WO2021135559A1 - 一种检测人类红细胞abo血型基因分型的引物组、试剂盒及应用 - Google Patents

一种检测人类红细胞abo血型基因分型的引物组、试剂盒及应用 Download PDF

Info

Publication number
WO2021135559A1
WO2021135559A1 PCT/CN2020/123604 CN2020123604W WO2021135559A1 WO 2021135559 A1 WO2021135559 A1 WO 2021135559A1 CN 2020123604 W CN2020123604 W CN 2020123604W WO 2021135559 A1 WO2021135559 A1 WO 2021135559A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
detecting
primers
sequence listing
primer pairs
Prior art date
Application number
PCT/CN2020/123604
Other languages
English (en)
French (fr)
Inventor
胡志超
胡智明
李颖龙
郭飞飞
程艳伟
王光辉
郭秀明
Original Assignee
河南兴龙生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 河南兴龙生物技术有限公司 filed Critical 河南兴龙生物技术有限公司
Publication of WO2021135559A1 publication Critical patent/WO2021135559A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the field of biotechnology, in particular to a primer set, kit and application for detecting ABO blood type genotyping of human red blood cells.
  • the ABO blood group system is the most immunogenic blood group system in the human blood group system, and is also a classic human genetic marker. Therefore, it plays an important role in blood transfusion medicine, paternity testing, anthropological research, and forensic material evidence testing.
  • the human ABO gene is located on chromosome 9 (9q34.1-9q34.2) and is controlled by three multiple alleles A, B and O. It contains 7 exons ranging in length from 28bp to 691bp and 6 with a length of about 19514bp. The DNA sequence of this intron is highly conserved. The product encoded by the gene is glycosyltransferase. Most of the coding sequences are located on the 6th and 7th exons. These transferases control the biosynthesis of ABO blood group antigens, thereby determining their blood group. . ABO blood group differences are mainly related to genetic changes. There are abundant single nucleotide polymorphisms in the ABO blood group gene. The ABO blood group gene's SNP sites can be detected by molecular biology methods to determine the A, B, O or AB phenotype.
  • ABO blood group detection There are two main methods for human ABO blood group detection: one is the classic serological method, and the other is the genotyping method.
  • the current routine detection method is a traditional serological method. This method is susceptible to factors such as antigen activity, antigen polymorphism, antibody specificity, autoantibodies, irregular antibodies, microorganisms, diseases, etc., making the phenotype identification inaccurate, and Abnormal expression and genetic analysis suffer from insurmountable limitations.
  • PCR-RFLP restriction fragment length polymorphism PCR
  • single-strand conformation polymorphism PCR single-strand conformation polymorphism PCR.
  • PCR-SSCP specific sequence primer PCR method
  • PCR-SSP specific sequence primer PCR method
  • RT-PCR real-time fluorescent quantitative PCR method
  • sequencing method etc.
  • the PCR-SSP method requires multi-tube amplification, multi-band electrophoresis, and there is a risk of typing errors caused by confusion in interpretation.
  • the biggest drawback of the PCR-SBT typing method is that the operation process is very cumbersome, the workload is large, and it is difficult to achieve high-throughput operations.
  • the amplification of different regions of the ABO gene has different amplification conditions, which not only requires a large sample volume, but also increases the consumption of reagents and consumables, and requires more equipment resources.
  • the first object of the present invention is to provide a primer set for detecting ABO blood type genotyping of human red blood cells;
  • the second object of the present invention is to provide a kit for detecting ABO blood type genotyping of human red blood cells and the same Preparation method;
  • the third purpose of the present invention is to provide a primer set or kit in the preparation of human red blood cell ABO blood group genotyping reagent application.
  • the present invention consults a large number of relevant literature and accumulated experimental data for the ABO blood group system, and determines the detected 10 common Chinese population ABO blood group subtypes, and the corresponding 24 multiple alleles SNP locus information A101, A102(467C>T), A201(467C>T,1061delC), A205(467C>T,1009A>G), B101(297A>G,526C>G,657C>T,703G>A ,796C>A,803G>C,930G>A,1096G>A), O01(261delG),
  • the present invention is based on competition Based on the principle of sex allele-specific PCR, 24 pairs of primer sets were designed to simultaneously detect the combination information of 24 genetic gene loci in the ABO blood group system of human red blood cells.
  • a primer set for detecting the ABO blood group genotyping of human red blood cells includes 24 sets, and the sequence list of the 24 sets of primer sets is shown in SEQ ID No. 1-72.
  • the 24 primer sets and their corresponding detection sites are:
  • the first set of primers is shown in SEQ ID No. 1-3 in the sequence list, and is used to detect the 106 (rs688976) locus in the genetic locus corresponding to the ABO blood type;
  • the second set of primers are used to detect the 188 (rs549446) locus in the genetic locus corresponding to the ABO blood group;
  • the third set of primers is shown in SEQ ID No. 7-9 in the sequence table, and is used to detect the 189 (rs549443) locus in the genetic locus corresponding to the ABO blood group;
  • the fourth set of primers is shown in SEQ ID No. 10-12 in the sequence list, and is used to detect the 220 (rs512770) locus in the genetic locus corresponding to the ABO blood group;
  • the fifth set of primers is shown in SEQ ID No. 13-15 in the sequence list, and is used to detect the 261 (rs8176719) locus in the genetic locus corresponding to the ABO blood group;
  • the sixth set of primers is shown in SEQ ID No. 16-18 in the sequence list, and is used to detect the 297 (rs8176720) locus in the genetic locus corresponding to the ABO blood group;
  • the seventh set of primers is shown in SEQ ID No. 19-21 in the sequence list, and is used to detect the 467 (rs1053878) locus in the genetic locus corresponding to the ABO blood group;
  • the eighth set of primers is shown in SEQ ID No. 22-24 in the sequence list, and is used to detect the 526 (rs7853989) locus in the genetic locus corresponding to the ABO blood group;
  • the ninth set of primers is shown in SEQ ID No. 25-27 in the sequence table, and is used to detect the 579 (rs55764262) locus of the genetic locus corresponding to the ABO blood group;
  • the tenth set of primers is shown in SEQ ID No. 28-30 in the sequence list, and is used to detect the 646 (rs8176740) locus in the genetic locus corresponding to the ABO blood type;
  • the eleventh set of primers is shown in SEQ ID No. 31-33 in the sequence list, and is used to detect the 657 (rs8176741) locus in the genetic locus corresponding to the ABO blood group;
  • the twelfth set of primers is shown in SEQ ID No. 34-36 in the sequence table, and is used to detect the 681 (rs8176742) locus in the genetic locus corresponding to the ABO blood group;
  • the thirteenth set of primers is shown in SEQ ID No. 37-39 in the sequence table, and is used to detect the 703 (rs8176743) locus in the genetic locus corresponding to the ABO blood group;
  • the fourteenth set of primers is shown in SEQ ID No. 40-42 in the sequence table, and is used to detect the 721 (rs781957267) locus in the genetic locus corresponding to the ABO blood group;
  • the fifteenth set of primers is shown in SEQ ID No. 43-45 in the sequence table, and is used to detect the 771 (rs8176745) locus in the genetic locus corresponding to the ABO blood group;
  • the sixteenth set of primers is shown in SEQ ID No. 46-48 in the sequence table, and is used to detect the 796 (rs8176746) locus in the genetic locus corresponding to the ABO blood group;
  • the seventeenth set of primers is shown in SEQ ID No. 49-51 in the sequence list, and is used to detect the 803 (rs8176747) locus in the genetic locus corresponding to the ABO blood group;
  • the eighteenth set of primers is shown in SEQ ID No. 52-54 in the sequence list, and is used to detect the 829 (rs8176748) locus in the genetic locus corresponding to the ABO blood group;
  • the nineteenth set of primers is shown in SEQ ID No. 55-57 in the sequence list, and is used to detect the 930 (rs8176749) locus in the genetic locus corresponding to the ABO blood type;
  • the twentieth set of primers is shown in SEQ ID No. 58-60 in the sequence list, and is used to detect the 1009 (rs566015043) locus in the genetic locus corresponding to the ABO blood group;
  • the twenty-first set of primers is shown in SEQ ID No. 61-63 in the sequence list, and is used to detect the 1061 (rs56392308) locus in the genetic locus corresponding to the ABO blood group;
  • the twenty-second set of primers is shown in SEQ ID No. 64-66 in the sequence table, and is used to detect the 1096 (rs8176751) locus in the genetic locus corresponding to the ABO blood group;
  • the twenty-third set of primers is shown in SEQ ID No. 67-69 in the sequence table, and is used to detect the 53 (rs55876802) locus in the genetic locus corresponding to the ABO blood group;
  • the twenty-fourth set of primers is shown in SEQ ID No. 70-72 in the sequence list, and is used to detect the 802 (rs41302905) locus in the genetic locus corresponding to the ABO blood type.
  • each of the 24 primer sets includes two primers designed based on the difference in allelic sequences and a common paired primer.
  • primer pairs for detecting 106G in the first set of primers are SEQ ID No. 1 and SEQ ID No. 3 in the sequence listing
  • primer pairs for detecting 106T are SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing;
  • the primer pairs for detecting 188G in the second set of primers are SEQ ID No. 4 and SEQ ID No. 6 in the sequence listing, and the primer pairs for detecting 188A are SEQ ID No. 5 and SEQ ID No. 6 in the sequence listing;
  • the primer pairs for detecting 189C in the third set of primers are SEQ ID No. 7 and SEQ ID No. 9 in the sequence listing, and the primer pairs for detecting 189T are SEQ ID No. 8 and SEQ ID No. 9 in the sequence listing;
  • the primer pairs for detecting 220C in the fourth set of primers are SEQ ID No. 10 and SEQ ID No. 12 in the sequence listing, and the primer pairs for detecting 220T are SEQ ID No. 11 and SEQ ID No. 12 in the sequence listing;
  • the primer pairs for detecting 261G in the fifth set of primers are SEQ ID No. 13 and SEQ ID No. 15 in the sequence listing, and the primer pairs for detecting 261delG are SEQ ID No. 14 and SEQ ID No. 15 in the sequence listing;
  • the primer pairs for detecting 297A in the sixth set of primers are SEQ ID No. 16 and SEQ ID No. 18 in the sequence listing, and the primer pairs for detecting 297G are SEQ ID No. 17 and SEQ ID No. 18 in the sequence listing;
  • the primer pairs for detecting 467C are SEQ ID No. 19 and SEQ ID No. 21 in the sequence listing
  • the primer pairs for detecting 467T are SEQ ID No. 20 and SEQ ID No. 21 in the sequence listing;
  • the primer pairs for detecting 526C in the eighth set of primers are SEQ ID No. 22 and SEQ ID No. 24 in the sequence listing, and the primer pairs for detecting 526G are SEQ ID No. 23 and SEQ ID No. 24 in the sequence listing;
  • the primer pairs for detecting 579T in the ninth set of primers are SEQ ID No. 25 and SEQ ID No. 27 in the sequence listing, and the primer pairs for detecting 579C are SEQ ID No. 26 and SEQ ID No. 27 in the sequence listing;
  • the primer pairs for detecting 646T in the tenth set of primers are SEQ ID No. 28 and SEQ ID No. 30 in the sequence listing, and the primer pairs for detecting 646A are SEQ ID No. 29 and SEQ ID No. 30 in the sequence listing;
  • the primer pairs for detecting 657C are SEQ ID No. 31 and SEQ ID No. 33 in the sequence listing
  • the primer pairs for detecting 657T are SEQ ID No. 32 and SEQ ID No. 33 in the sequence listing;
  • the primer pairs for detecting 681G in the twelfth set of primers are SEQ ID No. 34 and SEQ ID No. 36 in the sequence listing, and the primer pairs for detecting 681A are SEQ ID No. 35 and SEQ ID No. 36 in the sequence listing;
  • the primer pairs for detecting 703G are SEQ ID No. 37 and SEQ ID No. 39 in the sequence listing, and the primer pairs for detecting 703A are SEQ ID No. 38 and SEQ ID No. 39 in the sequence listing;
  • the primer pairs for detecting 721C in the fourteenth set of primers are SEQ ID No. 40 and SEQ ID No. 42 in the sequence listing, and the primer pairs for detecting 721T are SEQ ID No. 41 and SEQ ID No. 42 in the sequence listing;
  • the primer pairs for detecting 771C are SEQ ID No. 43 and SEQ ID No. 45 in the sequence listing
  • the primer pairs for detecting 771T are SEQ ID No. 44 and SEQ ID No. 45 in the sequence listing;
  • the primer pairs for detecting 796C are SEQ ID No. 46 and SEQ ID No. 48 in the sequence listing
  • the primer pairs for detecting 796A are SEQ ID No. 47 and SEQ ID No. 48 in the sequence listing;
  • the primer pairs for detecting 803G are SEQ ID No. 49 and SEQ ID No. 51 in the sequence listing
  • the primer pairs for detecting 803C are SEQ ID No. 50 and SEQ ID No. 51 in the sequence listing;
  • the primer pairs for detecting 829G are SEQ ID No. 52 and SEQ ID No. 54 in the sequence listing
  • the primer pairs for detecting 829A are SEQ ID No. 53 and SEQ ID No. 54 in the sequence listing;
  • the primer pairs for detecting 930G are SEQ ID No. 55 and SEQ ID No. 57 in the sequence listing
  • the primer pairs for detecting 930A are SEQ ID No. 56 and SEQ ID No. 57 in the sequence listing;
  • the primer pairs for detecting 1009G in the twentieth set of primers are SEQ ID No. 58 and SEQ ID No. 60 in the sequence listing, and the primer pairs for detecting 1009A are SEQ ID No. 59 and SEQ ID No. 60 in the sequence listing;
  • the primer pairs for detecting 1061C are SEQ ID No. 61 and SEQ ID No. 63 in the sequence listing, and the primer pairs for detecting 1061delC are SEQ ID No. 62 and SEQ ID No. 63 in the sequence listing;
  • the primer pairs for detecting 1096G in the 22nd set of primers are SEQ ID No. 64 and SEQ ID No. 66 in the sequence listing, and the primer pairs for detecting 1096A are SEQ ID No. 65 and SEQ ID No. 66 in the sequence listing;
  • the primer pairs for detecting 53G in the 23rd set of primers are SEQ ID No. 67 and SEQ ID No. 69 in the sequence listing, and the primer pairs for detecting 53T are SEQ ID No. 68 and SEQ ID No. 69 in the sequence listing;
  • the primer pairs for detecting 802G in the twenty-fourth set of primers are SEQ ID No. 70 and SEQ ID No. 72 in the sequence listing, and the primer pairs for detecting 802A are SEQ ID No. 71 and SEQ ID No. 72 in the sequence listing.
  • the SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 16, SEQ ID No. 19, SEQ ID No. .22, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 34, SEQ ID No. 37, SEQ ID No. 40, SEQ ID No. 43, SEQ ID No. 46 , SEQ ID No. 49, SEQ ID No. 52, SEQ ID No. 55, SEQ ID No. 58, SEQ ID No. 61, SEQ ID No. 64, SEQ ID No. 67, SEQ ID No. 70 carry universal Tag sequence GAAGGTCGGAGTCAACGGATT; the SEQ ID No.
  • SEQ ID No. 5 SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 26, SEQ ID No. 29, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 47.
  • SEQ ID No. 50, SEQ ID No. 53, SEQ ID No. 56, SEQ ID No. 59, SEQ ID No. 62, SEQ ID No. 65, SEQ ID No. 68, SEQ ID No. 71 carry The universal tag sequence GAAGGTGACCAAGTTCATGCT.
  • kits for detecting the ABO blood group genotyping of human red blood cells comprising at least 22 primer sets out of the 24 primer sets according to any one of claims 1 to 5.
  • the kit includes at least 26 chip reaction cells and at least 2 positioning points, and each of the chip reaction cells is coated with a set of primer sets.
  • the chip reaction for each sample is equipped with 1 internal control quality control, 1 blank control and 1 positive quality control.
  • the sum of the product of the three primers in the primer set in the chip reaction pool is equal to 0.05 ⁇ L.
  • a method for preparing a kit for detecting ABO blood group genotyping of human red blood cells including the following steps:
  • the PCR amplification reagents include universal probes carrying HEX and FAM fluorescent groups, dNTPs, Mg 2+ , DNA polymerase and reaction buffer.
  • the present invention utilizes microfluidic chip technology and KASP technology, and each detection index is set in a separate reaction pool, and firstly, the distinctive site information of the ABO blood group multiple allele is designed into a primer with a universal Tag tag sequence Then print the above primer set in the gene chip reaction pool.
  • the primer set and the probe, PCR amplification reagent, TE buffer, and template DNA form an independent PCR reaction system, and the amplification reaction After the end, the gene locus information is obtained by reading the fluorescent signal.
  • a reagent for detecting ABO blood group genotyping of human red blood cells comprising the primer set according to any one of claims 1 to 5 or the kit according to any one of claims 6 to 9.
  • the primers involved in the present invention have undergone a series of optimization processes during the design.
  • the primer set used to simultaneously detect 24 genetic gene loci of the human red blood cell ABO blood group system have been optimized and have similar annealing temperatures and can achieve 24
  • the set of primers are amplified simultaneously under the same PCR amplification conditions.
  • the volume of each PCR reaction system is only 1.15 ⁇ L, and the DNA template only needs 5-10ng, which greatly increases the detection efficiency and reaction sensitivity.
  • the use of this primer set for detection has The advantages of fast speed, low cost, high sensitivity and easy realization of multi-site joint detection;
  • the present invention can not only realize the uniform distribution of samples in different reaction chambers, prevent cross-contamination between different reaction holes, facilitate software interpretation, but also avoid sample waste and greatly save experimental costs, thereby achieving high throughput, Highly sensitive, stable and efficient multi-gene locus joint detection makes ABO blood group genotyping more accurate.
  • Figure 1 shows the detection information of 24 sites and the corresponding fluorescence expression map
  • Figure 2 is a diagram of the chip substrate of the kit
  • Figure 3 shows the fluorescence expression graphs of 4 samples of different ABO blood type combinations on the gene chip
  • Figure 4 is an example diagram of the genotyping for A101/O02 sample software interpretation and corresponding chip fluorescence expression
  • Figure 5 is an example diagram of the genotyping for A102/O01 sample software interpretation and corresponding chip fluorescence expression
  • Figure 6 is an example diagram of the software interpretation of the genotyping of A102/O02 samples and the fluorescence expression of the corresponding chip
  • Figure 7 is an example diagram of software interpretation of B101/B101 genotyping and corresponding chip fluorescence expression
  • Figure 8 is an example diagram of software interpretation of B101/O01 genotyping and corresponding chip fluorescence expression
  • Figure 9 is an example diagram of software interpretation of B101/O02 genotyping and corresponding chip fluorescence expression
  • Figure 10 is an example diagram of software interpretation of genotyping for O01/O01 samples and corresponding chip fluorescence expression
  • Figure 11 is an example diagram of software interpretation of genotyping for O01/O02 samples and corresponding chip fluorescence expression
  • Figure 12 is an example diagram of software interpretation for genotyping O02/O02 samples and corresponding chip fluorescence expression
  • Figure 13 is an example diagram of software interpretation of genotyping for A101/B101 samples and corresponding chip fluorescence expression
  • Figure 14 is an example diagram of the genotyping for A102/B102 sample software interpretation and corresponding chip fluorescence expression.
  • each pair of primer sets is designed for simultaneous detection of 24 human red blood cell ABO blood types Combination information of a genetic locus, and each set of primers contains two primers designed based on the differences in allele sequence positions and a common paired primer.
  • the gene sequences of the 24 primer sets are shown in SEQ ID No. 1-72.
  • the 24 sets of primers were used to prepare a kit for detecting the ABO blood group genotyping of human red blood cells.
  • the volume of the three primers in each primer set in the kit is equal to 0.05 ⁇ L, which are coated in the chip reaction pool in advance.
  • 0.25 ⁇ L of sample genomic DNA, 0.5 ⁇ L of PCR amplification reagent and 0.25 ⁇ L of 1*TE buffer constitute a PCR reaction System, the total volume of each PCR reaction system is 1.15 ⁇ L, in which the genomic DNA concentration is 10-50ng/ ⁇ L, and the A260/A280 OD value is 1.6-2.0;
  • PCR amplification reagents contain HEX or FAM fluorophores 2xMasterMix for universal probes, dNTPs, Mg 2+ , DNA polymerase and reaction buffer.
  • the samples can be genotyped by grouping according to the fluorescence signal.
  • the use of these primer sets can achieve multi-site joint detection, which can achieve the purpose of low-cost and high-efficiency detection of human red blood cell ABO blood group genotyping ( Figure 1 of the specification).
  • a kit for SNP typing based on the principle of competitive allele-specific PCR in which the PCR reaction is completed on a microfluidic chip (Beijing Boao Jingdian Biotechnology Co., Ltd., product number G020010, attached figure 2 in the manual),
  • the preparation method steps are:
  • Each chip can detect 4 samples. For each sample, there are 1 internal control quality control, 1 blank control and 1 positive quality control (ie: the internal control quality control, blank control and positive quality control in Table 1). control).
  • test kit equipped with a corresponding number of sealing films (1 piece/4 persons), reference substance (human genomic DNA 50 ⁇ L/tube/24 persons)
  • the sample for this kit is human genomic DNA extracted from whole blood.
  • the tested genomic DNA must meet a concentration of 10ng/ ⁇ L-50ng/ ⁇ L.
  • the extracted human genomic DNA needs to be subjected to concentration determination. If the concentration is higher than 50ng/ ⁇ L, it needs to be diluted to meet the above requirements before subsequent experiments can be carried out; if the concentration is less than 10ng/ ⁇ L, it needs to be re-extracted until it meets the requirements.
  • reagent storage and preparation area prepare a corresponding number of 0.2mL centrifuge tubes according to the number of samples, and mark the sample numbers on the tubes.
  • PCR amplification reagents Take out the PCR amplification reagents from the kit, fully thawed at room temperature (naturally thawed), vortex and shake to make it completely mixed, and then centrifuge to the bottom of the tube.
  • the sample DNA In the sample preparation area, the sample DNA needs to be thawed at room temperature before use, then mixed and centrifuged instantaneously; add 10 ⁇ L each of the sample DNA to be tested into a 0.2 mL centrifuge tube containing PCR amplification reagents, and the total volume of each PCR reaction system is 40 ⁇ L ,
  • the composition of the PCR amplification system for each sample is shown in Table 2.
  • centrifuge power supply fix the loaded chip on the centrifuge and balance it.
  • the chip reaction pool faces down and the corner is on the top. Centrifuge at 4000rpm for 60 seconds and then remove it. If there are still bubbles in the chip reaction pool, Prolong the centrifugation time appropriately until the bubbles disappear.

Abstract

本发明公开了一种检测人类红细胞ABO血型基因分型的引物组、试剂盒及其在试剂中的应用，所述引物组包括24组，所述的24组引物组的序列表如SEQ ID No.1-72所示；所述试剂盒包含所述的24组引物组中的至少22组引物组；所述试剂包括24组引物组或所述试剂盒。引物经过了优化设计，具有相似的退火温度，能够实现24组引物在相同的PCR扩增条件下同时扩增，每个PCR反应体系的体积只有1.15μL，DNA模板只需5～10ng，大大增加了检测效率和反应灵敏度；应用该引物组进行检测，具有速度快、成本低、灵敏性高及易于实现多位点联合检测等优点。

Description

一种检测人类红细胞ABO血型基因分型的引物组、试剂盒及应用 技术领域
本发明涉及生物技术领域，具体涉及一种检测人类红细胞ABO血型基因分型的引物组、试剂盒及应用。
背景技术
自1900年奥地利著名医学家Karl Landerstainer发现人类ABO血型系统后，截止到目前由国际输血协会(ISBT)认可的血型系统共有39种。ABO血型系统是人类血型系统中免疫原性最强的血型系统，也是经典的人类遗传标记，因此在输血医学、亲子鉴定、人类学研究、法医物证检验具有重要作用。
人类ABO基因位于9号染色体上(9q34.1-9q34.2)，由A、B和O三个复等位基因控制，包含28bp-691bp长度不等的7个外显子和约19514bp长度的6个内含子，其DNA序列高度保守，基因编码的产物是糖基转移酶，多数编码序列位于第6和第7外显子上，这些转移酶控制ABO血型抗原的生物合成，从而决定其血型。ABO血型差异主要与基因变化有关，ABO血型基因上存在丰富的单核苷酸多态性，通过分子生物学方法检测ABO血型基因的SNP位点，可以确定A、B、O或AB表型。
人类ABO血型检测方法主要有两种：一是经典的血清学方法，二是基因分型方法。目前常规检测方法是传统的血清学方法，该方法易受抗原活性、抗原多态性、抗体特异性、自身抗体、不规则抗体、微生物、疾病等因素影响，使得表型鉴定不准确，且在异常表达和遗传学分析时受到无法克服的局限性。随着分子生物学技术的发展，ABO血型基因分型的方法也越来越多，目前最主要的方法有限制性片段长度多态性PCR法(PCR-RFLP)、单链构象多态性PCR(PCR-SSCP)、特异序列引物PCR法(PCR-SSP)、实时荧光定量PCR法(RT-PCR)、测序法等。PCR-SSP方法需要进行多管扩增，多条带电泳，还存在判读易混淆所导致分型错误的分险。PCR-SBT分型方法最大的缺陷是操作过程非常繁琐，工作量大，难以实现高通量操作。ABO基因不同区域的扩增有不同的扩增条件，不仅需要的样本量大，同时增加了试剂耗材的消耗，且需要占用更多的仪器设备资源。综上所述，目前常见的这些方法存在以下几点问题：第一点实验操作复杂，步骤繁琐；第二点结果判读仍是人工方式；第三点样本通量较低；第四点成本较高。这些缺陷直接导致了ABO基因分型无法实现高通量的精准分型，难以适应将来越来越高的医疗需求。
因此，研发一种简单、便捷、快速、高通量、易于判读的ABO血型基因分型产品是具有重要意义的。
发明内容
本发明第一个目的在于提供一种用于检测人类红细胞ABO血型基因分型的引物组；本发明的第二个目的在于提供一种用于检测人类红细胞ABO血型基因分型的试剂盒及其制备方法；本发明的第三个目的在于提供引物组或试剂盒在制备用于检测人类红细胞ABO血型基因分型试剂中的应用。从而实现能够快速、低成本、高灵敏度、同时多位点联合检测地对人类血液进行ABO血型进行基因分型，适于临床应用。
本发明采用的技术方案如下：本发明针对ABO血型系统查阅大量相关文献及积累的实验数据，确定了所检测的10种常见的中国人群ABO血型亚型，及其对应的24个复等位基因SNP位点信息A101，A102(467C>T)，A201(467C>T,1061delC)，A205(467C>T,1009A>G)，B101(297A>G,526C>G,657C>T,703G>A,796C>A,803G>C,930G>A,1096G>A)，O01(261delG),
O02(106G>T,188G>A,189C>T,220C>T,261delG,646T>A,681G>A,771C>T,829G>A)，O03(53G>T,220C>T,297A>G,526C>G,802G>A)，O04(261delG,579)，O07(261delG,297A>G,646T>A,681G>A,721C>T,771C>T,829G>A)，本发明基于竞争性等位基因特异性PCR原理，设计了24对引物组，用于同时检测人类红细胞ABO血型系统的24个遗传基因位点组合信息。
一种检测人类红细胞ABO血型基因分型的引物组，所述引物组包括24组，所述的24组引物组的序列表如SEQ ID No.1-72所示。
进一步的，所述的24组引物组及其对应的检测位点分别为：
第一组引物如序列表SEQ ID No.1-3所示，用于检测ABO血型对应的遗传基因位点中的106(rs688976)位点；
第二组引物如序列表SEQ ID No.4-6所示，用于检测ABO血型对应的遗传基因位点中的188(rs549446)位点；
第三组引物如序列表SEQ ID N o.7-9所示，用于检测ABO血型对应的遗传基因位点中的189(rs549443) 位点；
第四组引物如序列表SEQ ID No.10-12所示，用于检测ABO血型对应的遗传基因位点中的220(rs512770)位点；
第五组引物如序列表SEQ ID No.13-15所示，用于检测ABO血型对应的遗传基因位点中的261(rs8176719)位点；
第六组引物如序列表SEQ ID No.16-18所示，用于检测ABO血型对应的遗传基因位点中的297(rs8176720)位点；
第七组引物如序列表SEQ ID No.19-21所示，用于检测ABO血型对应的遗传基因位点中的467(rs1053878)位点；
第八组引物如序列表SEQ ID No.22-24所示，用于检测ABO血型对应的遗传基因位点中的526(rs7853989)位点；
第九组引物如序列表SEQ ID No.25-27所示，用于检测ABO血型对应的遗传基因位点中的579(rs55764262)位点；
第十组引物如序列表SEQ ID No.28-30所示，用于检测ABO血型对应的遗传基因位点中的646(rs8176740)位点；
第十一组引物如序列表SEQ ID No.31-33所示，用于检测ABO血型对应的遗传基因位点中的657(rs8176741)位点；
第十二组引物如序列表SEQ ID No.34-36所示，用于检测ABO血型对应的遗传基因位点中的681(rs8176742)位点；
第十三组引物如序列表SEQ ID No.37-39所示，用于检测ABO血型对应的遗传基因位点中的703(rs8176743)位点；
第十四组引物如序列表SEQ ID No.40-42所示，用于检测ABO血型对应的遗传基因位点中的721(rs781957267)位点；
第十五组引物如序列表SEQ ID No.43-45所示，用于检测ABO血型对应的遗传基因位点中的771(rs8176745)位点；
第十六组引物如序列表SEQ ID No.46-48所示，用于检测ABO血型对应的遗传基因位点中的796(rs8176746)位点；
第十七组引物如序列表SEQ ID No.49-51所示，用于检测ABO血型对应的遗传基因位点中的803(rs8176747)位点；
第十八组引物如序列表SEQ ID No.52-54所示，用于检测ABO血型对应的遗传基因位点中的829(rs8176748)位点；
第十九组引物如序列表SEQ ID No.55-57所示，用于检测ABO血型对应的遗传基因位点中的930(rs8176749)位点；
第二十组引物如序列表SEQ ID No.58-60所示，用于检测ABO血型对应的遗传基因位点中的1009(rs566015043)位点；
第二十一组引物如序列表SEQ ID No.61-63所示，用于检测ABO血型对应的遗传基因位点中的1061(rs56392308)位点；
第二十二组引物如序列表SEQ ID No.64-66所示，用于检测ABO血型对应的遗传基因位点中的1096(rs8176751)位点；
第二十三组引物如序列表SEQ ID No.67-69所示，用于检测ABO血型对应的遗传基因位点中的53(rs55876802)位点；
第二十四组引物如序列表SEQ ID No.70-72所示，用于检测ABO血型对应的遗传基因位点中的802(rs41302905)位点。
进一步的，所述的24组引物组中每组引物组包含两条基于等位基因序列差异而设计的引物和一条共用配对引物。
更进一步的，第一组引物中检测106G的引物对为序列表SEQ ID No.1和SEQ ID No.3，检测106T的引物对为序列表SEQ ID No2和SEQ ID No.3；
第二组引物中检测188G的引物对为序列表SEQ ID No.4和SEQ ID No.6，检测188A的引物对为序列表SEQ ID No.5和SEQ ID No.6；
第三组引物中检测189C的引物对为序列表SEQ ID No.7和SEQ ID No.9，检测189T的引物对为序列表SEQ ID No.8和SEQ ID No.9；
第四组引物中检测220C的引物对为序列表SEQ ID No.10和SEQ ID No.12，检测220T的引物对为序列表SEQ ID No.11和SEQ ID No.12；
第五组引物中检测261G的引物对为序列表SEQ ID No.13和SEQ ID No.15，检测261delG的引物对为序列表SEQ ID No.14和SEQ ID No.15；
第六组引物中检测297A的引物对为序列表SEQ ID No.16和SEQ ID No.18，检测297G的引物对为序列表SEQ ID No.17和SEQ ID No.18；
第七组引物中检测467C的引物对为序列表SEQ ID No.19和SEQ ID No.21，检测467T的引物对为序列表SEQ ID No.20和SEQ ID No.21；
第八组引物中检测526C的引物对为序列表SEQ ID No.22和SEQ ID No.24，检测526G的引物对为序列表SEQ ID No.23和SEQ ID No.24；
第九组引物中检测579T的引物对为序列表SEQ ID No.25和SEQ ID No.27，检测579C的引物对为序列表SEQ ID No.26和SEQ ID No.27；
第十组引物中检测646T的引物对为序列表SEQ ID No.28和SEQ ID No.30，检测646A的引物对为序列表SEQ ID No.29和SEQ ID No.30；
第十一组引物中检测657C的引物对为序列表SEQ ID No.31和SEQ ID No.33，检测657T的引物对为序列表SEQ ID No.32和SEQ ID No.33；
第十二组引物中检测681G的引物对为序列表SEQ ID No.34和SEQ ID No.36，检测681A的引物对为序列表SEQ ID No.35和SEQ ID No.36；
第十三组引物中检测703G的引物对为序列表SEQ ID No.37和SEQ ID No.39，检测703A的引物对为序列表SEQ ID No.38和SEQ ID No.39；
第十四组引物中检测721C的引物对为序列表SEQ ID No.40和SEQ ID No.42，检测721T的引物对为序列表SEQ ID No.41和SEQ ID No.42；
第十五组引物中检测771C的引物对为序列表SEQ ID No.43和SEQ ID No.45，检测771T的引物对为序列表SEQ ID No.44和SEQ ID No.45；
第十六组引物中检测796C的引物对为序列表SEQ ID No.46和SEQ ID No.48，检测796A的引物对为序列表SEQ ID No.47和SEQ ID No.48；
第十七组引物中检测803G的引物对为序列表SEQ ID No.49和SEQ ID No.51，检测803C的引物对为序列表SEQ ID No.50和SEQ ID No.51；
第十八组引物中检测829G的引物对为序列表SEQ ID No.52和SEQ ID No.54，检测829A的引物对为序列表SEQ ID No.53和SEQ ID No.54；
第十九组引物中检测930G的引物对为序列表SEQ ID No.55和SEQ ID No.57，检测930A的引物对为序列表SEQ ID No.56和SEQ ID No.57；
第二十组引物中检测1009G的引物对为序列表SEQ ID No.58和SEQ ID No.60，检测1009A的引物对为序列表SEQ ID No.59和SEQ ID No.60；
第二十一组引物中检测1061C的引物对为序列表SEQ ID No.61和SEQ ID No.63，检测1061delC的引物对为序列表SEQ ID No.62和SEQ ID No.63；
第二十二组引物中检测1096G的引物对为序列表SEQ ID No.64和SEQ ID No.66，检测1096A的引物对为序列表序列表SEQ ID No.65和SEQ ID No.66；
第二十三组引物中检测53G的引物对为序列表SEQ ID No.67和SEQ ID No.69，检测53T的引物对为序列表SEQ ID No.68和SEQ ID No.69；
第二十四组引物中检测802G的引物对为序列表SEQ ID No.70和SEQ ID No.72，检测802A的引物对为序列表SEQ ID No.71和SEQ ID No.72。
优选地，所述SEQ ID No.1、SEQ ID No.4、SEQ ID No.7、SEQ ID No.10、SEQ ID No.13、SEQ ID No.16、SEQ ID No.19、SEQ ID No.22、SEQ ID No.25、SEQ ID No.28、SEQ ID No.31、SEQ ID No.34、SEQ ID No.37、SEQ ID No.40、SEQ ID No.43、SEQ ID No.46、SEQ ID No.49、SEQ ID No.52、SEQ ID No.55、SEQ ID No.58、SEQ ID No.61、SEQ ID No.64、SEQ ID No.67、SEQ ID No.70携带通用标签序列GAAGGTCGGAGTCAACGGATT；所述SEQ ID No.2、SEQ ID No.5、SEQ ID No.8、SEQ ID No.11、SEQ ID No.14、SEQ ID No.17、SEQ ID No.20、SEQ ID No.23、SEQ ID No.26、SEQ ID No.29、SEQ ID No.32、SEQ ID No.35、SEQ ID No.38、SEQ ID No.41、SEQ ID No.44、SEQ ID No.47、SEQ ID No.50、SEQ ID No.53、SEQ ID No.56、SEQ ID No.59、SEQ ID No.62、SEQ ID No.65、SEQ ID No.68、SEQ ID No.71携带通用标签序列GAAGGTGACCAAGTTCATGCT。
一种检测人类红细胞ABO血型基因分型的试剂盒，所述试剂盒包含权利要求1～5任一项所述的24组引物组中的至少22组引物组。
优选地，所述的试剂盒包括至少26个芯片反应池，至少2个定位点，每个所述芯片反应池中包被有一组引物组。针对每个样本的芯片反应均设有1个内对照质控、1个空白对照以及1个阳性质控。
优选地，所述芯片反应池中的引物组中三条引物积之和相等，均为0.05μL。
一种检测人类红细胞ABO血型基因分型的试剂盒的制备方法，包括以下步骤：
a)从24个位点中选检测人类红细胞ABO血型基因分型中的22个位点，将选的22个位点特异引物组制定孔位表，分别将22组等位基因引物按照孔位表包被至芯片基板22个芯片反应池中，得到芯片半成品；
b)制备PCR扩增试剂；所述PCR扩增试剂中包含携带有HEX和FAM荧光基团的通用探针、dNTPs、Mg 2+、DNA聚合酶和反应缓冲液。
c)配备相应数量封口膜、对照品；
d)将芯片半成品、PCR扩增试剂以及封口膜、对照品按照检测人份组装成一种用于检测人类红细胞ABO血型基因分型的试剂盒。
本发明利用微流控芯片技术和KASP技术，每个检测指标设置于单独的反应池，先将ABO血型复等位基因具有区别性的特异性位点信息设计成带有通用Tag标签序列的引物组，再将上述引物组印制在基因芯片反应池中，在每个反应池中，引物组与探针、PCR扩增试剂、TE缓冲液、模板DNA形成独立的PCR反应体系，扩增反应结束后通过读取荧光信号来获得基因位点信息。竞争性等位基因特异性PCR扩增结束后，对PCR产物进行荧光信号检测，根据读取的荧光信号判断对应位点等位基因信息，后根据多位点等位基因信息对样本进行基因型分型。以此实现多位点联合检测，可以达到低成本高效率的检测人类红细胞ABO血型基因分型的目的。
一种用于检测人类红细胞ABO血型基因分型试剂，包括权利要求1～5任一项所述的引物组或权利要求6～9任一项所述试剂盒。
相较于现有技术，本发明的有益效果是：
(1)本发明所涉及的引物在设计时经过了一系列优化过程，第一，为了确保引物特异性，在引物设计时以及引物设计后对引物进行分析，与基因组序列比对，保证设计的引物只和目标序列完全匹配，在基因组上没有相似扩增产物；第二，为了实现24组引物在相同条件下扩增，引物设计时综合考虑引物长度、GC含量、引物二级结构和引物之间互补性等因素，使24组引物在扩增时具有相似的退伙温度(Tm值)和扩增效率；第三，经过评估有一半的引物设计为正常的序列，无过多的GC含量或过少的GC含量，引物设计困难较低；另外一半左右位点有较多的GC含量，AT含量或引物设计处有较多的二级结构，这些位点需要经过详细的分析后才能进行引物设计和确认，针对这些位点通过多次尝试和优化，才获得效果良好的引物组；
(2)与现有技术相比，根据本发明的用于同时检测人类红细胞ABO血型系统24个种遗传基因位点的引物组，这些引物经过了优化设计，具有相似的退火温度，能够实现24组引物在相同的PCR扩增条件下同时扩增，每个PCR反应体系的体积只有1.15μL，DNA模板只需5～10ng，大大增加了检测效率和反应灵敏度；应用该引物组进行检测，具有速度快、成本低、灵敏性高及易于实现多位点联合检测等优点；
(3)本发明不仅能实现样本在不同反应腔体的均匀分配，杜绝不同反应孔之间的交叉污染，便于软件判读，而且避免了样品浪费，大大节约实验成本，从而实现了高通量、高灵敏度和稳定高效的多基因位点联合检测，使ABO血型基因分型更加精准化。
附图说明
图1为24个位点检测信息及对应的荧光表达图；
图2为试剂盒芯片基片图；
图3为4例不同ABO血型组合样本基因芯片荧光表达图；
图4为基因分型为A101/O02样本软件判读及对应芯片荧光表达示例图；
图5为基因分型为A102/O01样本软件判读及对应芯片荧光表达示例图；
图6为基因分型为A102/O02样本软件判读及对应芯片荧光表达示例图；
图7为基因分型为B101/B101样本软件判读及对应芯片荧光表达示例图；
图8为基因分型为B101/O01样本软件判读及对应芯片荧光表达示例图；
图9为基因分型为B101/O02样本软件判读及对应芯片荧光表达示例图；
图10为基因分型为O01/O01样本软件判读及对应芯片荧光表达示例图；
图11为基因分型为O01/O02样本软件判读及对应芯片荧光表达示例图；
图12为基因分型为O02/O02样本软件判读及对应芯片荧光表达示例图；
图13为基因分型为A101/B101样本软件判读及对应芯片荧光表达示例图；
图14为基因分型为A102/B102样本软件判读及对应芯片荧光表达示例图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
实施例1引物设计
依据美国国立生物技术信息中心基因库(NCBI Gene Bank)公开的ABO等位基因参考序列，通过查阅大量ABO血型系统相关文献及积累的实验数据，确定了所检测的10种ABO血型亚型，后根据血型抗原突变数据库中这10种ABO血型亚型对应的24个复等位基因SNP位点信A101，A102(467C>T)，A201(467C>T,1061delC)，A205(467C>T,1009A>G)，B101(297A>G,526C>G,657C>T,703G>A,796C>A,803G>C,930G>A,1096G>A)，O01(261delG)，O02(106G>T,188G>A,189C>T,220C>T,261delG,646T>A,681G>A,771C>T,829G>A)，O03(53G>T,220C>T,297A>G,526C>G,802G>A)，O04(261delG,579)，O07(261delG,297A>G,646T>A,681G>A,721C>T,771C>T,829G>A)进行引物设计。
基于竞争性等位基因特异性PCR原理，对SNPs或者特定位点上DNA片段缺失插入(InDels)进行精准的双等位基因判断，设计了24对引物组用于同时检测人类红细胞ABO血型的24个遗传基因位点组合信息，每组引物包含两条基于等位基因序列位点差异而设计的引物和一条共用配对引物。24对引物组的基因序列如SEQ ID No.1-72所示。
实施例2试剂盒制备
将这24组引物用于制备一种检测人类红细胞ABO血型基因分型的试剂盒。
试剂盒中每组引物组中三条引物积相等均为0.05μL，提前包被于芯片反应池中，样本基因组DNA0.25μL、PCR扩增试剂0.5μL以及1*TE缓冲液0.25μL组成一个PCR反应体系，每个PCR反应体系的总体积为1.15μL，其中基因组DNA浓度为10-50ng/μL，A260/A280 OD值为1.6-2.0；PCR扩增试剂为包含携带有HEX或FAM荧光基团的通用探针、dNTPs、Mg 2+、DNA聚合酶和反应缓冲液的2xMasterMix。
通过竞争性等位基因特异性PCR扩增，最后对PCR产物进行荧光信号检测，根据荧光信号分组即可对样本进行基因型分型。利用这些引物组能够实现多位点联合检测，可以达到低成本高效率的检测人类红细胞ABO血型基因分型的目的(说明书附图1)。
基于竞争性等位基因特异性PCR的原理进行SNP分型的试剂盒，其中PCR反应在微流控芯片(北京博奥晶典生物技术有限公司，产品编号G020010，说明书附图2)上完成，制备方法步骤为：
(1)从24个位点中优选了检测人类红细胞ABO血型基因分型中的22个位点，将优选的22个位点特异引物组制定孔位表(见表1)，分别将22组等位基因引物SEQ ID No.1-66按照孔位表包被至芯片基板22 个芯片反应池中，制成试剂盒中的芯片半成品。
每张芯片可检测4个样本，针对每个样本均设有1个内对照质控、1个空白对照以及1个阳性质控(即：表1中的内对照质控、空白对照以及阳性质控)。
表1芯片反应池对应检测指标
Figure PCTCN2020123604-appb-000001
(2)将多种普通生物化学试剂(探针、酶、dNTPs等)按一定量比例进行混合并充分混匀，形成PCR扩增试剂(PCR预混液、1×TE缓冲液)。依据试剂盒检测人份对试剂半成品进行分装，PCR扩增试剂900μL/管/24人份，1×TE缓冲液500μL/管/24人份；
(3)依据试剂盒检测人份配备相应数量封口膜(1张/4人份)、对照品(人类基因组DNA 50μL/管/24人份)
(4)将制备成的上述芯片半成品、试剂半成品以及封口膜、对照品按照检测人份组装成一种用于检测人类红细胞ABO血型基因分型的试剂盒。
实施例3 22个SNP基因位点检测
1.采集260位无血缘关系的献血者的血液，提取基因组DNA：本试剂盒适用的样品为从全血中提取的人基因组DNA。被检基因组DNA需满足浓度为10ng/μL-50ng/μL。提取后的人基因组DNA需进行浓度测定，如果浓度高于50ng/μL，需经稀释后满足上述要求方可进行后续实验；浓度低于10ng/μL需重新提取直至符合要求方可。
2.用上述所制备的专用试剂盒检测260人份22个SNP位点信息，按照使用操作流程进行如下操作：
2.1分装PCR扩增试剂
在试剂存储和准备区内，根据样品数目，准备相应数量的0.2mL离心管，并在管上标记样品编号。从试剂盒中取出PCR扩增试剂，室温使其充分融化(自然解冻)，涡旋振荡，使其完全混匀，瞬时离心至管底。将融化后混匀的PCR扩增试剂按30μL/管(20μL PCR扩预混液+10μL 1×TE缓冲液)分装到同一样品编号的0.2mL离心管中，然后将其转移至标本制备区。
2.2混样
在样本制备区，样本DNA在使用前需室温解冻，然后混匀并瞬时离心；向含有PCR扩增试剂的0.2mL离心管中加入待测样品DNA各10μL，每份PCR反应体系总体积为40μL，每份样品PCR扩增体系组成见表2。
表2反应体系
反应物成分 加样量(μL)
PCR扩增试剂 20
1×TE 10
DNA模板 10
总体积(μL) 40
2.3芯片加样
在样本制备区，从试剂盒中取出芯片使其恢复至室温。在洁净工作台内打开包装，将芯片水平放置，用移液器吸取38μL上述配制好的PCR扩增体系，从芯片右侧进样孔垂直将液体打入芯片，直至液体经进样通道到达左侧出样孔，此时应立即停止加样，然后用无尘纸擦去进出样孔残留的液体，最后用封口膜封闭进出样孔。
2.4芯片离心
打开离心机电源，将加样后的芯片固定在离心机上并配平，放置时，芯片反应池朝下，缺角在上，以4000rpm离心60秒后取下，如果芯片反应池中仍有气泡，可适当延长离心时间，直至气泡消失。
2.5芯片热封
打开热封仪电源，待温度稳定后，将托盘拉出，将芯片反应池朝上，芯片缺角位于左上角，插入托盘(每次可插入4张芯片)，推托盘进仓后，进行热封，待界面显示热封完毕后，即可取出芯片。
2.6 PCR扩增
将芯片置于PCR扩增仪中，按表3中的热循环程序进行PCR扩增反应。
表3.核酸扩增反应程序
Figure PCTCN2020123604-appb-000002
3.芯片扫描
使用ABO血型基因分型检测分析软件进行扫描、信号读取及结果判读。具体判读原则见表4。
根据结果判读标准，判读界面中内对照质控正常、空白对照正常以及阳性质控正常时，则芯片质控正常，表明本次检验结果有效。而判读界面中任一质控显示异常时，则判定本次样品检测结果无效，需要进行重检(说明书附图4)。
表4.荧光信号对应的ABO血型基因分型判读原则
Figure PCTCN2020123604-appb-000003
Figure PCTCN2020123604-appb-000004
Figure PCTCN2020123604-appb-000005
4、检测结果及基因分型结果
根据检测分析软件进行扫描、信号读取及结果判读(如图4-14)，可知260例样本中基因分型结果见表5，按照判读原则中的45种基因分型组合检出18种，另外对260例样本进行血清学检测以及基因测序检测，将结果进行对比后，通过基因分型结果判读的基因表型和血清学以及基因测序结果100％相符。
表5人类红细胞ABO血型基因分型结果
Figure PCTCN2020123604-appb-000006
以上所述实施例仅表达了本申请的具体实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请技术方案构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。

Claims (10)

  1. 一种检测人类红细胞ABO血型基因分型的引物组，其特征在于，所述引物组包括24组，所述的24组引物组的序列表如SEQ ID No.1-72所示。
  2. 根据权利要求1所述的一种检测人类红细胞ABO血型基因分型的引物组，其特征在于，所述的24组引物组及其对应的检测位点分别为：
    第一组引物如序列表SEQ ID No.1-3所示，用于检测ABO血型对应的遗传基因位点中的106(rs688976)位点；
    第二组引物如序列表SEQ ID No.4-6所示，用于检测ABO血型对应的遗传基因位点中的188(rs549446)位点；
    第三组引物如序列表SEQ ID N o.7-9所示，用于检测ABO血型对应的遗传基因位点中的189(rs549443)位点；
    第四组引物如序列表SEQ ID No.10-12所示，用于检测ABO血型对应的遗传基因位点中的220(rs512770)位点；
    第五组引物如序列表SEQ ID No.13-15所示，用于检测ABO血型对应的遗传基因位点中的261(rs8176719)位点；
    第六组引物如序列表SEQ ID No.16-18所示，用于检测ABO血型对应的遗传基因位点中的297(rs8176720)位点；
    第七组引物如序列表SEQ ID No.19-21所示，用于检测ABO血型对应的遗传基因位点中的467(rs1053878)位点；
    第八组引物如序列表SEQ ID No.22-24所示，用于检测ABO血型对应的遗传基因位点中的526(rs7853989)位点；
    第九组引物如序列表SEQ ID No.25-27所示，用于检测ABO血型对应的遗传基因位点中的579(rs55764262)位点；
    第十组引物如序列表SEQ ID No.28-30所示，用于检测ABO血型对应的遗传基因位点中的646(rs8176740)位点；
    第十一组引物如序列表SEQ ID No.31-33所示，用于检测ABO血型对应的遗传基因位点中的657(rs8176741)位点；
    第十二组引物如序列表SEQ ID No.34-36所示，用于检测ABO血型对应的遗传基因位点中的681(rs8176742)位点；
    第十三组引物如序列表SEQ ID No.37-39所示，用于检测ABO血型对应的遗传基因位点中的703(rs8176743)位点；
    第十四组引物如序列表SEQ ID No.40-42所示，用于检测ABO血型对应的遗传基因位 点中的721(rs781957267)位点；
    第十五组引物如序列表SEQ ID No.43-45所示，用于检测ABO血型对应的遗传基因位点中的771(rs8176745)位点；
    第十六组引物如序列表SEQ ID No.46-48所示，用于检测ABO血型对应的遗传基因位点中的796(rs8176746)位点；
    第十七组引物如序列表SEQ ID No.49-51所示，用于检测ABO血型对应的遗传基因位点中的803(rs8176747)位点；
    第十八组引物如序列表SEQ ID No.52-54所示，用于检测ABO血型对应的遗传基因位点中的829(rs8176748)位点；
    第十九组引物如序列表SEQ ID No.55-57所示，用于检测ABO血型对应的遗传基因位点中的930(rs8176749)位点；
    第二十组引物如序列表SEQ ID No.58-60所示，用于检测ABO血型对应的遗传基因位点中的1009(rs566015043)位点；
    第二十一组引物如序列表SEQ ID No.61-63所示，用于检测ABO血型对应的遗传基因位点中的1061(rs56392308)位点；
    第二十二组引物如序列表SEQ ID No.64-66所示，用于检测ABO血型对应的遗传基因位点中的1096(rs8176751)位点；
    第二十三组引物如序列表SEQ ID No.67-69所示，用于检测ABO血型对应的遗传基因位点中的53(rs55876802)位点；
    第二十四组引物如序列表SEQ ID No.70-72所示，用于检测ABO血型对应的遗传基因位点中的802(rs41302905)位点。
  3. 根据权利要求2所述的一种检测人类红细胞ABO血型基因分型的引物组，其特征在于，所述的24组引物组中每组引物组包含两条基于等位基因序列差异而设计的引物和一条共用配对引物。
  4. 根据权利要求3所述的一种检测人类红细胞ABO血型基因分型的引物组，其特征在于，
    第一组引物中检测106G的引物对为序列表SEQ ID No.1和SEQ ID No.3，检测106T的引物对为序列表SEQ ID No.2和SEQ ID No.3；
    第二组引物中检测188G的引物对为序列表SEQ ID No.4和SEQ ID No.6，检测188A的引物对为序列表SEQ ID No.5和SEQ ID No.6；
    第三组引物中检测189C的引物对为序列表SEQ ID No.7和SEQ ID No.9，检测189T 的引物对为序列表SEQ ID No.8和SEQ ID No.9；
    第四组引物中检测220C的引物对为序列表SEQ ID No.10和SEQ ID No.12，检测220T的引物对为序列表SEQ ID No.11和SEQ ID No.12；
    第五组引物中检测261G的引物对为序列表SEQ ID No.13和SEQ ID No.15，检测261delG的引物对为序列表SEQ ID No.14和SEQ ID No.15；
    第六组引物中检测297A的引物对为序列表SEQ ID No.16和SEQ ID No.18，检测297G的引物对为序列表SEQ ID No.17和SEQ ID No.18；
    第七组引物中检测467C的引物对为序列表SEQ ID No.19和SEQ ID No.21，检测467T的引物对为序列表SEQ ID No.20和SEQ ID No.21；
    第八组引物中检测526C的引物对为序列表SEQ ID No.22和SEQ ID No.24，检测526G的引物对为序列表SEQ ID No.23和SEQ ID No.24；
    第九组引物中检测579T的引物对为序列表SEQ ID No.25和SEQ ID No.27，检测579C的引物对为序列表SEQ ID No.26和SEQ ID No.27；
    第十组引物中检测646T的引物对为序列表SEQ ID No.28和SEQ ID No.30，检测646A的引物对为序列表SEQ ID No.29和SEQ ID No.30；
    第十一组引物中检测657C的引物对为序列表SEQ ID No.31和SEQ ID No.33，检测657T的引物对为序列表SEQ ID No.32和SEQ ID No.33；
    第十二组引物中检测681G的引物对为序列表SEQ ID No.34和SEQ ID No.36，检测681A的引物对为序列表SEQ ID No.35和SEQ ID No.36；
    第十三组引物中检测703G的引物对为序列表SEQ ID No.37和SEQ ID No.39，检测703A的引物对为序列表SEQ ID No.38和SEQ ID No.39；
    第十四组引物中检测721C的引物对为序列表SEQ ID No.40和SEQ ID No.42，检测721T的引物对为序列表SEQ ID No.41和SEQ ID No.42；
    第十五组引物中检测771C的引物对为序列表SEQ ID No.43和SEQ ID No.45，检测771T的引物对为序列表SEQ ID No.44和SEQ ID No.45；
    第十六组引物中检测796C的引物对为序列表SEQ ID No.46和SEQ ID No.48，检测796A的引物对为序列表SEQ ID No.47和SEQ ID No.48；
    第十七组引物中检测803G的引物对为序列表SEQ ID No.49和SEQ ID No.51，检测803C的引物对为序列表SEQ ID No.50和SEQ ID No.51；
    第十八组引物中检测829G的引物对为序列表SEQ ID No.52和SEQ ID No.54，检测829A的引物对为序列表SEQ ID No.53和SEQ ID No.54；
    第十九组引物中检测930G的引物对为序列表SEQ ID No.55和SEQ ID No.57，检测930A的引物对为序列表SEQ ID No.56和SEQ ID No.57；
    第二十组引物中检测1009G的引物对为序列表SEQ ID No.58和SEQ ID No.60，检测1009A的引物对为序列表SEQ ID No.59和SEQ ID No.60；
    第二十一组引物中检测1061C的引物对为序列表SEQ ID No.61和SEQ ID No.63，检测1061delC的引物对为序列表SEQ ID No.62和SEQ ID No.63；
    第二十二组引物中检测1096G的引物对为序列表SEQ ID No.64和SEQ ID No.66，检测1096A的引物对为序列表序列表SEQ ID No.65和SEQ ID No.66；
    第二十三组引物中检测53G的引物对为序列表SEQ ID No.67和SEQ ID No.69，检测53T的引物对为序列表SEQ ID No.68和SEQ ID No.69；
    第二十四组引物中检测802G的引物对为序列表SEQ ID No.70和SEQ ID No.72，检测802A的引物对为序列表SEQ ID No.71和SEQ ID No.72。
  5. 根据权利要求1～4任一项所述的一种检测人类红细胞ABO血型基因分型的引物组，其特征在于，所述SEQ ID No.1、SEQ ID No.4、SEQ ID No.7、SEQ ID No.10、SEQ ID No.13、SEQ ID No.16、SEQ ID No.19、SEQ ID No.22、SEQ ID No.25、SEQ ID No.28、SEQ ID No.31、SEQ ID No.34、SEQ ID No.37、SEQ ID No.40、SEQ ID No.43、SEQ ID No.46、SEQ ID No.49、SEQ ID No.52、SEQ ID No.55、SEQ ID No.58、SEQ ID No.61、SEQ ID No.64、SEQ ID No.67、SEQ ID No.70携带通用标签序列GAAGGTCGGAGTCAACGGATT；所述SEQ ID No.2、SEQ ID No.5、SEQ ID No.8、SEQ ID No.11、SEQ ID No.14、SEQ ID No.17、SEQ ID No.20、SEQ ID No.23、SEQ ID No.26、SEQ ID No.29、SEQ ID No.32、SEQ ID No.35、SEQ ID No.38、SEQ ID No.41、SEQ ID No.44、SEQ ID No.47、SEQ ID No.50、SEQ ID No.53、SEQ ID No.56、SEQ ID No.59、SEQ ID No.62、SEQ ID No.65、SEQ ID No.68、SEQ ID No.71携带通用标签序列GAAGGTGACCAAGTTCATGCT。
  6. 一种检测人类红细胞ABO血型基因分型的试剂盒，其特征在于，所述试剂盒包含权利要求1～5任一项所述的24组引物组中的至少22组引物组。
  7. 根据权利要求6所述的一种检测人类红细胞ABO血型基因分型的试剂盒，其特征在于，所述的试剂盒包括至少26个芯片反应池，至少2个定位点，每个所述芯片反应池中包被有一组引物组。
  8. 根据权利要求7所述的一种检测人类红细胞ABO血型基因分型的试剂盒，其特征在于，每个所述芯片反应池中的引物组中三条引物积之和相等。
  9. 一种检测人类红细胞ABO血型基因分型的试剂盒的制备方法，其特征在于，包括以下步骤：
    a)从24个位点中选检测人类红细胞ABO血型基因分型中的22个位点，将选的22个位点特异引物组制定孔位表，分别将22组等位基因引物按照孔位表包被至芯片基板22个芯片反应池中，得到芯片半成品；
    b)制备PCR扩增试剂；
    c)配备相应数量封口膜、对照品；
    d)将芯片半成品、PCR扩增试剂以及封口膜、对照品按照检测人份组装成一种用于检测人类红细胞ABO血型基因分型的试剂盒。
  10. 一种用于检测人类红细胞ABO血型基因分型试剂，其特征在于，包括权利要求1～5任一项所述的引物组或权利要求6～9任一项所述试剂盒。
PCT/CN2020/123604 2019-12-31 2020-10-26 一种检测人类红细胞abo血型基因分型的引物组、试剂盒及应用 WO2021135559A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911420155.XA CN110982895B (zh) 2019-12-31 2019-12-31 一种检测人类红细胞abo血型基因分型的引物组、试剂盒及应用
CN201911420155.X 2019-12-31

Publications (1)

Publication Number Publication Date
WO2021135559A1 true WO2021135559A1 (zh) 2021-07-08

Family

ID=70080190

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/123604 WO2021135559A1 (zh) 2019-12-31 2020-10-26 一种检测人类红细胞abo血型基因分型的引物组、试剂盒及应用

Country Status (2)

Country Link
CN (1) CN110982895B (zh)
WO (1) WO2021135559A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114507724B (zh) * 2022-03-29 2023-08-22 河南兰德施坦纳基因科技有限公司 一种检测人类红细胞Rh血型基因分型的引物组、试剂盒及应用
CN114561458B (zh) * 2022-04-20 2023-12-29 青岛市中心血站 一种与abo血型系统中b变异型相关的snp位点及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050009072A1 (en) * 2003-05-28 2005-01-13 Koshi Maeda Genetic testing kits and a method of bladder cancer
CN1900314A (zh) * 2006-07-19 2007-01-24 辽宁省刑事科学技术研究所 一种荧光标记abo基因分型的方法及其试剂盒
CN101403007A (zh) * 2008-11-03 2009-04-08 深圳市血液中心 Abo血型基因检测试剂及定型方法
CN101921834A (zh) * 2010-05-17 2010-12-22 浙江省血液中心 一种abo血型基因分型的pcr-sbt方法及试剂
CN108624663A (zh) * 2017-03-23 2018-10-09 上海市血液中心 一种人类红细胞abo血型抗原多重pcr分型方法及试剂盒

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1591534A1 (en) * 2004-04-01 2005-11-02 Stichting Sanquin Bloedvoorziening A method of genotyping blood cell antigens and a kit suitable for genotyping blood cell antigens
EP2778235B1 (en) * 2012-01-18 2019-09-18 Shanghai Blood Centre Multiplex pcr detection method for human rare blood types and kit
US20160010153A1 (en) * 2013-03-15 2016-01-14 Life Technologies Corporation Novel compositions, methods and kits for blood typing
KR101695477B1 (ko) * 2014-05-27 2017-01-12 주식회사 시선바이오머티리얼스 Pna 프로브 및 형광융해곡선분석을 이용한 혈액형 판별방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050009072A1 (en) * 2003-05-28 2005-01-13 Koshi Maeda Genetic testing kits and a method of bladder cancer
CN1900314A (zh) * 2006-07-19 2007-01-24 辽宁省刑事科学技术研究所 一种荧光标记abo基因分型的方法及其试剂盒
CN101403007A (zh) * 2008-11-03 2009-04-08 深圳市血液中心 Abo血型基因检测试剂及定型方法
CN101921834A (zh) * 2010-05-17 2010-12-22 浙江省血液中心 一种abo血型基因分型的pcr-sbt方法及试剂
CN108624663A (zh) * 2017-03-23 2018-10-09 上海市血液中心 一种人类红细胞abo血型抗原多重pcr分型方法及试剂盒

Also Published As

Publication number Publication date
CN110982895B (zh) 2023-02-14
CN110982895A (zh) 2020-04-10

Similar Documents

Publication Publication Date Title
Wu et al. Rapid and/or high-throughput genotyping for human red blood cell, platelet and leukocyte antigens, and forensic applications
US11542556B2 (en) Single nucleotide polymorphism in HLA-B*15:02 and use thereof
WO2002061122A2 (en) New method for genotype determination
Latini et al. A new strategy to identify rare blood donors: single polymerase chain reaction multiplex SNaPshot reaction for detection of 16 blood group alleles
BRPI0616323A2 (pt) mÉtodo para determinar se um fluido mamÁrio doado foi obtido a partir de um indivÍduo especÍfico
US10253366B2 (en) Discrimination of blood type variants
WO2021135559A1 (zh) 一种检测人类红细胞abo血型基因分型的引物组、试剂盒及应用
CN106755360B (zh) 用于检测人类cyp2d6基因多态性的核酸、试剂盒及方法
CN114507724B (zh) 一种检测人类红细胞Rh血型基因分型的引物组、试剂盒及应用
CN104651488A (zh) 检测染色体非整倍体数目异常的扩增组合物及快速检测试剂盒
CN111118138A (zh) 一种叶酸代谢能力基因mthfr和mtrr多态性检测试剂盒及方法
Huang et al. Reliable detection of trisomy 21 using MALDI-TOF mass spectrometry
CN108410972B (zh) 一种用于人类Rh血型23个基因位点的基因分型检测试剂盒
JP2011500062A (ja) 血液型群遺伝子の検出
CN105586392B (zh) 评估胎儿样本中母体细胞污染程度的方法
CN112592972B (zh) 弥漫性毒性甲状腺肿易感基因的早筛方法及试剂盒
CN108504731B (zh) 诊断脂质代谢相关疾病或阿尔茨海默病标志物的方法
WO2017087735A1 (en) Method for treating crohn's disease
US9139881B2 (en) Method for assessing breast cancer susceptibility
Kwok et al. Allele quantification Pyrosequencing® at designated SNP sites to detect allelic expression imbalance and Loss-of-Heterozygosity
CN110684832A (zh) 叶酸相关基因多态性检测的试剂盒及其使用方法
RU2805860C1 (ru) Способ генотипирования гена TLR2 по полиморфизму rs3804100 и набор олигонуклеотидных праймеров и зондов для его реализации
KR102511161B1 (ko) 한국인 특이적 snp 정보 및 최적화된 파이프라인을 이용한 hla-a 유전자형 분석 방법
CN108949949B (zh) 与抗结核药物性肝损伤发生相关的药物转运蛋白基因多态性位点及其应用
CN116891897A (zh) 用于预测卵巢高反应风险的生物标志物及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20909567

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20909567

Country of ref document: EP

Kind code of ref document: A1