CN117512085A - Primer group and kit for detecting HLA-DPB1 genotyping - Google Patents
Primer group and kit for detecting HLA-DPB1 genotyping Download PDFInfo
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Abstract
The invention relates to a primer group and a kit for detecting HLA-DPB1 genotyping, belonging to the field of biomedical clinical molecular detection. The primer group for detecting HLA-DPB1 genotyping comprises 1 pair of long fragment amplification primers designed according to the DPB1 gene specific sequence, and further comprises 6 exon sequencing primers, 2 specific single-strand sequencing primers and 5 specific intron sequencing primers, wherein the total number of the primers is 13. The invention has the following technical effects: the DPB1 gene specific amplification primer designed by combining ARMS with a double-mismatch specific base method has high specificity and high amplification efficiency, and can amplify all subtypes of the DPB1 gene, namely the 2/3/4 exons and the 2/3 introns through one-tube reaction; HLA-DPB1 gene subtype can be accurately distinguished by matching with the specific sequencing primer. The HLA-DPB1 genotyping of the experimental sample can be accurately judged by the kit. The operation is quick and simple, the cost is low, and the method has wide application prospect and clinical reference value.
Description
Technical Field
The invention relates to a primer group and a kit for detecting HLA-DPB1 genotyping, belonging to the field of biomedical clinical molecular detection.
Background
HLA is located in the 21.31 region of the short arm of human chromosome 6, contains about 360 ten thousand base pairs, and is the region of the human chromosome known at present with the highest gene density and the most abundant polymorphism, and is divided into HLA-I, II and III genes. Classical HLA-I genes include HLA-A, HLA-B and HLA-C, classical II genes generally refer to DR, DP and DQ, HLA-III genes are different from the first two, and comprise a plurality of non-immune related genes besides genes with immune related functions such as tumor necrosis factor (Tumour Necrosis Factor, TNF) genes, lymphotoxin alpha (lymphotoxin alpha, LTA) genes, heat shock protein genes and the like. HLA system plays an extremely important role in antigen recognition and presentation, immune response and regulation and the like, is one of key factors influencing the long-term survival of organ transplants and the success and failure of hematopoietic stem cell transplantation, and is closely related to various diseases, such as ankylosing spondylitis, rheumatoid arthritis, bezite's disease, celiac disease and the like.
The rapid and accurate detection of HLA-DPB1 genotyping has important significance for clinical disease auxiliary diagnosis, medical research, disease etiology research and the like. The currently commonly used HLA-DPB1 genotyping detection methods mainly comprise a PCR-SSP method, a SYBR Green I method, a Taqman fluorescent quantitative PCR method, a sequencing method and the like. The characteristics of each method are as follows:
PCR-SSP (sequence specific primer), namely a sequence specific primer-guided PCR reaction, is a detection method widely adopted at present, the basic method is to design a series of allele specific primers, amplify each allele specific DNA fragment through a specific PCR reaction system to generate corresponding specific amplified product strips, and detect the PCR products by agarose gel electrophoresis, the method has low cost, but the operation is complex, the result cannot be automatically obtained, and the accuracy is still to be improved.
SYBR Green I is a dye with Green excitation wavelength that binds to all dsDNA double helix minor groove regions, and its binding to DNA is nonspecific, and this method is simple and rapid to operate, but lacks specificity and is poorly accurate.
The Taqman fluorescent quantitative PCR method is simple and rapid to operate, but cannot realize high-resolution typing results.
Sequencing methods can obtain high resolution results, but are currently not widely used.
It can be seen that only sequencing methods can obtain high-resolution typing results, but specific amplification primers are required to be designed for PCR amplification, and the accuracy is affected due to the fact that HLA polymorphism is very high and false positive occurs easily. The prior Chinese patent application CN201910952019.9 discloses a group of primers and a detection method for HLA-DPB1 gene sequencing typing, which comprises 2 pairs of specific amplification primers and 5 oligonucleotide sequencing primers. The method comprises the steps of PCR amplification, electrophoresis detection of PCR products, PCR product purification, sequencing reaction, sodium acetate-ethanol precipitation purification of sequencing products, sequencing on machine and data analysis. However, in this method, only exons are sequenced, and more genotypes cannot be distinguished.
Therefore, there is an urgent need in the art for a detection method that is simple and rapid to operate and has high accuracy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a primer group and a kit for detecting HLA-DPB1 genotyping.
The design idea of the primer group of the invention is as follows: HLA-DPB1 gene specific amplification primers were designed based on known human leukocyte antigen gene sequences using amplification block mutation systems (Amplification refractory mutation system, ARMS) analysis combined with double mismatch specific base methods. When the primer sequence is perfectly matched with the target sequence to be detected, a polymerase chain reaction (Polymerase Chain Reaction, PCR) is performed. During the reaction, the target nucleic acid fragment will be replicated and amplified, indicating the presence of the exact same gene sequence as the specific primer in the sample, and vice versa. And detecting and analyzing the PCR reaction result by using an agarose gel electrophoresis method. When the gel is stained and analyzed by a gel imaging system, the nucleic acid fragments are distinguished by the size. The reaction amplicons initially identified by electrophoresis are purified and subjected to a further sequencing analysis to identify the sequences of the individual alleles, thereby achieving high resolution genotyping of HLA-DPB 1.
HLA contains about 360 ten thousand base pairs, and is the region of the highest gene density and most abundant polymorphism in the currently known human chromosome. The common primer design method has certain limitation on distinguishing HLA gene subtypes and has low accuracy. The inventor firstly uses a primer design method of combining ARMS with double mismatch specific bases, firstly finds the specific base sequence of the DPB1 gene in a database, wherein an upstream specific amplification primer is positioned at the tail end of an intron No. 1, and a downstream specific amplification primer is positioned at the front end of an intron No. 4, so that the exon No. 2, the intron No. 2, the exon No. 3, the intron No. 3 and the exon No. 4 of the DPB1 gene are accurately and specifically amplified in the first step, and the amplification efficiency is high. And according to the preliminary detection result, a mismatched base is introduced on the primer, so that the specificity of the primer is improved. The method has high accuracy, simple and convenient operation and wide application prospect.
In a first aspect of the present invention, there is provided a primer set for detecting HLA-DPB1 genotyping of human leukocyte antigen comprising 1 pair of long fragment amplification primers designed according to the specific sequence of the DPB1 gene, corresponding to 6 exon sequencing primers, 2 specific single-strand sequencing primers and 5 specific intron sequencing primers.
The specific PCR amplification primer is 1 pair of long fragment amplification primers designed according to HLA-DPB1 gene specific sequences. The amplification primer can accurately and specifically amplify the No. 2 exon, the No. 2 intron, the No. 3 exon, the No. 3 intron and the No. 4 exon of the DPB1 gene, and is used for specifically amplifying all subtypes of the DPB 1.
The nucleotide sequences of the 1 pair of specific PCR amplification primers are shown in the following table:
the PCR amplification primer is designed according to an improved amplification inhibition mutation system (Amplification refractory mutation system, ARMS) analysis method and combined with a double-mismatch specific base method, the 3' ends of the forward amplification primer and the reverse amplification primer are both DPB1 gene specific bases, and a mismatch base is additionally introduced into the primers, so that the detection specificity is improved.
The primer group for detecting HLA-DPB1 genotyping also comprises 6 exon sequencing primers, 2 specific single-strand sequencing primers and 5 specific intron sequencing primers, wherein the total number of the primers is 13.
The 13 sequencing primers, each primer nucleotide sequence is shown in the following table:
in the sequencing primer provided by the invention, 6 exon sequencing primers are conventional sequencing primers specific to the DPB1 gene, 2 specific single-stranded sequencing primers are single-stranded sequencing primers specific to the DPB1 gene, and then the primers are combined with 5 specific intron sequencing primers, so that high-resolution sequencing can be performed on 2/3/4 exons of the DPB1 gene, high-resolution sequencing can be performed on 2/3 introns, and single-stranded sequencing can be performed on 2 exons. The DPB1 genotype of the sample can be more accurately distinguished, and the ambiguous situation is reduced.
In a second aspect of the invention, there is provided the use of a primer set for detecting HLA-DPB1 genotyping of human leukocyte antigens as described in the first aspect in the preparation of a kit for detecting HLA-DPB1 genotyping.
In a third aspect of the present invention, there is provided a kit comprising the primer set for detecting human leukocyte antigen HLA-DPB1 genotyping as described in the first aspect, wherein the kit further comprises PCR reaction reagents.
Further, the PCR reaction reagent comprises PCR reaction liquid and high-fidelity Taq enzyme.
Further, the PCR reaction solution comprises: 0.5mM deoxynucleotide dNTP,40mM MgCl magnesium chloride 2 80mM potassium chloride KCl,60mM Tris-HCl,1mM TMAC, 0.6% v/v glycerol, 0.02% v/v cresol red and 5% v/v betaine.
In a fourth aspect of the present invention, there is provided a method for detecting HLA-DPB1 genotyping for non-disease diagnostic purposes using the primer set for detecting HLA-DPB1 genotyping as described in the first aspect, the method comprising an amplification reaction and sequencing.
Wherein, the amplification reaction system is as follows: the total volume is 12.4 mu L, including 6 mu L of PCR reaction solution, 0.4 mu L of enzyme, 3 mu L of amplification primer mixture and 3 mu L of DNA template; the PCR amplification reaction is carried out for 2 minutes at 95 ℃;93 ℃ for 10 seconds, 68 ℃ for 5 minutes, 30 cycles; 72℃for 5 min, 4℃until removal.
The invention has the following technical effects:
1. the DPB1 gene specific amplification primer designed by combining ARMS with a double-mismatch specific base method has high specificity, and can amplify all subtype 2/3/4 exons and 2/3 introns of the DPB1 gene.
2. The DPB1 gene specific amplification primer has high amplification efficiency, and can amplify the 2/3/4 exons and the 2/3 introns of all subtypes of the DPB1 gene by one tube reaction. The existing 201910952019.9 patent needs to carry out a plurality of amplification reactions, and only exons can be amplified, so that the experimental efficiency is low, and time and labor are wasted.
3. The sequencing primer of the invention comprises 6 exon sequencing primers, 2 specific single-chain sequencing primers and 5 specific intron sequencing primers, and 13 sequencing primers are used. The high-resolution sequencing of the 2/3/4 exon of the DPB1 gene, the high-resolution sequencing of the 2/3 intron and the single-chain sequencing of the 2 exon specificity can be performed. The existing 201910952019.9 patent only sequences exons, and more genotypes cannot be distinguished.
4. The kit containing the specific amplification primer and the universal sequencing primer can accurately judge HLA-DPB1 genotyping of the experimental sample. Can be used for detecting HLA-DPB1 genotyping of human leukocyte antigens.
5. Compared with the existing 201280036108.5, 201810331958.7, 202211530754.9 patents and the like, the existing methods use a second-generation sequencing method, a group of primers are designed to amplify the DPB1 gene, the amplified products are randomly broken, a library is constructed, and the second-generation sequencing is performed, and specific sequencing primers are not needed, but the accuracy is not high enough. In the Sanger sequencing method, long fragment amplification primers are designed aiming at the regions from the No. 2 exons to the No. 4 exons with highly concentrated polymorphism, specific sequencing primers are designed, and specific first-generation sequencing is carried out on the amplified products, so that the accuracy of the parting result is improved. The specific amplification primer designed by adopting the ARMS combined bispecific base primer design method has high specificity, and can accurately distinguish HLA-DPB1 gene subtypes by matching with the specific sequencing primer.
Drawings
FIG. 1-1 is a diagram of sample S1 aligned via an IMGT database sequence.
FIGS. 1-2 are single-stranded sequencing of exon 2 of the invention for sample S1.
FIGS. 1-3 are graphs of sample S2 aligned via the IMGT database sequence.
FIGS. 1-4 are graphs of sample S2 for the intron No. 2 sequencing of the present invention.
FIGS. 1-5 are graphs of sample S5 aligned via the IMGT database sequence.
FIGS. 1-6 are graphs of sample S6 aligned via IMGT database sequences.
FIGS. 1-7 are graphs of sample S5 for the intron No. 2 sequencing of the present invention.
FIGS. 1-8 are graphs of the intron sequence of sample S6 according to the present invention No. 2.
FIG. 2 is an electrophoretogram of a mismatch-free specific base set amplification primer, a Shan Cuopei specific base set amplification primer and a specific amplification primer of the invention, wherein the first behavior of the electrophoretogram is a mismatch-free specific base set amplification primer, the amplified product has poor specificity and many bands. The second behavior of the electrophoresis chart is that the single mismatch specific base group amplification primer has poor specificity of amplified products and more mixed bands. Third behavior of electrophoresis pattern of specific amplification primers of the present invention. The amplified product has single band, good specificity and no impurity band.
FIG. 3-1 shows a sample S1 sequencing, with genotypes DPB 1.times.05:01, DPB 1.times.09:01. The sequencing peak diagram has good result and accurate interpretation.
Fig. 3-2 is a sample S2 sequencing plot, genotype DPB1 x 02:01, DPB1 x 13:01. The sequencing peak diagram has good result and accurate interpretation.
Fig. 3-3 are sample S3 sequencing plots, genotype DPB1 x 04:01, DPB1 x 05:01. The sequencing peak diagram has good result and accurate interpretation.
Fig. 3-4 are sequencing charts of sample S4, with genotypes DPB1 x 03:01, DPB1 x 05:01. The sequencing peak diagram has good result and accurate interpretation.
Fig. 3-5 are sample S5 sequencing plots, genotype DPB1 x 02:01, DPB1 x 05:01. The sequencing peak diagram has good result and accurate interpretation.
Fig. 3-6 are sample S6 sequencing plots, genotype DPB1 x 02:01, DPB1 x 35:01. The sequencing peak diagram has good result and accurate interpretation.
FIGS. 3-7 are graphs of sample S7 sequencing, genotype DPB1 x 04:01, DPB1 x 135:01. The sequencing peak diagram has good result and accurate interpretation.
Fig. 3-8 are sample S8 sequencing plots, genotype DPB1 x 05:01, DPB1 x 38:01. The sequencing peak diagram has good result and accurate interpretation.
Detailed Description
The present invention will be further described in detail below with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent, and it is apparent that the described examples are only some of the examples of the present invention, but not all of the examples.
In the present invention, the main raw material list involved is as follows:
example 1
1 raw materials and equipment:
1.1 kit components:
1.1.1 the specific amplification primers of the present invention comprise 1 pair of long fragment amplification primers designed according to HLA-DPB1 gene specific sequences. According to the improved ARMS analysis method of the amplification inhibition mutation system, a double-mismatch specific base method is combined, a specific primer is designed, the forward amplification primer and the reverse amplification primer are both DPB1 gene specific bases, and a mismatch base is additionally introduced into the primer, so that the specificity of detection is improved. Can accurately and specifically amplify the No. 2 exon, the No. 2 intron, the No. 3 exon, the No. 3 intron and the No. 4 exon of the DPB1 gene, and is used for specifically amplifying all subtypes of the DPB 1.
The nucleotide sequences of the specific amplification primers are shown in the following table:
the sequencing primer of the invention comprises 6 exon sequencing primers, 2 specific single-chain sequencing primers and 5 specific intron sequencing primers, and 13 sequencing primers are used. The high-resolution sequencing can be carried out on the 2/3/4 exons of the DPB1 gene, and the high-resolution sequencing can be carried out on the 2/3 introns. The nucleotide sequence of each primer is shown in the following table:
| sequencing region | Sequencing direction | SEQ ID NO. | Primer probe sequence 5 '. Fwdarw.3' |
| Exon2 | Forward direction | #03 | ATTAGATGAGAGTGGCGCCT |
| Exon2 | Reverse direction | #04 | AAGGTCCCTTAGGCCAAC |
| Exon3,Intron3 | Forward direction | #05 | CAATCTCAAATGCTATTTCATTA |
| Exon3 | Reverse direction | #06 | AGTGGACCCCAGAGCCA |
| Exon4 | Forward direction | #07 | GACATGAGTAGGGATGCAGC |
| Exon4,Intron3 | Reverse direction | #08 | TGTGAATATGCCAGGGGAA |
| Exon2 | Forward single strand | #09 | CCGCAGAGAATTACCTTTT |
| Exon2 | Reverse single strand | #10 | GCAGGGTCACGGCCT |
| Intron2 | Forward direction 1 | #11 | GACTTTGGGTTGGGGATT |
| Intron2 | Forward direction 2 | #12 | GAAGCTGAGGGGAGGTGA |
| Intron2 | Forward direction 3 | #13 | GATTTTCTTGTTCCTGGAAAG |
| Intron2 | Forward direction 4 | #14 | AGGAGGGACCAGAGGCA |
| Intron2 | Forward direction 5 | #15 | AGAAGCTCTATGGGGAAGAA |
1.1.2 two sets of common amplification primers were designed
The first group is a specific amplification primer of a mismatch-free specific base group, and the sequence is as follows:
the second group is a single mismatch specific base group specific amplification primer, and the sequence is as follows:
1.1.3 primer formulation protocol
(1) Preparing each primer into a primer solution with the concentration of 12 OD/mL;
(2) preparation of amplification primer solution (AMP MIX)
(3) Preparation of sequencing primer solution (SEQ MIX)
1.1.4PCR reactant
DNA polymerase: is high-fidelity Taq polymerase;
PCR reaction solution: comprises 0.5mM deoxynucleotide dNTP and 40mM MgCl magnesium chloride 2 80mM potassium chloride KCl,60mM Tris-HCl,1mM TMAC, 0.6% v/v glycerol, 0.02% v/v cresol red and 5% v/v betaine.
1.2 sources of samples
1.2.1 blood sample collection
Blood samples can be collected using blood collection tubes containing the anticoagulant Sodium citrate (Sodium citrate) and ethylenediamine tetraacetic acid (EDTA) and fresh or freeze-preserved whole blood samples that have not been repeatedly freeze-thawed are used as experimental samples.
1.2.2 nucleic acid sample extraction
Nucleic acid extraction can be performed from a sample containing nuclear cells such as whole blood or a leukocyte layer by a precipitation method, a column method or a magnetic bead method to obtain a sufficient amount of nucleic acid with acceptable quality for polymerase chain reaction.
1.2.3 nucleic acid sample quantification
The extracted nucleic acid sample must be dissolved in sterile water or other suitable solution (e.g., TE Buffer) at a concentration of 10-40 ng/. Mu.l, and the nucleic acid sample cannot be dissolved in a solution containing more than 0.5mM of a chelating agent such as ethylenediamine tetraacetic acid (EDTA).
1.2.4 nucleic acid sample quality Specification
The A260/A280 ratio of the nucleic acid sample should be between 1.6 and 2.1.
1.3 required Experimental facility
PCR instrument, sequencer, pipettor of different ranges, small table centrifuge (8-pipe horizontal head).
2 genotyping procedure
8 EDTA anticoagulated whole blood samples are selected, and the specific amplification primer set of the primer set, the specific amplification primer set of the non-mismatched bispecific base set and the specific amplification primer set of the mismatched monospecificbase set are used for amplification respectively. Each sample was amplified with the DPB1, DPB1-23 sets of primers, the positive bands were sequenced, first with DPB1-E2F1, DPB1-E2R1, DPB1-E3F, DPB1-E3R, DPB1-E4F, DPB1-E4R 6 conventional exons, and DPB1-I2F1, DPB1-I2F2, DPB1-I2F3, DPB1-I2F4, DPB1-I2F55 specific introns were sequenced, and DPB1-E2F2, DPB1-E2R22 specific single strands were sequenced for samples with multiple possible typing results.
2.1 preparation of the reaction system: the reaction system is shown in the following table:
PCR reaction system
| Component name | Mu L/tube of addition |
| PCR reaction solution | 6 |
| Amplification primer mixture | 3 |
| Taq enzyme | 0.4 |
| Nucleic acid sample | 3 |
| Total volume of | 12.4 |
The reaction tube was covered and centrifuged briefly, and then placed in a fluorescent quantitative PCR apparatus.
2.2PCR reaction procedure: the following table shows:
PCR reaction procedure
2.3 electrophoresis
Running the gel at 8-10 volts/cm, 200V, about 10-20 minutes. The quality of the PCR product was confirmed by taking a photograph on an ultraviolet transilluminator.
2.4 PCR product purification
For wells for which sequencing is desired, 4. Mu.L of ExoSAP-IT is added TM To remove excess primer and DNA.
The purification step was started with reference to the following table set-up procedure. The total reaction time was about 1 hour.
ExoSap PCR reaction program setting
2.5 sequencing reactions
1.5. Mu.L BDT sequencing reagent was added to each reaction well;
2.5. Mu.L of sequencing primer was added to each reaction well;
mu.L of the purified PCR product was added to each reaction well.
2.6 sequencing product purification
Excess BDT was removed by ethanol precipitation.
2.7 on-machine sequencing
Before sequencing, 10 mu L of HiDi formamide can be optionally added, and a sequencer can be arranged after the PCR instrument is heated.
3 analysis of experimental results
3.1 the specific amplification primer of the invention has high specificity of the amplification product, no impurity band and completely correct negative and positive type. See third row of fig. 2 for electrophoreses.
3.2 No mismatch specific base group amplification primers, the amplification products have poor specificity and more bands, and the electrophoresis diagram is shown in the first row of FIG. 2.
3.3 Single mismatch specific base group amplification primers, the amplification product specificity is poor, the number of the mixed bands is large, and the electrophoresis diagram is shown in the second row of FIG. 2.
3.3 the specific amplification primer of the invention has the advantages that the sequencing result of the amplification product is completely correct, the HLA-DPB1 genotyping of the experimental sample can be accurately judged, and the sequencing diagrams are shown in figures 3-1 to 3-8.
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Conclusion: the specificity amplification primer provided by the invention has high specificity, and HLA-DPB1 gene subtypes can be accurately distinguished by matching with the specificity sequencing primer provided by the invention. The HLA-DPB1 genotyping of the experimental sample can be accurately judged by the kit.
Comparative example 1
A conventional method for HLA-DPB1 sequencing typing is by amplifying the exons of DPB 1E 2/E3/E4 separately and then sequencing the amplified products. The sample S1 of example 1 was sequenced in a conventional manner for the exon regions, giving the following two possible results:
possible 1-DPB 1. Times.05:01, DPB 1. Times.09:01;
2-DPB 1 x 22:01, DPB1 x 35:01.
Two possible results were obtained, since the 2/3/4 exons and the 2/3 introns of the above 2 combined alleles were identical, and further confirmation of single-stranded sequencing of the 2 exons was required by sequence alignment analysis in the IMGT database, see FIGS. 1-1. The final sequencing results using the exon 2 single-stranded sequencing primer of the invention were DPB 1:05:01, DPB 1:09:01, see FIGS. 1-2.
The sample S2 of example 1 was sequenced in a conventional manner for the exon regions, giving the following two possible results:
possible 1-DPB 1. Times.02:01, DPB 1. Times.13:01;
2-DPB 1 x 02:01, DPB1 x 107:01.
Two possible results were obtained, since the allele 2/3/4 exons of the above 2 combinations were identical. The identification point was at intron No. 2 7181, see fig. 1-3, by IMGT database sequence alignment analysis. The final sequencing results were DPB1 x 02:01, DPB1 x 13:01, see FIGS. 1-4.
The results of conventional exon region sequencing of sample S5 in example 1 were obtained using conventional methods, giving the following two possible results:
possible 1-DPB 1. Times.02:01, DPB 1. Times.05:01;
2-DPB 1 x 22:01, DPB1 x 105:01.
The results of conventional exon area sequencing of sample S6 in example 1 were obtained using conventional methods, giving the following two possible results:
possible 1-DPB 1. Times.02:01, DPB 1. Times.35:01;
possible 2-DPB 1. Times.09:01, DPB 1. Times.105:01.
The sample allele 2/3/4 exon sequences were identical and were analyzed by IMGT database sequence alignment, with the identification point at intron No. 2 6467, see figures 1-5 and 1-6. Sample S5 final sequencing results were dpb1×02:01, DPB1×05:01, sample S6 final sequencing results were dpb1×02:01, DPB1×35:01, see fig. 1-7 and 1-8.
Thus, if conventional methods are used to amplify and sequence only conventional 2/3/4 exons, the typing results may be likely to be not precisely typed. By using the amplification and sequencing primer and the method, the sample type can be accurately detected. The invention can carry out high-resolution sequencing on the 2/3/4 number of exons, can carry out high-resolution sequencing on the 2/3 number of introns, can carry out single-chain sequencing on the 2 number of exons, and improves the resolution capability of detection.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent modifications and variations are intended to be included within the scope of this invention.
Claims (7)
1. A primer set for detecting HLA-DPB1 genotyping, wherein said primer set comprises 1 pair of long fragment amplification primers designed according to HLA-DPB1 gene specific sequences, and wherein the nucleotide sequences of said primers are as follows:
the primer group also comprises 6 exon sequencing primers, 2 specific single-chain sequencing primers and 5 specific intron sequencing primers, 13 sequencing primers are used, and the nucleotide sequences of the 13 sequencing primers are shown in the following table:
。
2. The use of a primer set for detecting HLA-DPB1 genotyping according to claim 1 in the preparation of a kit for detecting HLA-DPB1 genotyping.
3. A kit comprising the primer set for detecting HLA-DPB1 genotyping according to claim 1, wherein the kit further comprises a PCR reagent.
4. The kit of claim 3, wherein the PCR reagent comprises a PCR reaction solution and high-fidelity Taq enzyme.
5. The kit of claim 4, wherein the PCR reaction solution comprises: 0.5mM deoxynucleotide dNTP,40mM MgCl magnesium chloride 2 80mM potassium chloride KCl,60mM Tris-HCl,1mM TMAC, 0.6% v/v glycerol, 0.02% v/v cresol red and 5% v/v betaine.
6. A method for detecting HLA-DPB1 genotyping for non-disease diagnosis purposes using the primer set for detecting HLA-DPB1 genotyping according to claim 1, which comprises an amplification reaction and sequencing.
7. The method of claim 6, wherein the amplification reaction system is: 12.4. Mu.L, including 6. Mu.L of PCR reaction solution, 0.4. Mu.L of enzyme, 3. Mu.L of amplification primer mixture and 3. Mu.L of DNA template; the PCR amplification reaction is carried out for 2 minutes at 95 ℃;93 ℃ for 10 seconds, 68 ℃ for 5 minutes, 30 cycles; 72℃for 5 min, 4℃until removal.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160060695A1 (en) * | 2013-05-09 | 2016-03-03 | Genodive Pharma Inc. | Method and kit for multiplex dna typing of hla gene |
| US20170029885A1 (en) * | 2013-11-27 | 2017-02-02 | Genodive Pharma Inc. | Simple Method and Kit for DNA Typing of HLA Genes by High-Throughput Massively Parallel Sequencer |
| US20170029886A1 (en) * | 2014-02-18 | 2017-02-02 | Etablissement Francais Du Sang | High-resolution hla typing |
| CN108588205A (en) * | 2018-04-13 | 2018-09-28 | 北京诺诗康瀛基因技术股份有限公司 | A kind of primer sets, kit and method for HLA-DRB1 High Resolution Gene Typings |
| US20190367983A1 (en) * | 2017-02-13 | 2019-12-05 | Kyoto University | Pcr primer set for hla gene, and sequencing method using same |
| CN111057706A (en) * | 2019-10-09 | 2020-04-24 | 西安市中心血站（陕西省血液中心） | Group of primers and detection method for HLA-DPB1 gene sequencing and typing |
-
2023
- 2023-11-21 CN CN202311551794.6A patent/CN117512085B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160060695A1 (en) * | 2013-05-09 | 2016-03-03 | Genodive Pharma Inc. | Method and kit for multiplex dna typing of hla gene |
| US20170029885A1 (en) * | 2013-11-27 | 2017-02-02 | Genodive Pharma Inc. | Simple Method and Kit for DNA Typing of HLA Genes by High-Throughput Massively Parallel Sequencer |
| US20170029886A1 (en) * | 2014-02-18 | 2017-02-02 | Etablissement Francais Du Sang | High-resolution hla typing |
| US20190367983A1 (en) * | 2017-02-13 | 2019-12-05 | Kyoto University | Pcr primer set for hla gene, and sequencing method using same |
| CN108588205A (en) * | 2018-04-13 | 2018-09-28 | 北京诺诗康瀛基因技术股份有限公司 | A kind of primer sets, kit and method for HLA-DRB1 High Resolution Gene Typings |
| CN111057706A (en) * | 2019-10-09 | 2020-04-24 | 西安市中心血站（陕西省血液中心） | Group of primers and detection method for HLA-DPB1 gene sequencing and typing |
Non-Patent Citations (4)
| Title |
|---|
| HIDENOBU SEGAWA ET AL.: "HLA genotyping by next-generation sequencing of complementary DNA", BMC GENOMICS, vol. 18, no. 1, 28 November 2017 (2017-11-28), pages 1 - 12, XP021250910, DOI: 10.1186/s12864-017-4300-7 * |
| YUKI OZAKI ET AL.: "Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing", BMC GENOMICS, vol. 16, no. 1, 18 April 2015 (2015-04-18), pages 1 - 12, XP021216791, DOI: 10.1186/s12864-015-1514-4 * |
| 赵霄晨 等: "下一代测序在白细胞抗原基因分型中的应用", 解放军医药杂志, vol. 29, no. 7, 28 July 2017 (2017-07-28), pages 14 - 17 * |
| 鲍自谦 等: "HLA-DPA1和-DPB1基因第2、第3外显子同步测序分型的研究", 临床血液学杂志, vol. 31, no. 12, 7 December 2018 (2018-12-07), pages 929 - 932 * |
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