CN117265090A - Primer set and kit for detecting HLA-DQA1 genotyping of human leukocyte antigen - Google Patents
Primer set and kit for detecting HLA-DQA1 genotyping of human leukocyte antigen Download PDFInfo
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- 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
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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- C—CHEMISTRY; METALLURGY
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to a primer group and a kit for detecting HLA-DQA1 genotyping of a human leukocyte antigen, belonging to the field of biomedical clinical molecular detection. The primer set for detecting HLA-DQA1 gene typing of the human leukocyte antigen comprises a specific PCR amplification primer set, wherein the specific PCR amplification primer set comprises 4 pairs of primers designed according to HLA-DQA1 gene subtype specific sequences; the 4 pairs of primers are used for amplifying all subtypes of DQA1 x 01, DQA1 x 02, DQA1 x 03 and DQA1 x 04/DQA1 x 05/DQA1 x 06 respectively. The primer group also comprises 5 DQA1 universal sequencing primers. The invention has the following technical effects: the HLA-DQA1 specific amplification primer designed by adopting the ARMS combined double mutation base method has high specificity, and can completely distinguish all subtypes of the HLA-DQA1 gene by matching with the DQA1 universal sequencing primer. The kit can accurately judge HLA-DQA1 genotyping of the experimental sample. 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-DQA1 genotyping of a human leukocyte antigen, 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 HLA-DQA1 genotyping is rapidly and accurately detected, and has important significance for clinical disease auxiliary diagnosis, medical research, disease etiology research and the like. The current commonly used methods for detecting the genes mainly comprise a PCR-SSP method, a SYBR Green I method, a Taqman fluorescent quantitative PCR method, a sequencing method and the like. 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. Thus, only sequencing methods can obtain high-resolution typing results, but specific amplification primers are needed to be designed for PCR amplification, and the accuracy of the typing results is affected due to the fact that HLA polymorphism is very high and false positive is easy to occur. There is an urgent need in the art for a solution for detecting HLA-DQA1 genotyping that is simple and rapid to operate and has high accuracy.
In addition, the Chinese patent application with the application number of 202210661530.5, which is named as a primer group for HLA-DQA1 genotyping and an analysis method, discloses a primer group for HLA-DQA1 genotyping, which is used for amplifying the DQA1 gene, and constructing a library after the amplified product is broken, and carrying out second-generation sequencing. The method uses a second generation sequencing method, a group of primers are designed to amplify the DQA1 genes, the genes are not typed, the amplified products are randomly broken to construct a library, and the second generation sequencing is performed without specific sequencing primers. Similarly, chinese patent application No. 201610589111.X, entitled DNA typing method and kit for HLA genes, discloses an upstream primer and a downstream primer for amplification of each locus of HLA, but not subtype. The amplified products were sequenced directly without specific sequencing primers. The typing methods of the two patents do not amplify the genes of each subtype of DQA1, and have poor specificity and can generate ambiguities and multiple combinations.
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-DQA1 genotyping of a human leukocyte antigen.
The primer set design principle of the invention is as follows: according to the known human leukocyte antigen gene sequence, an amplification inhibition mutation system (Amplification refractory mutation system, ARMS) analysis method is combined with a double mutation base method, and HLA-DQA1 subtype specific amplification primers are designed. 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 for further sequencing analysis to identify the sequences of the individual alleles, thereby achieving high resolution genotyping of HLA-DQA 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 an ARMS combined double mutation base primer design method, firstly finds out the specific mutation base of each subtype of the DQA1 gene in a database, wherein the upstream mutation base is positioned at the tail end of the No. 1 intron, and the downstream mutation base is positioned at the front end of the 3' UTR region, so that the specific fragments of each subtype can be amplified in the first step. And according to the preliminary detection result, other variant bases are introduced on each primer, so that the specificity of the primers 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-DQA1 genotyping of human leukocyte antigen, said primer set comprising a specific PCR amplification primer set comprising 4 pairs of primers designed according to HLA-DQA1 gene subtype specific sequences; the 4 pairs of primers are respectively used for amplifying all subtypes of DQA1 x 01, DQA1 x 02, DQA1 x 03 and DQA1 x 04/DQA1 x 05/DQA1 x 06;
the nucleotide sequences of the 4 pairs of specific PCR amplification primer groups are shown in the following table:
the PCR amplification primer group is designed into a specific primer according to an optimized ARMS analysis method, and mismatched bases are introduced into different positions of the primer, so that the detection specificity is improved;
the primer group for detecting HLA-DQA1 genotyping also comprises 5 DQA1 universal sequencing primers.
The 5 DQA1 universal sequencing primers, each primer nucleotide sequence is shown in the following table:
| sequencing region | SEQ ID NO. | Primer probe sequence 5 '. Fwdarw.3' |
| E2 | #09 | CATCATTTTGTGTATTAAGGTT |
| E2 | #10 | ATGGAAAGACCCTTGTATTAC |
| E3 | #11 | AGGTAAATAAGACCTCTTTGAC |
| E3 | #12 | TGAAGTGTGGAAAACAAGTT |
| E4 | #13 | TGAGTCTTTGCAGAGCCAAC |
。
In a second aspect of the invention, there is provided the use of a primer set for detecting HLA-DQA1 genotyping of human leukocyte antigens as described in the first aspect in the preparation of a kit for detecting HLA-DQA1 genotyping.
In a third aspect of the present invention, there is provided a kit comprising the primer set for detecting HLA-DQA1 genotyping of human leukocyte antigen 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-DQA1 genotyping for non-disease diagnostic purposes using the primer set for detecting HLA-DQA1 genotyping for human leukocyte antigens 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 12.2 mu L, including 6 mu L of PCR reaction solution, 0.2 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 5 minutes at 95 ℃;93 ℃ for 30 seconds, 60 ℃ for 40 seconds, 72 ℃ for 2 minutes and 30 seconds, 36 cycles; 72℃for 5 min, 4℃until removal.
The invention has the following technical effects:
1) Compared with the existing 202210661530.5, the existing method uses a second-generation sequencing method, a group of primers are designed to amplify the DQA1 genes, the DQA1 genes are not typed with each other, subtype specific primers are designed to specifically amplify each subtype in the first step, and the method can reduce the combination of the ambiguous alleles; in addition, the prior method breaks the amplified products randomly to construct a library, performs second generation sequencing, does not need specific sequencing primers, designs the specific sequencing primers of the DQA No. 12/No. 3/No. 4 exons, performs specific first generation sequencing on the amplified products, and improves the accuracy of parting results. The specific amplification primer designed by adopting the ARMS combined double mutation base primer design method has high specificity, and can completely distinguish all subtypes of HLA-DQA1 genes by matching with the universal sequencing primer.
2) The kit containing the specific amplification primer and the universal sequencing primer can accurately judge HLA-DQA1 genotyping of the experimental sample. Can be used for detecting HLA-DQA1 genotyping of human leukocyte antigens.
Drawings
FIG. 1 is an electrophoretogram of an amplification product using the specific amplification primers of the present invention. The amplified product has completely correct negative and positive, good specificity and no impurity band.
FIG. 2 is an electrophoretogram of amplification products using a common mismatch-free group-specific amplification primer. The amplified products have more nonspecific amplification, and part of samples have incorrect negative positivity.
FIG. 3-1 shows Sample 1 sequencing, genotype DQA 1.times.01:02, DQA 1.times.02:01. The two amplification products (DQA 1 x 01 and DQA1 x 02) gave good results with correct interpretation of the sequencing peak patterns.
FIG. 3-2 shows Sample 2 sequencing, genotype DQA 1.times.01:02, DQA 1.times.03:03. The two amplification products (DQA 1 x 01 set and DQA1 x 03 set) gave good results with correct interpretation of the sequencing peak patterns.
FIGS. 3-3 are Sample 3 sequencing charts, genotype DQA 1.times.03:03, DQA 1.times.05:05. The two amplification products (DQA 1 x 03 and DQA1 x 04, 05, 06) gave good peak patterns and were correctly interpreted.
FIGS. 3-4 are Sample 4 sequencing charts, genotype DQA 1.times.04:01, DQA 1.times.05:05. One amplified product (DQA 1 x 04, x 05, x 06 groups) gave good results with correct interpretation.
FIGS. 3-5 are Sample 5 sequencing charts, genotype DQA 1.times.06:01, DQA 1.times.01:03. The two amplification products (DQA 1 x 01 and DQA1 x 04, 05, 06) gave good results with correct interpretation.
FIGS. 3-6 are Sample 6 sequencing charts, genotype DQA 1.times.02:01, DQA 1.times.05:01. The two amplification products (DQA 1 x 02 and DQA1 x 04, 05, 06) gave good peak patterns and were correctly interpreted.
FIGS. 3-7 are Sample 7 sequencing charts, genotype DQA 1.times.06:01, DQA 1.times.03:03. The two amplification products (DQA 1 x 03 and DQA1 x 04, 05, 06) gave good peak patterns and were correctly interpreted.
FIGS. 3-8 are Sample 8 sequencing charts, genotype DQA 1.times.04:01, DQA 1.times.01:02. The two amplification products (DQA 1 x 01 and DQA1 x 04, 05, 06) gave good results with correct interpretation.
FIGS. 4-1 and 4-2 are schematic illustrations of the sequencing of the amplification products of a conventional mismatch-free group-specific amplification primer. The sequencing map has more miscellaneous peaks, and the result cannot be interpreted.
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 reagent composition:
1) The specific amplification primers of the present invention comprise 4 pairs of primers designed based on HLA-DQA1 gene subtype specific sequences. The 4 pairs of primers are respectively used for amplifying all subtypes of DQA1 x 01, DQA1 x 02, DQA1 x 03 and DQA1 x 04/DQA1 x 05/DQA1 x 06, and the sequences are SEQ ID NO: #01 to #08; the universal sequencing primer comprises 5 DQA1 universal sequencing primers, and the sequence of the universal sequencing primer is SEQ ID NO: #09 to #13.
The nucleotide sequences of the 4 pairs of specific PCR amplification primer groups are shown in the following table:
the nucleotide sequences of the sequencing primers are shown in the following table:
| sequencing region | SEQ ID NO. | Primer probe sequence 5 '. Fwdarw.3' |
| E2 | #09 | CATCATTTTGTGTATTAAGGTT |
| E2 | #10 | ATGGAAAGACCCTTGTATTAC |
| E3 | #11 | AGGTAAATAAGACCTCTTTGAC |
| E3 | #12 | TGAAGTGTGGAAAACAAGTT |
| E4 | #13 | TGAGTCTTTGCAGAGCCAAC |
2) Common amplification primers, wherein DQA1 x 01, DQA1 x 02 and DQA1 x 03 are specific amplification primers of mismatch free double base mutant groups, DQA1 x 04/05/06 are specific amplification primers of mismatch single base mutant groups, and the sequences are as follows:
3) PCR reaction reagent
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) 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.
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.
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).
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
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 | 0.2 |
| Nucleic acid sample | 3 |
| Total volume of | 12.2 |
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.
2.4PCR 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. The electrophoresis diagram is shown in fig. 1.
3.2 common non-mismatch group specific amplification primers, the specificity of the amplified products is poor, the number of the mixed bands is large, part of samples are wrong in negative and positive types, and an electrophoresis chart is shown in 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-DQA1 genotyping of the experimental sample can be accurately judged, and the sequencing diagrams are shown in figures 3-1 to 3-8.
3.4 general mismatch-free group-specific amplification primers, whose amplification products have a disordered or no signal sequencing pattern, cannot read HLA-DQA1 genotype, and typical patterns are shown in FIGS. 4-1 and 4-2, for example.
Conclusion: the specificity amplification primer provided by the invention has high specificity, and can completely distinguish all subtypes of HLA-DQA1 genes by matching with the universal sequencing primer provided by the invention. The kit can accurately judge HLA-DQA1 genotyping of the experimental sample.
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-DQA1 genotyping of a human leukocyte antigen, characterized in that the primer set comprises a specific PCR amplification primer set comprising 4 pairs of primers designed according to HLA-DQA1 subtype specific sequences; the 4 pairs of primers are respectively used for amplifying all subtypes of DQA1 x 01, DQA1 x 02, DQA1 x 03 and DQA1 x 04/DQA1 x 05/DQA1 x 06;
the nucleotide sequences of the 4 pairs of specific PCR amplification primer groups are shown in the following table:
the PCR amplification primer group is designed according to an amplification inhibition mutation system (Amplification refractory mutation system, ARMS) combined double mutation base method;
the primer group for detecting HLA-DQA1 genotyping of the human leukocyte antigen also comprises 5 DQA1 universal sequencing primers; the 5 DQA1 universal sequencing primers, each primer nucleotide sequence is shown in the following table:
2. the use of the primer set for detecting HLA-DQA1 genotyping of human leukocyte antigen as claimed in claim 1 in the preparation of a kit for detecting HLA-DQA1 genotyping.
3. A kit comprising the primer set for detecting HLA-DQA1 genotyping of human leukocyte antigen according to claim 1, wherein said kit further comprises PCR reagents.
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 3, wherein the PCR reaction solution comprises: 0.5mM deoxynucleotide dNTP,40mM MgCl magnesium chloride 2 80mM potassium chloride KCl,60mM trimethylammoniumMethyl chloride Tris-HCl,1mM tetramethyl ammonium chloride TMAC, glycerol 0.6% v/v, cresol red 0.02% v/v and betaine 5% v/v.
6. A method for detecting HLA-DQA1 genotyping for non-disease diagnosis purposes using the primer set for detecting HLA-DQA1 genotyping for human leukocyte antigens according to claim 1, wherein the method comprises an amplification reaction and sequencing.
7. The method according to claim 1, wherein the amplification reaction system is as follows: the total volume is 12.2 mu L, including 6 mu L of PCR reaction solution, 0.2 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 5 minutes at 95 ℃;93 ℃ for 30 seconds, 60 ℃ for 40 seconds, 72 ℃ for 2 minutes and 30 seconds, 36 cycles; 72℃for 5 min, 4℃until removal.
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