical. Genetic testing can effectively address the shortcomings of serology. Different throughput blood type genetic testing technologies have their own applicable scopes, and high-throughput genetic screening platforms demonstrate broad application prospects in blood type genetic testing. While the application of molecular biology techniques still has certain limitations, as the ability of genotypes to predict blood type phenotypes improves, high-resolution typing of clinically significant blood type antigens will become increasingly common.

[0005] Currently, the molecular mechanisms of most red blood cell (RBC) blood group systems have been elucidated. While the ABO and Rh blood group systems are relatively complex, polymorphisms in other blood group systems are almost exclusively due to single nucleotide polymorphisms (SNPs). This characteristic of the RBC blood group system provides the molecular biological basis for blood group antigen detection. Polymerase chain reaction (PCR) has been widely used in RBC blood group screening. By detecting either a single allele or multiple alleles (i.e., multiplex PCR), high-frequency antigen alleles are specifically amplified, and the amplification results are analyzed to determine the blood group phenotype. Blood typing, based on molecular biological detection techniques, offers advantages such as simple operation, high sensitivity, standardized result interpretation, and freedom from sample immunological limitations. However, current technologies lack unified specifications, standards, and quality control products for the quality management of RBC blood group molecular detection, making it difficult to determine the reliability of sample test results. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems raised in the above-mentioned background technology and to provide a quality control product for blood type gene testing and a preparation method thereof. The quality control product prepared can be used for effective quality control of human blood type gene testing, and the accuracy and stability of the quality control product meet the industry requirements for quality control products.

[0007] A first aspect of the present invention provides a method for preparing a quality control product for blood type gene testing, comprising the following steps: (1) Preparing AB type and O type suspensions: confirming the blood type of human blood materials with AB type heterozygous and O type homozygous blood types respectively; collecting the blood materials after blood type confirmation into leukocyte filters respectively, backwashing the leukocyte filters with blood preservation fluid, and collecting the flushing fluid aseptically to obtain AB type flushing fluid and O type flushing fluid respectively; mixing the AB type flushing fluid and the O type flushing fluid in equal amounts to form AB type and O type suspensions; (2) Preparation A 205 DNA: From blood type A 205 A subtype was extracted from blood samples 205 DNA or according to A 205 Sequence synthesis A 205 DNA plasmid, thereby obtaining A 205 DNA; (3) Prepare quality control product: 205 The DNA is mixed with the AB type and O type suspension to form a quality control product; In step (1), before mixing equal amounts of the AB-type flushing solution and the O-type flushing solution, the DNA concentrations in the AB-type flushing solution and the O-type flushing solution are controlled and confirmed so that the DNA content in the flushing solution of the quality control product is ≥800 ng in a single use amount; In step (2), control and confirm A 205The concentration of DNA is such that the DNA content in a single-use solution of the quality control product is ≥800 ng; In step (3), control A 205 The ratio of the DNA to the AB type and O type suspensions is such that the DNA content of the quality control product is ≥800 ng in a single use of the quality control product.

[0008] Optionally, the blood type confirmation in step (1) includes: when the blood type result meets the following conditions, the blood material is used: Blood type AB: serological typing: A4+, B4+, Ac-, Bc-, phenotype is AB, genotype is A, B; blood type O: serological typing: A-, B-, Ac4+, Bc4+, phenotype is O, genotype is O 261Gdel , O1, O2.

[0009] Optionally, in step (1), before mixing equal amounts of the AB-type washing solution and the O-type washing solution, the DNA concentrations in the AB-type washing solution and the O-type washing solution are controlled and confirmed so that the DNA absorbance 260 / 280 ratio is between 1.7 and 2.0.

[0010] Optionally, control and confirm the A prepared in step (2) 205 The concentration of DNA should be adjusted so that the DNA absorbance 260 / 280 ratio is between 1.7 and 2.0.

[0011] Optionally, the mixing method described in step (1) and step (3) includes slight inversion to avoid the formation of air bubbles.

[0012] The second aspect of the present invention provides a quality control product prepared by the above preparation method.

[0013] The third aspect of the present invention provides a method for blood type gene testing using the above-mentioned quality control product, comprising performing blood type gene testing on the above-mentioned quality control product and the sample to be tested according to the same operation, the operation including DNA extraction, amplification and analysis; the DNA extraction includes using a DNA extraction kit, and the single initial sample amount recorded in the instructions for use of the DNA extraction kit is the single usage amount of the quality control product; in the test results, if the quality control product is out of control, the test results of the sample to be tested will not be accepted.

[0014] Optionally, the out-of-control quality control product refers to that the detection value of the quality control product is inconsistent with the reference value of the corresponding item in the quality control product reference value; the quality control product reference value includes a genotyping reference value and a gene sequencing reference value; The genotyping reference values ​​are as follows: 1.1 ABO: A, A2, B, O 261Gdel , O1, O2; 1.2 RHD: E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, 845, 1227, GALC; 1.3 RHCE: C (RH2), E (RH3), c (RH4), e (RH5), CW (RH8), V (RH10), hrS (RH19), VS (RH20), hrB (RH31); 1.4 Kell: K (KEL1), k (KEL2), Kpa (KEL3), Kpb (KEL4), Jsa (KEL6), Jsb (KEL7); 1.5 Kidd: Jka (JK1), Jkb (JK2); 1.6 Duffy: Fya (FY1), Fyb (FY2); 1.7 MNS: M (MNS1), N (MNS2), S (MNS3), s (MNS4), U (MNS5), Mia (MNS7); 1.8 Diego: Dia (DI1), Dib (DI2); 1.9 Dombrock: Doa (DO1), Dob (DO2), Hy (DO4), Joa (DO5); 1.10 Colton: Coa (CO1), Cob (CO2); 1.11 Cartwright: Yta (YT1), Ytb (YT2); 1.12 Lutheran: Lua (LU1), Lub (LU2); The gene sequencing reference values ​​are as follows: 2.1 ABO: Exon3 c.106G>T, Exon4 c.188G>A, Exon4 c.189C>T, Exon5 c.220C>T, Exon6 c.261delG, Exon6 c.297A>G, Exon7 c.467C>T, Exon7 c.526C>G, Exon7 c.646T>A, Exon7 c.657C>T, Exon7 c.681G>A, Exon7 c.703G>A, Exon7 c.771C>T, Exon7 c.796C>A, Exon7 c.803G>C, Exon7 c.829G>A, Exon7 c.930G>A; 2.2 RhD: no mutation; 2.3 RhCE: Exon1 c.48G>C; 2.4 Kadd: Exon3 c.130G>A, Exon6 c.588A>G, Exon8 c.838G>A; 2.5 FUT1: Exon4 c.35C>T; FUT2: Exon2 c.357C>T, Exon2 c.385A>T; FUT3: Exon3c.59T>G, Exon3 c.508G>A; 2.6 A4GALT: Exon1 c.903C>G; 2.7 B3GALNT1: No mutation.

[0015] Optionally, the quality control product and the sample to be tested are subjected to blood type gene testing according to the same operation, including: each DNA extraction plate is provided with one negative control well, one positive control well, and the quality control product is added to the positive quality control well, and extraction is performed in the same manner as the sample to be tested; each reaction well of each DNA amplification plate is provided with an internal control; each DNA amplification plate is provided with one negative control well, one positive control well, and a positive quality control well, and the quality control product is added to the positive quality control well, and amplification is performed in the same manner as the sample to be tested; the positive quality control material (i.e., the quality control product) in the positive quality control well is analyzed and sequenced in the same manner as the sample to be tested.

[0016] Optionally, when the quality control product is out of control, when the reference value of the item is negative and the detection value of the quality control product in this item is a false positive, the samples with positive test results in this experiment will be retested; when the reference value of the item is positive and the detection value of the quality control product in this item is a false negative, the samples with negative test results in this experiment will be retested.

[0017] The present invention successfully constructs a quality control product that can be used for blood type gene detection. The quality control product contains 12 alleles such as ABO, RhD, RhCE, Kell, and Kadd, and can be used as a positive quality control product for blood type gene detection, such as a multiplex PCR system, and can also be used as a quality control product for sequencing analysis. By optimizing parameters such as the DNA concentration content of each intermediate reagent component and the finished quality control product during the preparation process, and experimentally exploring and verifying the effect of the quality control product, the obtained quality control product has high effectiveness and stability in quality control, good accuracy, and relatively ideal quality control effectiveness; and can be applied to various different blood type gene detection technologies and detection items. Utilizing the quality control product of the present invention, it is possible to standardize the detection process, improve the credibility and accuracy of the detection results, and promote the standardized development and wider clinical application of blood type gene detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is the DNA extraction layout diagram of Example 2 of the present invention.

[0019] Figure 2 This is the DNA amplification layout diagram of Example 2 of the present invention.

[0020] Figure 3 This is the melting curve diagram of Example 2 of the present invention.

[0021] Figure 4 This is a result interpretation table of Example 2 of the present invention.

[0022] Figure 5 This is the DNA extraction layout diagram of Example 3 of the present invention.

[0023] Figure 6 This is the DNA amplification layout diagram of Example 3 of the present invention.

[0024] Figure 7 This is the electrophoresis diagram of RhD genotyping (PCR-SSP) in Example 3 of the present invention.

[0025] Figure 8 This is the RhD genotyping result interpretation table of Example 3 of the present invention.

[0026] Figure 9 This is a diagram of the ABO gene sequencing results of Example 4 of the present invention. DETAILED DESCRIPTION

[0027] In clinical practice, the existing technology for quality management related to molecular red blood cell typing lacks unified standards and quality control products. The quality control products prepared by the present invention play an important role in laboratory work. They are used to evaluate and monitor the accuracy of laboratory analysis processes and results. They can not only verify the accuracy of laboratory testing methods and instruments, but also help laboratories identify potential problems and take corrective measures in a timely manner. Without the quality control products of the present invention, machine or human operating errors or mistakes during actual testing are difficult to define and eliminate.

[0028] This study successfully constructed quality control products containing 12 alleles, including ABO, RhD, RhCE, Kell, and Kadd, which can serve as positive quality controls for blood type gene testing, including multiplex PCR systems. Sequencing analysis quality control products for ABO, RhD, RhCE, Kadd, FUT1-3, A4GALT, and B3GALNT1 have also been successfully constructed. By utilizing these quality control products, the entire blood type molecular biology testing process is standardized across key steps influencing test results, including the testing process (including sample pretreatment, nucleic acid extraction, nucleic acid amplification, product analysis, and nucleic acid sequencing), as well as result evaluation and processing. This approach standardizes the testing process, improves result accuracy, and promotes the standardized development and broader clinical application of this technology.

[0029] The quality control product of the present invention is a mixture of human blood, including red blood cells, white blood cells, DNA plasmids, etc. The human red blood cells and white blood cells are from two different samples with blood type and target gene type AB and O; DNA plasmids use A 205 Subtype DNA reflects blood type gene information more comprehensively.

[0030] Specifically, the preparation method of quality control products includes: (1) Preparation of AB type and O type suspensions: Human blood materials with AB heterozygous type and O homozygous type are screened using monoclonal blood group antibodies, human typed red blood cells, genotyping, and SANGER sequencing technology to confirm the blood type; the blood materials (whole blood) after blood type confirmation are collected into leukocyte filters, the leukocyte filters are backwashed with blood preservation fluid, and the leukocyte filter flushing fluid is aseptically collected to obtain AB type flushing fluid and O type flushing fluid respectively; the AB type flushing fluid and the O type flushing fluid are mixed in equal amounts to form AB type and O type suspensions.

[0031] The blood preservation solution may be a commonly used one in the art, such as ACD solution, CPD solution (CPDA-1), etc. When mixing equal amounts of AB and O type blood flushing solutions, gently invert and mix thoroughly to avoid the formation of air bubbles.

[0032] (2) Preparation A 205 DNA: From blood type A 205 A subtype was extracted from blood samples 205 DNA or according to A 205 Sequence synthesis A 205 DNA plasmid, i.e. A 205 Type plasmid, thereby obtaining A 205 DNA. A 205 The sequences of the subtypes are known in the art.

[0033] Among them, blood type A 205 Subtype A 205 DNA is preferably artificially synthesized based on known published sequences. 205 DNA manipulation is more feasible and convenient, and the final reagent effect is more stable and controllable.

[0034] (3) Prepare quality control product: 205 The DNA is mixed with the AB type and O type suspensions to form a quality control product.

[0035] When mixing, slightly invert the mixture to avoid forming bubbles, otherwise it will affect the effect of the quality control product. In addition, the mixing ratio of the two should be controlled so that A 205The nucleic acid content of DNA in the quality control product reaches a certain concentration.

[0036] One of the key challenges in preparing quality control products for blood type gene testing is the rational design of the DNA content of each component. Improper DNA content can prevent the product from reaching the expected target value and render it ineffective. Through empirical reference, experimental exploration, and verification, the present invention provides a solution that includes: In step (1), before mixing the AB type washing solution and the O type washing solution in equal amounts, the DNA concentrations in the AB type washing solution and the O type washing solution are controlled and confirmed, so that the DNA content of each is ≥800 ng in the washing solution of the quality control product used once; in step (2), the DNA concentrations in the AB type washing solution and the O type washing solution are controlled and confirmed, so that the DNA content of each is ≥800 ng in the washing solution of the quality control product used once; 205 The concentration of DNA is adjusted so that the DNA content in the single-use solution of the quality control product is ≥800ng; in step (3), control and confirm A 205 The ratio of DNA to the AB and O suspensions is such that the DNA content of the quality control product is ≥ 800 ng per single use of the quality control product. Under this DNA content, the effectiveness of the quality control product is ideal.

[0037] More specifically, for the single use amount of the quality control product described in each of the above steps: When using the control sample, blood type gene testing is performed using the same procedures as the test sample, including DNA extraction from both the control sample and the test sample. The single-use volume of the control sample is the initial sample volume, i.e., the initial volume or mass, used in the DNA extraction procedure, for example, 200 µL. Furthermore, as is commonly done in the art, DNA extraction can be performed using a DNA extraction kit. In other words, the single-use volume of the control sample is the initial sample volume specified in the kit's instructions; for example, in some kits, the sample volume is 200 µL. Therefore, in blood type gene testing, the DNA content below the single-use volume of the control sample can be understood as the amount of nucleic acid extracted from whole blood in a single use. The preparation method for this control sample must ensure that, when used, it can extract at least 800 ng of DNA in a single DNA extraction. Different DNA extraction kits or blood type gene testing techniques may require different initial sample volumes, i.e., the single-use volume of the control sample, for DNA extraction. In the present invention, through experimental exploration and repeated verification, it was determined that the DNA content in a single use of the quality control product is limited to ≥800 ng, which can ensure the effectiveness of the quality control effect of the final quality control product. In some embodiments, ≥800 ng is further preferably 800-1000 ng, 1000-2000 ng, or 2000-4000 ng.

[0038] The DNA content of each component, as well as the DNA content of the combined components, must be controlled and confirmed within the aforementioned limits. By limiting the DNA content of each component during the preparation of the control product, a control product with a reasonable DNA distribution, good applicability, and superior quality control results can be produced.

[0039] Furthermore, in some embodiments, the control of DNA content further comprises: before mixing equal amounts of the AB type washing solution and the O type washing solution in step (1), controlling and confirming that the DNA absorbance (a) 260 / 280 ratio in each of the AB type washing solution and the O type washing solution is between 1.7 and 2.0; controlling and confirming that the DNA content in the A type washing solution prepared in step (2) is 205 The DNA absorbance (a) 260 / 280 ratio is between 1.7 and 2.0. This confirmation of DNA purity further ensures that the DNA content in the different components of the quality control product is within the appropriate range.

[0040] The use of the quality control product described in the present invention includes subjecting the quality control product to the same testing procedures as the sample to be tested, including extraction, amplification, analysis, sequencing, etc.; if the quality control product is out of control in the test results, the test results of the sample to be tested will not be accepted. The out-of-control quality control product refers to a discrepancy between the test value of the quality control product and the reference value of the corresponding item in the quality control product reference value; the quality control product reference value includes a genotyping reference value and a gene sequencing reference value. The present invention provides reference values ​​for numerous items of blood type gene testing, including various alleles such as ABO, RhD, RhCE, Kell, and Kadd, as well as reference values ​​for gene sequencing analysis such as ABO, RhD, RhCE, Kadd, FUT1-3, A4GALT, and B3GALNT1.

[0041] The quality control product of the present invention is applicable to detection methods including not only multiplex PCR systems, but also other gene detection techniques, including real-time PCR, PCR-SSP, SANGER Sequencing and other gene sequencing methods.

[0042] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0043] Example 1 Preparation of quality control products 1. Preparation of AB+O type suspension (1) Confirm the blood type. Blood type AB: Serological typing: A4+, B4+, Ac-, Bc-. Phenotype is AB; Genotype is A, B. Blood type O: Serological typing: A-, B-, Ac4+, Bc4+. Phenotype is O; Genotype: O 261Gdel , O1, O2.

[0044] (2) Preparation of suspension. Collect and confirm the blood type AB and O and collect several leukocyte filters. Backwash the leukocyte filter (biosafety cabinet) with 50-100 ml of blood preservation solution (CPDA-1) and collect the leukocyte filter rinse aseptically. Take 2 ml of the rinse solution to extract DNA and determine the DNA concentration threshold to ensure that the DNA absorbance (a) 260 / 280 ratio is between 1.7 and 2.0 and the amount of nucleic acid extracted from whole blood in a single time is ≥800 ng. Mix equal amounts of blood type AB and blood type O rinse solutions and gently invert to mix well without forming air bubbles. The amount of nucleic acid extracted from whole blood in a single time described in this example is the DNA content below the single use amount of the quality control product.

[0045] 2.A 205 DNA plasmid preparation. Synthesize A according to the designed sequence. 205 For DNA plasmids, the DNA concentration threshold should be determined as follows: the DNA absorbance (a) 260 / 280 ratio should be between 1.7 and 2.0, and the amount of nucleic acid extracted from whole blood should be ≥800 ng.

[0046] 3. Preparation of genotype quality control products. 205 The DNA plasmid was mixed with the AB+O suspension in a certain proportion and slightly inverted to mix well without forming air bubbles.

[0047] 4. Genotype quality control product typing and sequencing: ABO, RHD, RHCE, Kell, Kadd, Duffy, MNS, Diego, Dombrock, Colton, Cartwright, Lutheran and other genotyping, ABO, RhD, RhCE, Kadd, FUT1-3, A4GALT, B3GALNT1 and other gene sequencing.

[0048] 5. Aliquot the quality control sample: 2 ml per tube (sterile, enzyme-free, and contaminant-free). Label with the quality control sample name, code, preparation, and expiration date.

[0049] 6. Storage conditions: 4°C for 72 hours, -20°C for 8 weeks, -80°C for long-term storage.

[0050] 7. Reference values ​​of quality control products 7.1 Genotyping 7.1.1 ABO: A, A2, B, O 261Gdel , O1, O2.

[0051] 7.1.2 RHD: E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, 845, 1227, GALC.

[0052] 7.1.3 RHCE: C (RH2), E (RH3), c (RH4), e (RH5), CW (RH8), V (RH10), hrS (RH19), VS (RH20), hrB (RH31).

[0053] 7.1.4 Kell: K (KEL1), k (KEL2), Kpa (KEL3), Kpb (KEL4), Jsa (KEL6), Jsb (KEL7).

[0054] 7.1.5 Kidd: Jka (JK1), Jkb (JK2).

[0055] 7.1.6 Duffy: Fya (FY1), Fyb (FY2).

[0056] 7.1.7 MNS: M (MNS1), N (MNS2), S (MNS3), s (MNS4), U (MNS5), Mia (MNS7).

[0057] 7.1.8 Diego: Dia (DI1), Dib (DI2).

[0058] 7.1.9 Dombrock: Doa (DO1), Dob (DO2), Hy (DO4), Joa (DO5).

[0059] 7.1.10 Colton: Coa (CO1), Cob (CO2).

[0060] 7.1.11 Cartwright: Yta (YT1), Ytb (YT2).

[0061] 7.1.12 Lutheran: Lua (LU1), Lub (LU2).

[0062] 7.2 Gene Sequencing 7.2.1 ABO: Exon3 c.106G>T, Exon4 c.188G>A, Exon4 c.189C>T, Exon5 c.220C>T, Exon6 c.261delG, Exon6 c.297A>G, Exon7 c.467C>T, Exon7 c.526C>G, Exon7 c.646T>A, Exon7 c.657C>T, Exon7 c.681G>A, Exon7 c.703G>A, Exon7 c.771C>T, Exon7c.796C>A, Exon7 c.803G>C, Exon7 c.829G>A. Exon7 c.930G>A.

[0063] 7.2.2 RhD: No mutation.

[0064] 7.2.3 RhCE: Exon1 c.48G>C.

[0065] 7.2.4 Kadd: Exon3 c.130G>A, Exon6 c.588A>G, Exon8 c.838G>A.

[0066] 7.2.5 FUT1: Exon4 c.35C>T. FUT2: Exon2 c.357C>T, Exon2 c.385A>T. FUT3: Exon3 c.59T>G, Exon3 c.508G>A.

[0067] 7.2.6 A4GALT: Exon1 c.903C>G.

[0068] 7.2.7 B3GALNT1: No mutation.

[0069] 8. Usage and Effects 8.1 The quality control products are tested in the same manner as the specimens through the processes of mixing, extraction, amplification, analysis, and sequencing. Each DNA extraction plate is equipped with one negative control well, one positive control well, and one positive quality control well, and the testing is performed in the same manner as the specimens. Each reaction well of each amplification plate is equipped with an internal control; each amplification plate is equipped with one negative control well, one positive control well, and one positive quality control well. The quality control products of the present invention are added to the above-mentioned positive quality control wells. The testing is performed in the same manner as the specimens. When performing product analysis and sequencing, the positive quality control materials are tested in the same manner as the specimens.

[0070] 8.2 The laboratory shall use real-time PCR for testing. This is a qualitative test, and non-statistical methods shall be used for quality control. In-house quality control charts shall be established based on the Ct values ​​of each measurement of the quality control product. In-house quality control charts shall be established based on the target values ​​of each measurement of the quality control product for product analysis and sequencing.

[0071] 8.3 In-control and test validity standards. If any of the conditions such as the quality control reference value are not met, the experiment is considered out of control.

[0072] (1) If the quality control product is out of control and is predicted to be negative in a certain item (i.e. the reference value is negative), and the test value shows a false positive result, the positive sample of this experiment should be retested.

[0073] (2) If the quality control product is out of control and it is predicted to be positive in a certain item (i.e. the reference value is positive), but the test value shows a false negative result, the negative sample of this experiment should be retested.

[0074] In Examples 2-4 below, different blood type tests were performed using different genetic testing methods. Sequencing results indicate that the quality control products of the present invention all achieved the expected values ​​and maintained their effectiveness. This demonstrates the stability, effectiveness, and broad applicability of the quality control products prepared by the present invention in various experimental testing scenarios.

[0075] Example 2: ABO Genotyping (Real-time PCR) 1. Specimen Preparation 1.1. Select five EDTA-anticoagulated whole blood quality control samples with known genotypes.

[0076] 1.2. DNA concentration should be 40-70 ng / µL, and the A260 / A280 purity should be 1.6-2.0.

[0077] 2. Reagent Preparation 2.1. Reagent composition PCR reagents: rTap enzyme; purified water; ABO primer plate: ABO primers, 4× Tris-HCl.

[0078] Fluorescent PCR buffer: 10× PCR Reaction Buffer I, dNTPs, SYBR Green I.

[0079] Nucleic acid extraction reagents: proteinase K, lysis buffer, binding buffer.

[0080] 2.2. Preparation of PCR working solution Take out 1 tube of fluorescent PCR buffer (500µL) from the refrigerator and thaw it at room temperature. Then take 180µL of fluorescent PCR buffer and add it to a labeled centrifuge tube. Then add 180µL of purified water and 3.6µL of rTaq enzyme. Vortex to mix and then centrifuge briefly (1000rpm for 10 seconds).

[0081] 2.3. Nucleic acid extraction reagent packaging Take 3 centrifuge tubes and fill them with 120ul proteinase K, 2100ul nucleic acid lysis buffer, and 2100ul nucleic acid binding buffer respectively and label them.

[0082] 3. Equipment Preparation 3.1. FQD-96A fluorescence quantitative polymerase chain reaction (PCR) detection system (Hangzhou Bioray).

[0083] 3.2. BE-6100 plate centrifuge (Haimen Qilin Bell).

[0084] 3.3. XH-T vortex mixer (Baita Xinbao Instrument Factory, Jintan District).

[0085] 3.4. ZK-96 fully automatic nucleic acid extraction and purification instrument (Nanjing Zhongke Baier).

[0086] 3.5. 10μl-1000μl micropipette (Thermo Fisher Scientific).

[0087] 4. DNA Extraction Sample processing (1) Mix the EDTA anticoagulant whole blood quality control product.

[0088] (2) Take five 1.5ml centrifuge tubes numbered 1-5, add 20ul proteinase K, 200uL of the corresponding quality control products numbered 1-5, and 350ul lysis buffer to the centrifuge tubes in sequence, and shake and mix for 1 minute. The 200uL here refers to the single use amount of the aforementioned quality control product, which is also the single initial sample volume recorded in the instructions for use of the DNA extraction kit. In the 200uL quality control product, its DNA content is ≥800ng, which also means that the amount of nucleic acid extracted from whole blood in a single use is ≥800ng.

[0089] (3) Place the centrifuge tube on a 65°C constant temperature shaker and shake at 1000 rpm for 30 minutes.

[0090] (4) After the incubation is completed, centrifuge briefly to discard the tube cap and liquid on the side wall into the solution in the tube.

[0091] (5) Transfer all the liquid in the tube to the effective wells of the deep well plate 1 sample plate. See the DNA extraction layout diagram Figure 1 .

[0092] (6) Add 350ul of binding solution to the effective sample wells of plate 1, and then place it on the automatic nucleic acid extraction instrument for nucleic acid extraction.

[0093] (7) Take 5 centrifuge tubes numbered 1-5, transfer the nucleic acid from the 6th sample well of the deep-well plate into the centrifuge tubes, and measure the nucleic acid concentration using a micro-spectrophotometer.

[0094] The evaluation of DNA concentration is shown in Table 1 below.

[0095] Table 1

[0096] The DNA sample concentration was tested to be in the range of 40-70 ng / µL and the purity A260 / A280 value was 1.6-2.0, and the next step of the experiment could be carried out.

[0097] 4.2 Sample addition (real-time PCR) (1) Take 6 centrifuge tubes numbered 1-6, add 65µL of PCR working solution to each centrifuge tube, add 6.5µL of the corresponding DNA samples 1-5 to tubes 1-5, and add 6.5µL of the negative reference to tube 6, mix them separately, shake and mix thoroughly, and then centrifuge briefly (1000rpm centrifuge for 10s). (2) Perform 6-well PCR reaction for each sample, namely A, A2, B, OT, O1, O2. The ABO primer plate can be cut and numbered according to the number of samples. See the DNA amplification plate diagram for details. Figure 2 .

[0098] Add 10µL of the mixture of the PCR working solution and the DNA sample to each well, and then centrifuge briefly using a plate centrifuge to allow the residual liquid on the tube wall to gather at the bottom of the tube.

[0099] (3) Add 20µL of paraffin oil to each well to completely cover the PCR reaction solution.

[0100] 5. PCR Amplification 5.1. Program Selection Log in to the '9600Plus' operating system, click 'Classification Templates', select the 'ABO Typing' program template, enter the settings page, change the experiment name to 'ABO Typing Test', click 'Run', confirm the heated lid temperature, liquid addition volume, and increment parameters based on the sample reaction conditions, then confirm execution and save the program. After the run is complete, click 'Analyze' to view the results. For amplification curves and melting curves, click the reaction wells to view the corresponding well results, record them, and analyze them.

[0101] 5.2. Check parameters Heated lid temperature: 105°C; liquid volume: 10 μl. Value-added parameters are shown in Table 2.

[0102] Table 2

[0103] 5.3 Result Analysis (Analysis and Interpretation of Amplification Curves and Melting Curves) Interpretation criteria: Positive: The sample is considered positive when it has both the internal control positive judgment value range Tm and the characteristic Tm of the PCR positive judgment value range, and the peak fluorescence derivative is not less than 50.

[0104] Negative: It is judged as negative when it only has the internal control positive judgment value Tm and the peak fluorescence value is not less than 50. There is no melting characteristic peak in the PCR positive judgment value.

[0105] Table 3 below is a comparison table of positive reference values. Figure 3 .

[0106] Table 3 Positive reference value comparison table

[0107] According to the experimental amplification curve, all the results were positive, the internal control and specific amplification peaks were within the Tm judgment value range, and the peak fluorescence derivative was not less than 50.

[0108] 6. Interpretation of results a. Use the original real-time fluorescence quantitative PCR software to open the raw data and use the positive and negative Tm judgment value range to determine that all the sample wells are positive.

[0109] b. Use the interpretation table ( Figure 4 ) The ABO allele results were interpreted as shown in Table 4 below. Because the samples used in the experiment were positive quality control samples, the results were all positive. This is completely consistent with the expected values.

[0110] Table 4 ABO genotyping (real-time PCR) results

[0111] 7. Analysis of factors causing adverse experimental results and re-examination 7.1 If the nucleic acid extraction concentration does not meet the requirements, it can be adjusted by dilution or concentration. The concentration and purity of DNA will affect the efficiency and success rate of amplification. High DNA concentration is usually accompanied by a large number of PCR inhibitors, while too low a concentration may cause amplification failure.

[0112] 7.2 The experimental process must strictly adhere to the principles of aseptic operation to avoid product contamination.

[0113] 7.3 Positive and negative controls must be set up for the experiment. Any abnormal results must be further verified and confirmed, and retested if necessary.

[0114] Example 3: RhD genotyping (PCR-SSP) 1. Specimen Preparation 1.1. Select five EDTA-anticoagulated whole blood quality control samples with known genotypes.

[0115] 1.2. DNA concentration should be 40-70 ng / µL, and the A260 / A280 purity should be 1.6-2.0.

[0116] 2. Reagent Preparation PCR reagents: dNTP-Buffer working solution, rTap enzyme, PCR reaction plate (primer plate).

[0117] Nucleic acid extraction reagents: proteinase K, lysis buffer, binding buffer.

[0118] 3. Equipment Preparation 3.1. TC-96 / G / H(b)C polymerase chain reaction (PCR) detection system; Hangzhou Biori Technology Co., Ltd.

[0119] 3.2. BE-6100 plate centrifuge (Haimen Qilin Bell).

[0120] 3.3. XH-T vortex mixer (Baita Xinbao Instrument Factory, Jintan District).

[0121] 3.4. ZK-96 fully automatic nucleic acid extraction and purification instrument (Nanjing Zhongke Baier).

[0122] 3.5. 10μl-1000μl micropipette (Thermo Fisher Scientific).

[0123] 4. DNA Extraction Sample processing (1) Mix the EDTA anticoagulant whole blood quality control product.

[0124] (2) Take five 1.5ml centrifuge tubes numbered 1-5 and add 20ul of proteinase K, 200ul of the corresponding quality control sample numbered 1-5 (i.e., quality control sample suspension), and 350ul of lysis buffer to the centrifuge tubes in sequence. Vortex and mix for 1 minute. Place the centrifuge tubes on a 65℃ constant temperature shaker and shake at 1000 rpm for 30 minutes.

[0125] After incubation, centrifuge briefly to discard the tube cap and side liquid into the solution in the tube.

[0126] (3) Transfer all the liquid in the tube to the effective wells of the deep well plate 1 sample plate. See the DNA extraction layout diagram Figure 5 .

[0127] (4) Add 350ul of binding solution to the effective sample well of plate 1, and then place it on the automatic nucleic acid extraction instrument for nucleic acid extraction.

[0128] (5) Take 5 centrifuge tubes numbered 1-5, transfer the nucleic acid from the 6th sample well of the deep-well plate into the centrifuge tubes, and measure the nucleic acid concentration using a micro-spectrophotometer.

[0129] The evaluation of DNA concentration is shown in Table 5 below.

[0130] Table 5

[0131] The DNA sample concentration was tested to be in the range of 40-70 ng / µL, and the purity A260 / A280 value was 1.6-2.0, so the next step of the experiment could be carried out.

[0132] 4.2 Sample addition (PCR-SSP method) (1) Prepare dNTP-Buffer working solution: 510 μl concentrated dNTP-Buffer + 650 μl sterile water = 1160 μl dNTP-Buffer working solution.

[0133] (2) Prepare the buffer-enzyme-sample mixture: The amount per person is: 80 μl dNTP-Buffer working solution + 0.8 μl Taq enzyme + 10 μl DNA = 90.8 μl mixture.

[0134] (3) Vortex the buffer-enzyme-sample mixture and centrifuge briefly.

[0135] (4) Add 10 μl of the above mixture to each primer well (wells 1-8).

[0136] (5) Add 15-20 μl of paraffin oil to each well, or seal the PCR reaction plate (primer plate) with sealing film.

[0137] (6) Place the primer plate into the PCR instrument with the cycling parameters set, and use the appropriate plate holder adapter.

[0138] (7) Place a PCR sealing pressure pad on the top of the reaction plate to prevent liquid evaporation.

[0139] See the DNA amplification layout diagram for Figure 6 .

[0140] 5. PCR Amplification Log in to the TC-96 / G / H(b)C operating system, set the PCR cycle parameters based on the sample reaction conditions, and start the PCR program until the cycle is complete. After the run is complete, click "Analyze" to view the amplification curve. Click the reaction well to view the corresponding well results, record them, and analyze them.

[0141] Table 6

[0142] 6. Product Analysis Remove the PCR reaction plate. If electrophoresis is to be performed immediately, gently remove the sealing film and proceed with electrophoresis. If not, store at 4°C for up to 48 hours. Transfer each PCR reaction product (5-10 μl) to a well of a 2.5% agarose gel according to the sequence of the primer well map. Run electrophoresis at 140-150V for 15-20 minutes until the internal reference band and the positive band are clearly separated. Observe the results under UV light and photograph them. Figure 7 ).

[0143] 7. Interpretation of results According to the result typing table provided by the kit ( Figure 8 ) Explain the typing results.

[0144] Table 7. RhD genotyping (PCR-SSP) results

[0145] 8. Analysis of factors causing adverse experimental results and re-examination.

[0146] 8.1 If the nucleic acid extraction concentration does not meet the requirements, it can be adjusted by dilution or concentration. The concentration and purity of DNA will affect the efficiency and success rate of amplification. High DNA concentration is usually accompanied by a large number of PCR inhibitors, while too low a concentration may cause amplification failure.

[0147] 8.2 The experimental process must strictly adhere to the principles of aseptic operation to avoid product contamination.

[0148] 8.3 Positive and negative controls must be set up for the experiment. Abnormal results must be further verified and confirmed, and retested if necessary.

[0149] Example 4. ABO Blood Type Gene Sequencing (SANGER Sequencing) 1. Using the AxyPrep DNA Gel Recovery Kit, mix gel melting agent DE-A and binding buffer DE-B in a ratio of 3:2. Add 200ul of the mixture to each sample well and let it stand for two minutes. Then transfer the liquid to an adsorption column (labeled in advance), let it stand for two minutes, and centrifuge for 1 minute (for tapping samples, first add 300ul of solution A, then add 200ul of solution B, dissolve in a 72°C water bath, transfer to the adsorption column, let it stand and centrifuge for 1 minute). Finally, add 500ul of wash buffer (wash the tapping samples twice), centrifuge for 1 minute, discard the waste liquid, and centrifuge again for 1 minute.

[0150] 2. Transfer the adsorption columns to new pre-labeled centrifuge tubes in order, then vacuum dry them for 2 hours, add 50ul of pure water (half the amount to 25ul for weaker ones) to the membrane of each adsorption column, let it stand for 3 minutes, then centrifuge for 1 minute and discard the adsorption column.

[0151] 3. In a new 96-well PCR plate, first add 2 μl of purified water to each well and centrifuge for 15 seconds. Then add 1 μl of the corresponding primer according to the sample reaction table and centrifuge for 15 seconds. Then add 1 μl of the corresponding DNA (DNA extracted from the quality control sample) and centrifuge for 15 seconds. Finally, add 1 μl of BDT and centrifuge for 15 seconds. Cover with a silicone pad and proceed with PCR amplification.

[0152] 4. After amplification, centrifuge for 2 minutes, add 2.5ul EDTA to each reaction well, and centrifuge for 15 seconds. Then transfer the sample to a new reaction plate and centrifuge for 15 seconds. Add 18ul of alcohol to each well, seal the film with disposable sealing paper, shake well with a mixer, and then centrifuge at 4000rpm at 4°C for 30 minutes. Remove the film and centrifuge upside down for 15 seconds to remove the supernatant.

[0153] Finally, add 40ul of chilled alcohol, seal the membrane, centrifuge at 4000rpm at 4°C for 15min, tear the membrane upside down and centrifuge again for 15s to remove the supernatant, and dry in a vacuum drying oven for 20min.

[0154] 5. Add 10 μl of HIDI (highly deionized-formamide, ABI) to each well, seal the membrane and centrifuge. Denature the tube using a PCR instrument at 95°C for 5 min - 10°C for 10 s, and then transfer to the machine.

[0155] 6. Turn on the ABI3500 sequencer. After the sequencer self-test completes (green light remains on), enter the computer power-on password. Prepare the setup sheet according to the template, open the sequencer data collection software, and enter the DC software password. Import the setup sheet and follow the operating procedures to click Start to begin the run. At the end of the run, shut down the DC software, the sequencer, and the computer.

[0156] The gene sequencing results are shown in Table 8 and Figure 9 .

[0157] Table 8. ABO gene sequencing results (SANGER Sequencing)

[0158] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. A method for preparing a quality control product for blood type gene detection, characterized in that: The following steps are involved: (1) Preparing AB type and O type suspensions: confirming the blood type of human blood materials with AB type heterozygous and O type homozygous blood types respectively; collecting the blood materials after blood type confirmation into leukocyte filters respectively, backwashing the leukocyte filters with blood preservation fluid, and collecting the flushing fluid aseptically to obtain AB type flushing fluid and O type flushing fluid respectively; mixing the AB type flushing fluid and the O type flushing fluid in equal amounts to form AB type and O type suspensions; (2) Preparation A 205 DNA: From blood type A 205 A subtype was extracted from blood samples 205 DNA or according to A 205 Sequence synthesis A 205 DNA plasmid, thereby obtaining A 205 DNA; (3) Prepare quality control product: 205 The DNA and the AB type and O type suspensions are mixed to form a quality control product; In step (1), before mixing equal amounts of the AB-type flushing solution and the O-type flushing solution, the DNA concentrations in the AB-type flushing solution and the O-type flushing solution are controlled and confirmed so that the DNA content in the flushing solution of the quality control product is ≥800 ng in a single use amount; In step (2), control and confirm A 205 The concentration of DNA is such that the DNA content in a single-use solution of the quality control product is ≥800 ng; In step (3), control A 205 The ratio of the DNA to the AB type and O type suspensions is such that the DNA content of the quality control product is ≥800 ng in a single use of the quality control product.

2. The preparation method according to claim 1, characterized in that The blood type confirmation in step (1) includes: when the blood type result meets the following conditions, the blood material is used: Blood type AB: Serological positive and negative typing: A4+, B4+, Ac-, Bc-, phenotype is AB, genotype is A, B; Blood type O: Serological typing: A-, B-, Ac4+, Bc4+, phenotype is O, genotype is O 261Gdel , O1, O2.

3. The preparation method according to claim 1, characterized in that In step (1), before mixing equal amounts of the AB-type washing solution and the O-type washing solution, the DNA absorbance 260 / 280 ratio in each of the AB-type washing solution and the O-type washing solution is controlled and confirmed to be between 1.7 and 2.

0.

4. The preparation method according to claim 1, characterized in that Control and confirm the A prepared in step (2) 205 The DNA absorbance 260 / 280 ratio is between 1.7 and 2.

0.

5. The preparation method according to claim 1, characterized in that The mixing method described in step (1) and step (3) includes slight inversion to avoid the formation of air bubbles.

6. A quality control product obtained by the preparation method according to any one of claims 1 to 5.

7. A method for blood type gene testing using the quality control product according to claim 6, characterized in that: The method comprises performing blood type gene testing on the quality control product and the sample to be tested according to the same operation, wherein the operation includes DNA extraction, amplification and analysis; the DNA extraction includes using a DNA extraction kit, and the single initial sample volume recorded in the instructions for use of the DNA extraction kit is the single usage volume of the quality control product; in the test result, if the quality control product is out of control, the test result of the sample to be tested will not be accepted.

8. The method according to claim 7, characterized in that The out-of-control quality control product refers to the fact that the test value of the quality control product is inconsistent with the reference value of the corresponding item in the quality control product reference value; the quality control product reference value includes the genotyping reference value and the gene sequencing reference value; The genotyping reference values ​​are as follows: 1.1 ABO:A、A2、B、O 261Gdel 、O1、O2; 1.2 RHD: E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, 845, 1227, GALC; 1.3 RHCE: C (RH2), E (RH3), c (RH4), e (RH5), CW (RH8), V (RH10), hrS (RH19), VS (RH20), hrB (RH31); 1.4 Kell: K (KEL1), k (KEL2), Kpa (KEL3), Kpb (KEL4), Jsa (KEL6), Jsb (KEL7); 1.5 Kidd: Jka (JK1), Jkb (JK2); 1.6 Duffy: Fya (FY1), Fyb (FY2); 1.7 MNS: M (MNS1), N (MNS2), S (MNS3), s (MNS4), U (MNS5), Mia (MNS7); 1.8 Diego: Dia (DI1), Dib (DI2); 1.9 Dombrock: Doa (DO1), Dob (DO2), Hy (DO4), Joa (DO5); 1.10 Colton: Coa (CO1), Cob (CO2); 1.11 Cartwright: Yta (YT1), Ytb (YT2); 1.12 Lutheran: Lua (LU1), Lub (LU2); The gene sequencing reference values ​​are as follows: 2.1 ABO: Exon3 c.106G>T, Exon4 c.188G>A, Exon4 c.189C>T, Exon5 c.220C>T, Exon6c.261delG, Exon6 c.297A>G, Exon7 c.467C>T, Exon7 c.526C>G, Exon7 c.646T>A, Exon7c.657C>T, Exon7 c.681G>A, Exon7 c.703G>A, Exon7 c.771C>T, Exon7 c.796C>A, Exon7c.803G>C, Exon7 c.829G>A, Exon7 c.930G>A; 2.2 RhD: no mutation; 2.3 RhCE: Exon1 c.48G>C; 2.4 Kadd: Exon3 c.130G>A, Exon6 c.588A>G, Exon8 c.838G>A; 2.5 FUT1: Exon4 c.35C>T; FUT2: Exon2 c.357C>T, Exon2 c.385A>T; FUT3: Exon3c.59T>G, Exon3 c.508G>A; 2.6 A4GALT: Exon1 c.903C>G; 2.7 B3GALNT1: No mutation.

9. The method according to claim 7, characterized in that The blood type gene test is performed on the quality control product and the sample to be tested according to the same operation, including: each DNA extraction plate is provided with a negative control well, a positive control well, and a positive quality control well, the quality control product is added to the positive quality control well, and extraction is performed in the same manner as the sample to be tested; each reaction well of each DNA amplification plate is provided with an internal control; each DNA amplification plate is provided with a negative control well, a positive control well, and a positive quality control well, the quality control product is added to the positive quality control well, and amplification is performed in the same manner as the sample to be tested; the positive quality control material in the positive quality control well is analyzed and sequenced in the same manner as the sample to be tested.

10. The method according to claim 8, characterized in that When the quality control product is out of control, when the reference value of the item is negative and the detection value of the quality control product in this item is false positive, the samples with positive test results in this experiment will be retested; when the reference value of the item is positive and the detection value of the quality control product in this item is false negative, the samples with negative test results in this experiment will be retested.

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