CN114032295A - Human leukocyte antigen ABCDB 1DQB1 genotyping detection method and kit - Google Patents
Human leukocyte antigen ABCDB 1DQB1 genotyping detection method and kit Download PDFInfo
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Abstract
An HLA genotyping detection method comprises the following steps: step 1, processing a sample; step 2, taking the processed sample product as a template, and carrying out amplification on gene long products of different sites of HLA through a specific primer group; carrying out sequencing reaction by taking the long fragment amplification product as a template to obtain a sequencing reaction product; 3, taking the sequencing reaction product as a reaction hole to be tested, and taking a first-generation sequencer as a platform to perform sequencing reaction; step 4, analyzing the sequencing reaction data to obtain HLA different site genotype results, wherein the HLA different site genotype results comprise a specific universal amplification primer group and a specific amplification primer group combination mode; designing a method for detecting primers aiming at HLA genotyping; aiming at an HLA genotyping detection kit and an HLA genotyping detection single-tube amplification 5-site tube-by-tube segmented sequencing method, the detection of a plurality of sites is rapidly completed at a time; the detection result is the high-variability result in 4 bits.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a genotyping detection method and a kit for human leukocyte antigen ABCDB 1DQB 1.
Background
The success of organ transplantation and bone marrow transplantation is not only determined by the well-done surgical techniques, but also by the compatibility of immune systems between donor and recipient, which is a major influencing factor.
Human leukocyte antigen molecules play an extremely important role in the specific identification of T cell immunity, during the transplantation of organs or bone marrow, the degree of compatibility of human leukocyte antigen molecules of donors and recipients has a considerable influence on the transplantation result, therefore, the identification test of both human leukocyte antigen molecular types is an absolutely necessary step before transplantation, and in the past, the identification test of human leukocyte antigen molecular types is performed serologically, the gene coding human leucocyte antigen is HLA, and the human leucocyte antigen molecule gene with HLA located on the short arm of the sixth pair of chromosomes has very high diversity, including A, B, C, D (DR, DQ, DP), E, F and other genes, a, B, C, DRB1 and DQB1 are the genes which are most related to immune rejection reaction during transplantation and are necessary to be typed before transplantation. After the gene sequence of the human leukocyte antigen molecule is decrypted, the difference of only few or even only one amino acid among most human leukocyte antigen molecules is found. The traditional method for performing antigen molecule identification test in a serological mode cannot meet clinical requirements, so that the detection of HLA genotyping is completed by designing specific amplification primers and sequencing primers aiming at HLA genes A, B, C, DRB1 and DQB1 and a reagent method with high efficiency and specificity based on a first generation sequencing (Sanger method) technology.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a genotyping detection method and a kit for human leukocyte antigen ABCDB 1DQB 1.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: an HLA genotyping detection method comprises the following steps:
3, taking the sequencing reaction product as a reaction hole to be tested, and taking a first-generation sequencer as a platform to perform sequencing reaction;
and 4, analyzing the sequencing reaction data to obtain HLA different site genotype results.
Further, the sample treatment mode is a nucleic acid treatment mode, and the product is treated by a nucleic acid releasing agent.
Further, the accounting processing mode specifically includes: extracting and purifying the product of nucleic acid by a purification kit; a product of nucleic acid release from a sample with a nucleic acid releasing agent; nucleic acid products processed in other ways without taking the kit include physical heating, snap freezing, grinding or sample dilution.
Further, the specific primer group consists of an HLA A site specific amplification primer group, a B site specific amplification primer group, a C site specific amplification primer group, a DRB1 site specific amplification primer group and a DQB1 site specific amplification primer group.
Further, methods of amplifying long fragment products with specific amplification primer sets are also included, including but not limited to one or more of:
1)5, single-tube amplification of multiple amplicons of a site-specific amplification primer group;
6) an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, a molecular amplification primer group MIX and DRB1 and a DQB1 amplification group MIX, so that two-tube amplification of HLA 5-site long fragment products is realized;
7) an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, an amplification primer group with site specificity DQB1 and an amplification primer group with site specificity DRB1 independently amplify long products, and 5-site independent amplification is realized;
8) an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, an amplification primer group with site specificity DQB1, an amplification primer group with site specificity 2 tubes for segmented amplification and a amplification primer group with site specificity DRB1 for 2 tubes for segmented amplification and a growing segment, wherein 6-7 independent amplicons with 5 sites are realized;
9) a, B, C, DRB1 and DQB15 locus multiple amplicon single tube amplification to realize 5 locus multiple PCR.
Further, the long fragment product is subjected to a sequencing reaction, and the long fragment product template includes, but is not limited to, one or more of a single-site single-tube amplification primer set product, a multi-site single-tube amplification primer set product, and a multi-site multi-tube amplification product.
Further, the long fragment product template is a purified product; purification means include, but are not limited to, one or more of, preferably enzymatic digestion; bead purification methods, magnetic beads include, but are not limited to, the following: XP beads, SPRI beads; the enzyme digestion method comprises 0.1-100U shrimp alkaline phosphatase and 0.1-100U EXO I.
Furthermore, the sequencing reaction consists of a sequencing primer of a specific site and a sequencing reaction reagent, the sequencing primer performs hole division and independent sequencing reaction according to different sites and different determined regions, the sequencing reaction is linear PCR amplification of a single-side single primer, and the sequencing reaction reagent consists of a BigDYE reagent, DNA polymerase, PCR reaction buffer, Mg2+ and the like.
An HLA ABCDQB1DRB1 high resolution genotyping detection kit, the reagent consists of amplification reagent, sequencing reagent, nucleic acid polymerase and amplification buffer solution; the amplification reagent consists of a general amplification primer group reaction solution and a group specificity primer group reaction solution, and the sequencing reagent consists of different sequencing primers and Tris-HCl buffer solutions which are designed according to different sites and different quantities of concerned exons; nucleic acid polymerases have a high fidelity long product amplification DNA polymerase combination, including but not limited to EZ-Taq enzyme.
The amplification reagent comprises amplification primers, dNTP and the like, the amplification buffer solution comprises buffer solution, metal coenzyme and specificity enhancer, and the sequencer applicable to the detection kit provided by the invention comprises but is not limited to: one or more of ABI3130, ABI 3500, ABI 3730.
(III) advantageous effects
Compared with the prior art, the invention provides a genotyping detection method and a kit for human leukocyte antigen ABCDB 1DQB1, which have the following beneficial effects:
comprises a combination mode of a specific universal amplification primer group and a specific amplification primer group; designing a method for detecting primers aiming at HLA genotyping; aiming at an HLA genotyping detection kit and an HLA genotyping detection single-tube amplification 5-site tube-by-tube segmented sequencing method, the detection of a plurality of sites is rapidly completed at a time; the detection result is the high-variability result in 4 bits.
Drawings
FIG. 1 is a detection map of site A;
FIG. 2 is a detection map of B site;
FIG. 3 is a detection map of C site;
FIG. 4 is a detection map of DRB1 site;
fig. 5 is a detection map of DQB1 site.
Detailed Description
An HLA genotyping detection method comprises processing a sample; taking the processed sample product as a template, and carrying out amplification on the gene long products of different sites of the HLA through a specific primer group; performing sequencing reaction by using the long fragment amplification product as a template; taking a sequencing reaction product as a reaction hole to be detected, and taking a first-generation sequencer as a platform to perform sequencing reaction; analyzing the sequencing reaction data to obtain HLA different site genotype results;
further, the sample treatment means includes, but is not limited to, one or more of the following, preferably nucleic acid treatment means is a nucleic acid releasing agent treatment product; :
1. extracting and purifying the product of nucleic acid by a purification kit;
2. a product of nucleic acid release from a sample with a nucleic acid releasing agent;
3. nucleic acid products processed by other modes of the hand-free kit comprise heating, quick freezing, grinding, sample diluting and the like by a physical method;
further, the specific primer group consists of an HLA A site specific amplification primer group, a B site specific amplification primer group, a C site specific amplification primer group, a DRB1 site specific amplification primer group and a DQB1 site specific amplification primer group;
the invention provides a method for amplifying long fragment products by using a specific amplification primer group, which comprises but is not limited to one or more of the following, preferably 5-site specific amplification primer group multi-amplicon single-tube amplification;
1. an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, a molecular amplification primer group MIX and DRB1 and a DQB1 amplification group MIX, so that two-tube amplification of HLA 5-site long fragment products is realized;
2. an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, an amplification primer group with site specificity DQB1 and an amplification primer group with site specificity DRB1 independently amplify long products, and 5-site independent amplification is realized;
3. an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, an amplification primer group with site specificity DQB1, an amplification primer group with site specificity 2 tubes for segmented amplification and a amplification primer group with site specificity DRB1 for 2 tubes for segmented amplification and a growing segment, wherein 6-7 independent amplicons with 5 sites are realized;
4. a, B, C, DRB1 and DQB15 locus multiple amplicon single tube amplification to realize 5 locus multiple PCR.
The invention provides a method for carrying out sequencing reaction on a long fragment product;
further, the long fragment product template includes, but is not limited to, one or more of a single-site single-tube amplification primer set product, a multi-site multi-tube amplification product, preferably a multi-site single-tube amplification primer product;
further, the long fragment product template is a purified product; purification means include, but are not limited to, one or more of, preferably enzymatic digestion;
1. bead purification methods, magnetic beads include, but are not limited to, the following: XP beads, SPRI beads, preferably XP beads;
2. an enzyme digestion method in which the enzyme is composed of 0.1 to 100U of shrimp alkaline phosphatase and 0.1 to 100U of EXO I, preferably 1U of shrimp alkaline phosphatase and 10U of EXO I enzyme;
further, the sequencing reaction consists of a sequencing primer of a specific site and a sequencing reaction reagent;
furthermore, the sequencing primer carries out hole division and independent sequencing reaction according to different sites and different determined regions;
further, the sequencing reaction is linear PCR amplification of a single primer;
furthermore, the sequencing reaction reagent comprises a BigDYE reagent, DNA polymerase, PCR reaction buffer, Mg2+ and the like;
furthermore, the sequencing reaction consists of 0.1-50 ng of long fragment product, 0.5-100 uM of sequencing primer and BigDYE reaction solution, and the preferential reaction system is as follows: 2.5uM sequencing primer, 2-5 ng long fragment product and 1.5ul BigDYE reaction solution.
The invention also provides a specific amplification primer and a sequencing primer design method aiming at the HLA gene analysis and detection;
further, the specific sequencing primer is designed for one or more pairs of primer groups aiming at specific detection target regions of different HLA gene loci;
further, the characteristic amplification primer group consists of a universal amplification primer group and a group-specific primer group;
further, the universal amplification primer set covers most of the typing, and the main design region is the 3 'UTR or 5' UTR region or the intron region;
further, the specific amplification primer group or the group specific primer group consists of a complete complementary pairing base group with the template and 0-5 non-complementary pairing bases;
further, the specific amplification primer has Tm regulation function, and the primers are modified by 0-5 LANs;
further, the 3' penultimate base of the specific amplification primer is subjected to thio modification;
further, the group-specific primer group is a specific primer designed for typing specific within the group;
further, the detection target regions of the different HLA gene loci comprise regions from A locus exon2 to exon 6; the B site comprises regions exon 2-exon 5, the C site comprises regions exon 2-exon 5, the DRB1 site comprises regions exon 2-exon 5, and the DQB1 site comprises regions exon 2-exon 5.
The invention provides a HLA ABCDQB1DRB1 high-resolution genotyping detection kit, wherein the kit comprises an amplification reagent, a sequencing reagent, nucleic acid polymerase and an amplification buffer solution;
further, the amplification reagent consists of a general amplification primer group reaction solution and a group specificity primer group reaction solution;
further, the amplification reagent consists of an amplification primer, dNTP and the like;
furthermore, the sequencing reagent consists of different sequencing primers and Tris-HCl buffer solutions which are designed according to different sites and different numbers of concerned exons;
further, the nucleic acid polymerase has a high fidelity long-product amplified DNA polymerase combination, including but not limited to EZ-Taq enzyme;
further, the amplification buffer solution consists of a buffer solution, a metal coenzyme and a specificity enhancer.
The types of samples targeted by the detection kit provided by the invention include but are not limited to: oral exfoliative cells, whole blood, hair, skin, body fluids.
The sequencer applicable to the detection kit provided by the invention comprises but is not limited to: one or more of ABI3130, ABI 3500, ABI 3730.
For a further understanding of the method of the invention, reference is made to the following description;
firstly, extracting and purifying nucleic acid from a whole blood sample by using a nucleic acid extraction kit, amplifying a purified product according to a single-site single-tube amplification mode, and digesting the amplified product by using an enzyme digestion mode to remove redundant amplification primers and dNTPs; and (3) carrying out sequencing reaction on the enzyme digestion product, carrying out ethanol precipitation purification or magnetic bead purification on the sequencing reaction product, sequencing the purified product, and carrying out data analysis on the sequencing result to obtain a data typing result.
Example 1HLA ABCDB 1DQB1 primer design and screening
1.1 primer design
Designing A B C DRB1DQB1 corresponding site-specific amplification primers (universal amplification primer and group-specific amplification primer) and sequencing primer according to IWHG database in hla database
Universal amplification primer set
A A_L_01:GCCGACCCAGTTCTCACTCC
A_L_02:GCGGACCCAGTTCTCACTCC
A_L_03:AAGAAACCCCATAGCAGAGCT
A_L_04:AAGAAACCCCATAGCACAGCT
B
B_L_01:GTCCTTCTTCCGGGATACTCG
B_L_02:GGTCCTTCTTCCAGGATACTCG
B_L_03:ACAACAGTCATGAACAAATTGTGG
C
C_L_01:GTCCTTCTTCCTTGATACTC
C_L_02:GATCTTGAAGTCGTGAGGAG
C_L_03:AGTTCTTGAAGTCAGTTGAGGAG
DRB1
DRB1_L1_01CGTACTTGAGCGCACGTTTCTTGGAGTACTC
DRB1_L1_02CGTACTTGAGCGCACGTTTCTTGGAGGAGGT
DRB1_L1_03CGTACTTGAGCGCACGTTTCTTGTGGCAGCT
DRB1_L1_04CGTACTTGAGCGCACGTTTCCTGTGTCAGCC
DRB1_L1_05TCAGCACAGTGCCACGTTAGTTGGAGCAGGT
DRB1_L1_06TCAGCACAGTGCCACGTTTCCTGGGGCAGGG
DRB1_L1_07TCAGCACAGTGCCACGTTTCTTGAAGCAGGA
DRB1_L1_08CAGTGACCAGCAGGCGTGCTCACCTCGCCGCT
DRB1_L1_09CAGTGACCAGCAGGCATGCTCACCTCGCCTCT
DRB1_L2_01CTTGCCTGCTTCTCTGG
DRB1_L2_02CTTGCCTGCTGCTCTGG
DRB1_L2_03CTCACCTGCTCCTCTGG
DRB1_L2_04AGAATTTGGGATCCATA
DRB1_L2_05AATAAGTTGGGATCCAA
DRB1_L3_01ATGATTTGGGATCCATA
DRB1_L3_02TTGACAATGCATGACA
DRB1_L3_03CTTGATGTAAGGCAAG
DQB1_L1_01GCCGCCGGGCCGATCCTAG
DQB1_L1_02CCGCCGGTTGGATCATAA
DQB1_L1_03CAGGTTCCAGGTGTAACGA
DQB1_L1_04GGCTCAGGTTCCACGGTGATT
DQB1_L1_05GCTCAGGTTCCACAGTCCG
DQB1_L1_06CTCAGGTTTCGGTGATGCCGC
DQB1_L1_07GTGGAATGAAACCTTAGGCTT
DQB1_L1_08GGTGGAATGACCCGGCTTAGA
DQB1_L1_09GTTTTCCTGTCTGAAGCTGCC
DQB1_L1_10TTTGGGTGTGTGTTACTGCCC
DQB1_L1_11TCAGGGATAACTGCTGGTAAGG
Group-specific amplification primer set
A01_01TTCTCCCTCTCCCAACCTAC
A01_02GTCGGCCCATGATTCTGGAAG
A02_01 CGTGTCTTCGCGGTCGCTC
A02_02GTCGGCCCATGATTCTGGAAG
A03_01CTGCTACTCTCGCGGGCT
A03_02CCAGTGATCACAGCTCCAAAG
A04_01CCCAGGCGTGCCTCTCAGA
A04_02TAGGCGCATGATTGTGGAC
A05_01GACAGAGCGCTTGGCACAGA
A05_02TGATGCCCACCATGTGGAC
A06_01CCCTGACCCAGACCTGGGCA
A06_02ACCCCTTCTCCACCTCAGCTG
A07_01CGCAGTTTCTTTTCTGCCTCTG
A07_02 CCACCCTTCATCCCTTCA
A08_01 GGTGTATGGATTGGGCAGTCA
A08_02 ACTCCCTCCTTTTCTATCTGTGA
A09_01 GGGCTCAGGGCGAAGTA
A09_02CAGGTCCCATCACCGCCA
A10_01 GCAGTTTCTTTTCTCCCTCTC
A10_02 CCCAGGCTGGGTAGGCTC
A11_01ATGCCGAGCGTTTCTCCCA
A11_02CCAGTGATCACAGCTCCAAAG
A12_01 GACATTGAGACAGAGCGCTTC
A12_02 GGTCCTAGAGGAACGTCGTAGC
B01_01 CCCACTCCCATTGGGTATTG
B01_02 CGTACTTGAGCGCACGTTTCTTGGAGTACTC
B02_01 ACCCACCCGGACTCAGAG
B02_02 CCCCAACACCACAAGCAC
B03_01 GGTCTCAGGCTCCGAGAGCCTT
B03_02 TCAGCACAGTGCCACGTTTCCTGGGGCAGGG
B04_01 AGGATACTCGTGACGCA
B04_02 CGTACTTGAGCGCACGTTTCTTGGAGGAGGT
B05_01 CAGAGTCTCCTCACACGCCA
B05_02 CGTACTTGAGCGCACGTTTCTTGGAGGAGGT
B06_01 CTGCTGCTCTCGGGA
B06_02 CAACCATCAAGGCGATACATCT
B07_01 ATTGGGGACGCGCAGCA
B07_02 GGAACCTCCTCGTCTTGGAA
B08_01 CTCCCATTGGCTGTCGGGTG
B08_02 CGTACTTGAGCGCACGTTTCTTGTGGCAGCT
B09_01 GGTCTCAGGCTCCGAGAGCCTT
B09_02 TGGGAAAGGAGGCGAAGAC
B10_01 GGTCTCAGGCTCCGAGAGCCTT
B10_02 CTCCACCTCCTCACATTAG
B11_01 CTCCCATTGGCTGTCGGGTG
B11_02 CGTACTTGAGCGCACGTTTCTTGGAGTACTC
B12_01 CCAATCAGCCTCGCCGT
B12_02 CCCCAACACCACAAGCAC
B13_01 ACCCACCCGGACTCAGAA
B13_02GCTCCTCCACACTCCCG
B14_01 CCAATCAGCCTCGCCGT
B14_02 GGACCTGGTCAGAACCCT
C
C01_01 TCTGCGGGGGGGAACAAGGT
C01_02 AAAGCAGACTTGGGTTATGA
C02_01 CTTCTTCTCCGCGTTTGCGA
C02_02 GATCTTGAAGTCGTGAGGAG
C03_01 CTTCTTCTCCGCGTTTGCGA
C03_02 CTACACATCACTGTAGCCAT
C04_01 TCTCCGCAGTCCTGGATTCTG
C04_02 GGTTGCGTCATGGTAAGTGATC
C05_01 AGGGAACCCTGGCCTATGG
C05_02 GGCTCTTGAATTTACAAATGA
C06_01 CCTGAGTTTCACTTCATGVTATT
C06_02 GATCTTGAAGTCGTGAGGAG
C07_01 CCAGTCACCCAGTGGAACTCG
C07_02 TACACAACTGACAAAAGCCAT
C08_01 AGTCACCCACTGGGATTCAC
C08_02 CACACTCGAGTGCTGTCCCA
C09_01 GTCCTTCTTCCTTGATACTC
C09_02 GGGAAAGGCTGGCTGTAGGTC
C10_01 CCAGTCACCCAGTGGAACTCG
C10_02 TGCTAACAGGGTGGTAGACG
C11_01 TCTCCGCAGTCCTGGATTCTG
C11_02 TCTCTCCAACTTGCCTCAT
C12_01 ACTCACAAACTCCGTTAGAG
C12_02 AAAGCAGACTTGGGTTATGA
DRB1
DRB1_01_01CCCAGTGCCCGCACCCT
DRB1_01_02CACACTCAGATTCTCCGCTT
DRB1_02_01CGGTGGGTGCTCTTGAAGGT
DRB1_02_02 ACACACACACACTCAGATTCCCA
DRB1_03_01 GCACTAAGGAAGGGTTCAG
DRB1_03_02 ACACACACACACTCAGATTCCCA
DRB1_04_01 CTTGGGATCAGAGGTACTTTTT
DRB1_04_02 ACACACACACACACTCAGATTCTCC
DRB1_05_01 GCGTCGCTGTCAGTGTT
DRB1_05_02 AGATTCCCAGCTCGGAGA
DRB1_06_01 CGCAGGCCACGCACAAA
DRB1_06_02 ATTCTAAATGCTCACAGATGG
DRB1_07_01TCAGTTAAGGTTCGAGTGCCA
DRB1_07_02 ACACACACACACTCAGATTCCCA
DRB1_08_01GCGTTGCGGGTCGGCG
DRB1_08_02 ACACACACACTCAGATTCCCA
DRB1_09_01 AACAGGCTGGAGGTAGGGAC
DRB1_09_02 ACACACACACACTCAGATTCCCA
DRB1_10_01 GGTGGGCGTTGCGGCG
DRB1_10_02 ACACACACACACTCAGATTCCCA
DRB1_11_01GGGCGTTGCGCCGGC
DRB1_11_02 ACACACACACACTCAGATTCCCA
DQB1
DQB1_01_01CGGGCCGGCCCGAACTTCT
DQB1_01_02GTCAGGTTCCAGTCGGCTACG
DQB1_02_01GGGAGGACTTCAAGTCTGC
DQB1_02_02TCAGGTTCCCAGAGGATTCGC
DQB1_03_01CAGGAACCGGGATTCCC
DQB1_03_02AAGGATGGGCCATGCAGAC
DQB1_04_01GCAGGAACGCGGATTCCG
DQB1_04_02AGGATGAAGCTTGCGGAC
DQB1_05_01GGGAGGATCGGAGGTTTGGG
DQB1_05_02AGACTAGAGGTCGGTGCTAAC
DQB1_06_01GCCGCCGGGATTATCCTAG
DQB1_06_02GCCGCCGGGCCGATCCTAG
DQB1_06_03GGTGGAATGACCCGGCTTAGA
Sequencing primer:
A
A1F GTGTCGGGTTTCCAGAG
A2F TTGGGGACGGGGCTG
A3F GTTTCATTTTCAGTTTAGGC
A4F GTGTCTGGGTTCTGTGCTC
A2R CGGACCCGGAGACTGT
A3R TGTTGGTCCCAATTGTCTC
A4R ATAGAGGCTCCTGCTTTCC
A5F RGACCTTTAGCAGGGTCAG
B
B1F GTCCCAGTTCTAAAGTCCC
B2F01 GGGTCTCAGCCCCTCCTC
B2F02 GGGTCTCAGCCCCTCCTG
B2F03 GGGTTTCAGCCCCTCCTC
B2F04 TGGGTCTCAGCCCCTCCTT
B2F05 CAGGAACCGGGATTCCC
B2F06 CTGGGGACTGGGCTGAC
B3F01TTGGGGACGGGGCTGAC
B3F02 CTGGGGACGGGGCTGAC
B3F03 CTGGGGACGGTGCTGAC
B3F04 TTGGGGACTGGGCTGAC
B4F CATGGGTGGTCCTAGGGTG
B2R GTCGTGACCTGCGCCC
B3R GAGGGCGACATTCTAGCG
B4R GGCTCCTGCTTTCCCTG
B5R AGACCCCACCCCTCAC
C
C1R ATGCTGAGACCCCCCCGAC
C2F GCGCAGGACCCCCGGGAGAG
C2R AGGCCGTCCGTCCGGGATG
C3F GGGGGACGGGAAGCTGAC
C3R CGGGGGACGGAAGCTGAC
C4F01 GGGGGACCGGAAGCTGAC
C4F02 GGGGGACTGGAAGCTGAC
C4R01 GCGGGACGGGAAGCTGAC
C4R02 ATTTTCCTCCCCTCCTCG
C4R03TTCTCAGGATGGCATGGG
C4R04 TTCTCAGGATAGCATGGG
C4R05 GCTGAAGGGCAGGCGGAC
C4R06 GAAGGGCTCCGCCGGCC
C5R GAAGGGCTCCCGGCCAGGAC
C6F CACACAGGGTGGCCAGGC
C7R01 GACTAGGACCGGTTCCCC
C7R02 CACACTCGAGTGCTGTCCCA
C7R03 CACACTCGGGGC GTCCCA
C7R04 ACACATTCGGGGCGTCCCT
DRB2F01 TCGTGTCCCCACAGCA
DRB2F02 TCTTGTCCCCCCAGCA
DRB2F03 TTGTGCCCCCACAGCA
DRB2R01GCTCACCTCGCCGCTG
DRB2R02 GCTTACCTCGCCTCTG
DRB2R03 GCTCACCTCGTCGCTG
DRB2R04 GCTCACCTCGCCTCTG
DRB2R-86 CACTGTGAAGCTCTCCAC
DRB1F CCTGGTCCTGTCCTGTT
DRB1R CTGGGCACAATGTTAAC
DRB3F CACTATCAAGATTAAG
DQB1
DQB2F01 CTGACTGGGGCGTGATTC
DQB2F02 CTGACTGGGGGGTGATTC
DQB2F03 CTGACCGGGGGGTGATTC
DQB2R01 GGCGACGAGCCCTCACCTC
DQB2R02 GTCAACCAGCCCTCACCTC
DQB3F01CCCTGTCTGAAACTGGCCTC
DQB3F02 TCCTGTCTGAAACTGGCCTT
DQB3R01 AACAGAAACTCTTTATCTGCTTAC
DQB3R02 AACAGAAACTCTTTATCCGCTTAC
1.2 the following HLA standard cell line DNA was purchased from IHWG, the genotype of HLA ABCDB 1DQB1 of the IHWG standard is shown in the table: the negative quality control product adopts DNA of HLA defective cell strain K5602, and non-human genome DNA (zebra fish, Arabidopsis)
1.3 reagent preparation
1.3.1 preparation of primers
Combining the A site universal amplification primers according to A01A03, A01A 04, A02A03, A02A04, A01A03A 04A 02A03A04 and A01A02A03A04, mixing the primer concentrations according to equal mole numbers, and screening a primer group;
combining B site universal amplification primers according to B01B02, B01B03 and B01B02B03, mixing the primer concentrations according to equal mole numbers, and carrying out primer screening;
combining the C site universal amplification primers according to C01C02, C01C03 and C01C02C03, mixing the primer concentrations according to equal mole numbers, and screening the primers;
mixing DRB1 universal amplification primers according to the equal molar number of DRBL101020304, and naming the mixture as DRBL1_01, DRBL1050607, and naming the mixture as DRBL1_02, DRBL10809, and naming the mixture as DRBL1_ 03; then DRBL1_01 and DRBL1_02, DRBL1_01 and DRBL1_03, DRBL1_01, DRBL1_02 and DRBL1_03 are mixed in equal molar number for primer screening;
mixing DRB1 universal amplification primers according to the equal molar number of DRBL2010203, and naming the mixture as DRBL2_01, and mixing the equal molar number of DRBL10405, and naming the mixture as DRBL2_ 02; then DRBL2_01 and DRBL2_02 are mixed in equal molar number for primer screening;
combining DRBL30102, DRBL30103 and DRBL3010203 with DRB1 universal amplification primers, and performing primer screening;
mixing DQB1 universal amplification primers according to the equal molar number of DRBL1010203, and naming the mixture as DQBL1_01, and mixing DQBL10405 according to the equal molar number of DQBL1_ 02; the mixed mole number of the DQBL1060708 is named DQBL1_ 03; the mixed mole number of DQBL10091011 is named DQBL1_ 04; then, the DQBL1_01 and DQBL1_03, DQBL1_01 and DQBL1_04, DQBL1_02 and DQBL1_03, DQBL1_02 and DQBL1_04, DQBL1_01, DQBL1_02 and DQBL1_03, DQBL1_01, DQBL1_02 and DQBL1_04, DQBL1_01, DQBL1_02 and DQBL1_03 and DQBL1_04 are combined, and the mixture with equal molar number is mixed to carry out primer screening;
1.3.2 preparation of reaction solution
And carrying out amplification reaction according to EZ-Taq enzyme and the reaction solution, and testing, screening and amplifying the universal primer.
| Composition of | Dosage of |
| EZ-Taq enzyme 5U/ul | 0.25 |
| Reaction solution | |
| 5 | |
| Amplification primers MIX of |
1 |
| Standard DNA template | 50ng |
| NF-H2O | Supplement to 12.5ul |
Preparing a reaction system according to the table, uniformly mixing the PCR reaction system, placing the mixed reaction system on a PCR instrument, and carrying out PCR amplification according to the following reaction procedures:
after the reaction is finished, detecting the product by adopting 1 percent agarose gel electrophoresis
1.3.3 results of the experiment
Obtained according to the screening test of the primer groups at different sites, and the following experimental results are obtained:
| ID | B01B02 | B01B03 | B01B02B03 |
| IHW09009 | single stripe, brighter stripe | With non-specific bands | Single stripe, brighter stripe |
| IHW09014 | With non-specific bands | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09024 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09053 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09103 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09297 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09368 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09372 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09373 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09374 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09398 | With non-specific bands | Single stripe, brighter stripe | Single stripe, brighter stripe |
| IHW09462 | Single stripe, brighter stripe | Single stripe, brighter stripe | Single stripe, brighter stripe |
| K5602 | Without strips | Without strips | Without strips |
| Arabidopsis DNA | Without strips | Without strips | Without strips |
| Zebra fish DNA | Without strips | Without strips | Without strips |
Example 2 optimization testing of different reaction systems
2.1 preparation of different reaction buffer systems
In a typical PCR detection system, the PCR buffer used for HLA detection must have several characteristics: (1) the PCR reaction is carried out without using mineral oil insulation, so that slight differences in concentration due to evaporation of a small part of the volume must be tolerated; provides the reaction conditions suitable for PCR of the primers with high GC content and high similarity. In order to meet the above conditions, we tested the ratios of the components of 10 different buffers, and the specific information is shown in the experimental results of Table 7, wherein number # 6 is the most suitable buffer condition for the product.
| | # | 1 | #2 | #3 | #4 | #5 | #6 | #7 | #8 | #9 | #10 |
| KCl(mM) | 100 | 100 | 100 | 100 | 100 | 50 | 75 | 50 | 75 | 50 | |
| TrispH8.5 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | |
| dNTP(mM) | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | |
| MgCl2(mM) | 2.5 | 2.5 | 2.0 | 2.0 | 2.0 | 2.0 | 1.5 | 1.5 | 1.5 | 1.0 | |
| BSA(％) | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | |
| Betaine(M) | 1 | 1.5 | 1 | 1.5 | 0.5 | 1 | 0.5 | 1 | 1 | 1 | |
| Glycerol(％) | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 |
2.2 preparation of PCR reaction System
Preparing a reaction system according to the optimal primer universal primer group screened in the example 1, and preparing a PCR reaction system according to the step of the example 1;
2.3 Standard detection
Selecting corresponding standard substance for standard substance detection according to the standard substance in the example 1
2.4 results of the experiment
The specific experimental structure is subjected to the attached drawings of the specification.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. An HLA genotyping detection method, comprising the steps of:
step 1, processing a sample;
step 2, taking the processed sample product as a template, and carrying out amplification on gene long products of different sites of HLA through a specific primer group; carrying out sequencing reaction by taking the long fragment amplification product as a template to obtain a sequencing reaction product;
3, taking the sequencing reaction product as a reaction hole to be tested, and taking a first-generation sequencer as a platform to perform sequencing reaction;
and 4, analyzing the sequencing reaction data to obtain HLA different site genotype results.
2. The method for detecting HLA genotyping according to claim 1, wherein the sample treatment is a nucleic acid treatment and the product of the nucleic acid release treatment.
3. The method according to claim 2, wherein the accounting process specifically comprises: extracting and purifying the product of nucleic acid by a purification kit; a product of nucleic acid release from a sample with a nucleic acid releasing agent; nucleic acid products processed in other ways without taking the kit include physical heating, snap freezing, grinding or sample dilution.
4. The HLA genotyping detection method of claim 3, wherein the specific primer set comprises HLA A site specific amplification primer set, B site specific amplification primer set, C site specific amplification primer set, DRB1 site specific amplification primer set, and DQB1 site specific amplification primer set.
5. The method for detecting HLA genotyping according to claim 4, further comprising a method for amplifying the long fragment products with a specific amplification primer set, including but not limited to one or more of the following:
1)5, single-tube amplification of multiple amplicons of a site-specific amplification primer group;
2) an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, a molecular amplification primer group MIX and DRB1 and a DQB1 amplification group MIX, so that two-tube amplification of HLA 5-site long fragment products is realized;
3) an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, an amplification primer group with site specificity DQB1 and an amplification primer group with site specificity DRB1 independently amplify long products, and 5-site independent amplification is realized;
4) an amplification primer group with site specificity A, an amplification primer group with site specificity B, an amplification primer group with site specificity C, an amplification primer group with site specificity DQB1, an amplification primer group with site specificity 2 tubes for segmented amplification and a amplification primer group with site specificity DRB1 for 2 tubes for segmented amplification and a growing segment, wherein 6-7 independent amplicons with 5 sites are realized;
5) a, B, C, DRB1 and DQB15 locus multiple amplicon single tube amplification to realize 5 locus multiple PCR.
6. The method of claim 5, wherein the long fragment products are subjected to a sequencing reaction, and the long fragment product templates include, but are not limited to, one or more of single-site single-tube amplification primer set products, multi-site single-tube amplification primer set products, and multi-site multi-tube amplification products.
7. The method of claim 6, wherein the long fragment product template is a purified product; purification means include, but are not limited to, one or more of, preferably enzymatic digestion; bead purification methods, magnetic beads include, but are not limited to, the following: XP beads, SPRIbeads; the enzyme digestion method comprises 0.1-100U shrimp alkaline phosphatase and 0.1-100U EXO I.
8. The HLA genotyping detection method of claim 7, wherein the sequencing reaction comprises sequencing primers of specific sites and sequencing reaction reagents, the sequencing primers are subjected to separate hole sequencing reaction according to different sites and different determined regions, the sequencing reaction is linear PCR amplification of a single side and a single primer, and the sequencing reaction reagents comprise BigDYE reagent, DNA polymerase, PCR reaction buffer, Mg2+ and the like.
9. An HLA ABCDQB1DRB1 high resolution genotyping detection kit, the reagent consists of amplification reagent, sequencing reagent, nucleic acid polymerase and amplification buffer solution; the amplification reagent consists of a general amplification primer group reaction solution and a group specificity primer group reaction solution, and the sequencing reagent consists of different sequencing primers and Tris-HCl buffer solutions which are designed according to different sites and different quantities of concerned exons; nucleic acid polymerases have a high fidelity long product amplification DNA polymerase combination, including but not limited to EZ-Taq enzyme.
10. The amplification reagent comprises amplification primers, dNTP and the like, the amplification buffer solution comprises buffer solution, metal coenzyme and specificity enhancer, and the sequencer applicable to the detection kit provided by the invention comprises but is not limited to: one or more of ABI3130, ABI 3500, ABI 3730.
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