CN113736849B - Method for simultaneously detecting TCR and HLA genotyping and application thereof - Google Patents
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Abstract
The invention relates to a method for simultaneously detecting TCR and HLA genotyping and application thereof, belonging to the technical field of gene detection. The method comprises the following steps: sample extraction: taking a biological sample to be detected, and extracting DNA (deoxyribonucleic acid) in the biological sample; and (3) PCR amplification: taking the DNA, simultaneously adding a TCR amplification primer and an HLA amplification primer, and simultaneously amplifying a TCR target region and an HLA target region, wherein the HLA target region is the 2,3,4 exon region of an HLA gene; library construction: taking the amplification products to construct a library; sequencing on a machine: taking the library, and performing high-throughput sequencing on the machine; data analysis: and processing and analyzing the sequencing data to obtain the genotyping conditions of the TCR and the HLA. By adopting the method, HLA-I genes can be amplified and identified in a typing manner by using the HLA primer group in the same PCR reaction system under the condition of not increasing the library building step, and the typing result of the HLA is provided while TRB information is obtained, so that the health state of a sample provider is evaluated by using immune group library information.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a method for simultaneously detecting TCR and HLA genotyping and application thereof.
Background
T lymphocyte surface receptors (TCRs) are heterodimers of molecular structures that T cells specifically recognize and bind antigen peptide-MHC molecules, consisting of two distinct subunits. 95% -99% of T cell receptors are composed of alpha and beta subunits, and 1% -5% are composed of gamma and delta subunits, and the ratio varies with the development of individuals and the health state of the body.
TRB (T cell receptor β locus, T Cell Receptor Beta Locus) is the locus encoding the β peptide chain in a TCR molecule. CDR3 of TRB consists of four gene regions of V (Variable), D (Diversity), J (training) and C (Constant), each of which contains several alleles. Gene rearrangement during lymphocyte maturation forms various recombinant sequence fragments, and DNA base insertion and mutation form T cell diversity. The beta chain in the TCR has priority over the alpha chain of the TCR in an allelic exclusion manner, and can better reflect the characteristics of the T cell TCR. Therefore, the method has very important significance for the research of TRB diversity. The α and β peptide chains of TCRs are each composed of Variable (Variable), constant (Diversity) transmembrane and cytoplasmic regions, and the like. The variable region consists of three complementarity determining regions (Complementarity Determining Region, CDRs) CDR1, CDR2, CDR3, with the greatest variation in CDR3 directly determining the antigen binding specificity of the TCR.
The immune group library (Immune Repertoire) sequencing technology taking the T/B lymphocyte surface receptor as a research target can deeply evaluate the immune state of the organism and explore the relationship between the immune state of the human body and diseases. With the development of high-throughput sequencing, large-scale sequencing analysis such as immune repertoire and the like can be realized. By sequencing the CDR3 region of TRB in T cells, parameters such as TRB diversity related to human health can be obtained.
In addition, the human leukocyte antigen system HLA (human leukocyte antigen, HLA) genes are located on the short arm of human chromosome 6, the most diverse and important immune genetic system in human genome, and are the main gene system that regulates human specific immune responses and determines disease-susceptible individual differences. The diversity of HLA determines the body's resistance to infectious diseases, autoimmune diseases, genetic diseases and various tumors. HLA system plays an important role in antigen recognition, antigen presentation, immune response and regulation, destruction of foreign antigen target cells and the like, and is a main material foundation for causing immune rejection reaction.
HLA genes fall into three categories: class I, class II and class III genes, wherein class I comprises: HLA-A, HLA-B, HLA-C genes. Both class I and class II antigens on the surface of the graft cells are potent transplantation antigens, and humoral and cellular immunity are involved in rejection of the graft, whether xenogenic organs or tissues or cells are transplanted, and HLA matching between recipients is critical to success. With the development of medicine, new genetic techniques such as leukemia and thalassemia can be used for typing detection, and then suitable donors are searched for transplantation treatment.
At present, by means of HLA high-resolution parting, the peripheral blood stem cell transplantation technology can greatly improve the parting effect, and the higher the matching degree of HLA related genes of both a donor and a receptor is, the higher the separation rate is, the higher the transplantation success rate is, so that the recovery of a patient is faster and the better guarantee is provided.
In addition, HLA typing can also assist in diagnosing certain diseases and preventing serious adverse drug reactions. More than 200 diseases have been found to occur in association with HLA, including some autoimmune and infectious diseases, and more studies have found HLA and cancer susceptibility to be associated.
The diversity of HLA has close relation with the resistance of organisms to infectious diseases, autoimmune diseases, genetic diseases and various tumors, so HLA typing information can be used for assisting in diagnosing diseases and preventing serious adverse drug reactions, and becomes a basic reference basis for treating diseases and tracing the types.
HLA typing methods include serological typing methods, cytological typing methods and molecular biological typing methods. With the development of molecular biology and DNA sequencing technologies, traditional serological and cytological typing methods have gradually been replaced by molecular biology methods. Currently, the HLA molecular typing method mainly comprises the following steps: polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction-sequence specific primer (PCR-SSP), polymerase chain reaction single strand conformational polymorphism analysis (PCR-SSCP), polymerase chain reaction oligonucleotide probe hybridization (PCR-SSO), sequencing typing (PCR-SBT), and the like.
The serology method mainly analyzes the HLA type of a subject by an HLA micro lymphotoxin experiment method, separates lymphocytes from whole blood or lymphoid tissues, incubates with a micro-pore plate coated with HLA-specific antibodies, adds complement, and enables the specific antibodies which are combined with HLA antigens on the surface of the lymphocytes to activate the complement through a classical pathway, so that target cells are hydrolyzed (lymphocytotoxicity). There are many limitations to this method, such as the need for extensive screening work to determine HLA-specific antibodies, some HLA antigens are difficult to serologically identify, HLA cross-reactivity affects the accuracy of the typing results, and difficulty in determining further subtypes.
The cytological typing method is mainly through homozygous parting cells and sensitized lymphocyte experiment detection, and the basic principle of the cosmetic method is to judge the proliferation reaction of lymphocytes after identifying non-HLA antigenic determinants. The method is being phased out due to the difficult source of parting cells and complicated operation means.
PCR-FRLP (Restriction fragment length polymorphism) refers to the steps of cutting DNA into fragments with different sizes under the action of restriction enzyme, and analyzing target genes by using the difference of electrophoretic mobility and through the technologies of electrophoretic separation, blotting, probe hybridization and the like.
PCR-SSP (Sequence-specific primer) is designed as a set of allele-specific primer, and DNA fragments obtained by PCR amplification are distinguished by different molecular weights in the electrophoresis process.
In PCR-SSCP (Single strand conformational polymorphism), the PCR product is denatured into DNA single strands, and separated by gel electrophoresis, only one nucleotide is mutated, and the electrophoresis migration is dry-stir-fried, so that the PCR product can be used for polymorphism analysis.
PCR-SSO (Sequence specific oligonucleotide) was hybridized with HLA gene fragments amplified by PCR using artificially synthesized HLA type-specific oligonucleotide sequences as probes, thereby determining the HLA type.
PCR-SBT (Sequence based typing) is a method for obtaining a target sequence of HLA genes by PCR, obtaining HLA type results by sequencing analysis, and is widely used along with the development of NGS and is also a "gold standard" recommended by the world health organization at present.
In the technical scheme, the complicated operation of a serum method and a cell method is eliminated, and the gel electrophoresis separation experimental results are used as the parting basis in the process for all the PCR-FRLP, the PCR-SSP and the PCR-SSCP, so that larger uncertainty is brought, and the accuracy of HLA parting is greatly influenced. Therefore, further optimization of HLA primers to obtain accurate typing results by second generation sequencing analysis is required in SBT methods.
In addition, the conventional library construction scheme separately constructs TRB and HLA, only one of the results can be analyzed in the sequencing results, and one-step library construction sequencing and analysis are needed when the HLA is needed to assist diagnosis, so that the time waste and the cost increase are caused.
Disclosure of Invention
Based on the above, it is necessary to provide a method for simultaneously detecting TCR and HLA genotyping, by which TCR diversity and HLA information can be obtained at one time, and the HLA genotyping result and TCR immune status are combined, which is beneficial to comprehensive evaluation and provides more accurate in-place key information for subsequent adjuvant therapy.
A method for simultaneously detecting TCR and HLA genotypes comprising the steps of:
sample extraction: taking a biological sample to be detected, and extracting DNA (deoxyribonucleic acid) in the biological sample;
and (3) PCR amplification: taking the DNA, simultaneously adding a TCR amplification primer and an HLA amplification primer, and simultaneously amplifying a TCR target region and an HLA target region, wherein the HLA target region is the 2,3,4 exon region of an HLA gene;
library construction: taking the amplification products to construct a library;
sequencing on a machine: taking the library, and performing high-throughput sequencing on the machine;
data analysis: and processing and analyzing the sequencing data to obtain the genotyping conditions of the TCR and the HLA.
The present inventors have found in the prior investigation that, although there are also conventional techniques of library construction and detection schemes for TRB alone and HLA alone, library construction of TRB and HLA in the same reaction is difficult, such as interaction between multiple primers and coverage of A, B, C genes by HLA.
In HLA typing research, the total length of HLA is about 3.9kb, and the conserved introns have no typing reference value and mainly adopt exon research with rich diversity. HLA-I exons are 7 in total, and the most diversified 2,3,4 exons are mainly used in the typing process, so that the 2,3,4 exons are used as typing genes to have more accurate reference value.
On the basis, the inventor designs the amplification primer of the HLA genes to be only aimed at the 2,3,4 exons with the most abundant diversity by repeatedly fumbling and trying, reduces the target area of the HLA genes under the condition of not influencing HLA typing, shortens the amplification length of the HLA exons, and can realize the simultaneous acquisition of HLA type information in the sequencing of an immune repertoire TRB library.
In one embodiment, in the PCR amplification step, the TCR target region is the V and J regions of CDR3 in TRB;
the HLA target area is HLA-A gene No. 2-4 exon, HLA-B gene No. 2-4 exon and HLA-C gene No. 2-4 exon, and the fragment length of the target product is 200-500bp.
By selecting a specific TCR target region and an HLA target region and controlling the size of a target product of an HLA gene to be 200bp-500bp (preferably 296bp-360 bp), the multiplex PCR amplification effect is further improved, and a foundation is laid for subsequent high-quality library establishment.
In one embodiment, in the PCR amplification step, the TCR amplification primers comprise the sequences shown as SEQ ID NO.1-SEQ ID NO. 41; the HLA amplification primer comprises a sequence shown as SEQ ID NO.42-SEQ ID NO. 60.
The primer design of the present invention must include amplification of almost all HLA types to be suitable for HLA type confirmation of different people; primer binding sites should avoid non-conserved regions, and the best possible approach is to find the appropriate specific primer sites near the upstream and downstream of the A, B, C exon; because of the read length design of PE150, fewer polymorphisms should be present in the gap region that fail to obtain sequence by sequencing in the amplified product of greater than 300bp to improve HLA typing resolution. On the basis, the primer sequence is adopted, so that better typing resolution can be achieved.
In one embodiment, in the PCR amplification step, the TCR amplification primers are used in the following proportions:
the HLA amplification primers were used in the following proportions:
considering the problems of small amplification coverage range and the like possibly occurring in multiplex PCR (polymerase chain reaction) caused by the difference between sample individuals and the difference caused by the abundance of HLA-I type exons, which may cause insufficient or distorted data required by subsequent typing, the inventor adjusts the proportion of the primers through repeated experiments, and the effective data of each exon of HLA is adjusted through the proportion of the primers to reach the data volume which can be used for typing; the problems are successfully solved, the test shows that the primers for TRB library construction and the HLA library construction primers have no mutual influence, and the data size of A, B, C genes is properly distributed in the HLA library construction process, so that the wider coverage and the establishment of a diversity amplification system are realized.
In one embodiment, in the PCR amplification step, the total molar ratio of the TCR amplification primers to the HLA amplification primers is 8-12:1.
In the same DNA sequencing library, in order to ensure that the TRB primer group obtains data quantity higher than HLA and reaches HLA typing data quantity, better typing effect is achieved, the proportion of the two primer groups can be properly adjusted, and finally, the ideal data duty ratio can be obtained in the proportion of 8-12:1.
In one embodiment, the total concentration of the TCR amplification primers and the HLA amplification primers in the PCR amplification step is 0.3-0.5mM.
Because the number of different primers in the primer group in the multiplex PCR reaction system is large, the primer amount in the reaction system needs to be increased to 0.3-0.5mM in order to obtain an ideal PCR product, and the amplification effect is good.
The invention also discloses application of the method for simultaneously detecting the genotyping of the TCR and the HLA in preparation of reagents and/or equipment for the genotyping of the TCR and the HLA.
The invention also discloses a kit for simultaneously detecting the TCR and HLA genotyping, which comprises a TCR amplification primer and an HLA amplification primer for simultaneously amplifying a TCR target area and an HLA target area.
In one embodiment, the TCR target regions are the V and J regions of CDR3 in TRB; the HLA target region is HLA-A gene exon 2-4, HLA-B gene exon 2-4 and HLA-C gene exon 2-4.
The invention also discloses a system for simultaneously detecting TCR and HLA genotyping, which comprises:
the detection device is used for detecting according to the method to obtain sequencing data;
the analysis device is used for acquiring the sequencing data to perform analysis treatment and obtain TCR and HLA genotyping conditions;
and the output device is used for outputting the genotyping conditions of the TCR and the HLA.
Compared with the prior art, the invention has the following beneficial effects:
according to the method for simultaneously detecting the TCR and HLA genotyping, disclosed by the invention, the HLA-I genes are amplified by the HLA primer group in the same PCR reaction system for genotyping identification under the condition that a database building step is not added, and the HLA genotyping result is provided while TRB information is obtained, so that the method can be used as information references for disease tracing and organ transplantation, and the like, and the immune database information is obtained.
The method can acquire TCR diversity and HLA information at one time, reduce HLA typing cost, screen disease sources in the sample in combination with HLA typing results and TCR immune states, and can better perform immunodetection evaluation, thereby providing more accurate in-place key information for subsequent adjuvant therapy.
In addition, through design and experimental verification, two sets of multiple PCR primer groups which are not mutually influenced in respective target areas are obtained, the amplification of various TRB and HLA genes can be realized in one reaction by adopting a multiple PCR method, TRB and HLA libraries are quickly built through enrichment, the composition of TRB and the typing of HLA in a sample are analyzed by using an NGS sequencing technology, and the method has important significance in revealing human health and disease analysis.
Drawings
FIG. 1 is a technical scheme of example 1;
FIG. 2 is a statistical chart showing the effective data rate before adjusting the ratio of each exon primer of HLA-I gene A, B, C in example 1;
FIG. 3 is a statistical chart showing the effective data rate after the ratio of each exon primer of HLA-I gene A, B, C in example 1 is adjusted;
FIG. 4 is a graph showing the difference in preference before TRB primer scaling in example 1;
FIG. 5 is a graph showing the difference in preference after TRB primer ratio adjustment in example 1;
FIG. 6 is a gel electrophoresis chart of HLA (upper) and TRB (lower) gene fragments obtained under the same multiplex PCR reaction in example 1;
FIG. 7 is a CDR3 length distribution of the TRB gene of example 1;
FIG. 8 is a CDR3 abundance distribution of the TRB gene of example 1;
FIG. 9 is a CDR3 histogram (left) Top10 CDR3 histogram (right) of example 1;
FIG. 10 is a V-J pairing three-dimensional map of the TRB gene in example 1.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the examples below, unless otherwise specified, are all conventional.
Example 1
A method for simultaneously detecting TCR and HLA genotyping comprises the following steps:
1. sample collection
Peripheral blood (5 ml) was withdrawn using EDTA tubes, and the tubes were turned upside down several times to prevent clotting.
2. Sample extraction
Blood was taken in 2ml to EP tube, and DNA of the sample was obtained using a blood DNA extraction kit (Hipure Blood DNAMidi Kit I, D312-02, magen) and operated as described, and quantified using Q-bit for use as DNA library construction.
3. PCR amplification
1.2. Mu.g of the DNA was taken and added with both TCR and HLA amplification primers in the proportions shown in Table 1 below, and the 2,3,4 exon regions of the TCR VJ region and HLA-A, B, C were captured and enriched by multiplex PCR.
In the earlier stage work, the primer input amount is adjusted and optimized in the step, BWA comparison is carried out by the following machine data, and the proportion of the primer of each internal standard sequence before and after adjustment is determined. FIGS. 2 to 3 show examples of TRB and HLA amplification distribution before the adjustment of the primer ratio, and FIGS. 4 to 5 show examples of TRB and HLA amplification distribution after the adjustment of the primer ratio, respectively.
The amplification primers are equally added according to the mode that the molar ratio is 1, and serious amplification deviation exists in the multiplex PCR process, as shown in fig. 2, the abscissa of the graph is respectively different samples and different genes (the same depth is the same sample, namely, each group of data is sequentially from left to right, namely, samples 1 to 4), the ordinate is the effective data occupation ratio after amplification of each gene, the abscissa of the graph is respectively different samples and different genes, the ordinate is the effective data occupation ratio after amplification of each gene, and the serious amplification deviation exists as can be seen from the graph 2 to 3 according to the mode of equal addition.
By continuously optimizing and adjusting the primer system by using the internal standard sequence, the primers are added according to the optimized proportion shown in the following table 1, and finally most amplification deviation is reduced to be within one time, as shown in fig. 4-5, and the real distribution condition of the sample is reflected more accurately.
TABLE 1 TRB primer set and adjustment ratio
TABLE 2 HLA-I type A, B, C amplification primer set
Because the number of different primers in the primer group in the multiplex PCR reaction system is large, in order to obtain a more ideal PCR product, experiments prove that the primer amount in the reaction system needs to be increased to 0.4mM, the reaction system is configured according to the following table 3, the shaking and mixing are carried out uniformly, and the mixture is placed in a PCR instrument for reaction according to the following table 4 after instantaneous centrifugation.
TABLE 3 multiplex PCR reaction System
TABLE 4 multiplex PCR reaction System
4. Library construction
The amplified product was recovered by magnetic column purification, subjected to a second round of PCR, and introduced with sequencing adaptors and Index of library tags, and the PCR system was configured as shown in Table 5 below.
TABLE 5 library addition linker reaction System
Shaking and mixing uniformly, centrifuging instantaneously, and placing in a PCR instrument to operate according to the following conditions in Table 6.
TABLE 6 PCR reaction conditions with added sequencing tags
After the reaction is finished, the detection is carried out by gel electrophoresis, as shown in FIG. 6, the magnetic bead purification is carried out on a plurality of strip samples with obviously different sizes from 250bp to 500bp in the same lane, namely the sequencing library on the machine.
5. Sequencing on machine
The sequencing library NGS constructed above was run on a bench (Illumina, novaseq6000 here) with a sequencing type of PE150.
6. Data analysis
The sequencing data were analyzed for immune repertoires, processed using IMONITOR software, and analytical procedures were described in published articles (IMONITOR: A Robust Pipeline for TCR and BCR Repertoire analysis, "Genetics201 (2015). DOI:10.1534/Genetics 115.176735).
The TRB analysis processing method comprises the following steps:
taking a healthy human sample as an example for illustration, the sequencing data processed by the analysis flow is partially shown in a chart result after standard data analysis of the data by using an IMonitor, as shown in fig. 7-10, fig. 7 is a schematic diagram of CDR3 length distribution, fig. 8 is CDR3 abundance distribution, fig. 9 is a schematic diagram of CDR3 frequency distribution and a schematic diagram of Top10 CDR3 frequency distribution, and fig. 10 is a schematic diagram of TRB V-J pairing three-dimensional distribution. And combining information such as the distribution of the inserts of the Reads, the saturation curve, the distribution of the length of the CDR 3V (D) J gene, the distribution of the length of the V (D) J gene insertion, the distribution of the high-frequency mutation rate (base) of the V (D) J gene, the distribution of the usage of the V gene and the like. The diversity of the immune repertoire TRB of the sample can be reflected, and the immunity condition of the sample can be evaluated. The sample data shown in FIGS. 7-10 are healthy human blood, so the CDR3 composition of the sample is rich and the immune repertoire TRB diversity is high.
The HLA analysis and treatment method comprises the following steps:
the HLA data is firstly positioned on corresponding reference sequences (reference sequence source IMGT/HLA databases) through bwa software, homology sequences of the databases are constructed, DNA in the databases are screened and error-correcting in sequencing is carried out, finally, comparison results in the corrected DNA sequences and the databases are combined with frequency information of each exon 2,3 and 4 of A, B, C genes, and ranking results are comprehensively analyzed, so that HLA type information of the sample can be obtained.
The HLA-A, B, C type information obtained by techniques such as genome sequencing and the like before the sample is taken as the accurate target type in the experiment, namely the known type (A.times.24:02:01, B.times.35:01:01 and C.times.03:03:01/C.times.03:04:01) is taken as a control, the DNA sequence information is obtained by NGS sequencing through the same system library establishment method of TRB and HLA in the invention, and HLA typing results are shown in the following table 7.
TABLE 7 sample typing results
/>
Note that: "READS" refers to the number of READS sequenced, "RATIO" refers to the RATIO of all the typing categories, "RANK (Fre)" refers to the typing RANK (RATIO. Times. Asian category frequency), "EX2" refers to the number of exon READS 2, "EX3" refers to the number of exon READS 3, and "EX4" refers to the number of exon READS 4.
The results show that the type information of Top10 with highest sample homology is obtained by using bwa software for comparison, and the first or second type information ranked in the list is consistent with the known type information in the sample, so that the HLA primer group in the experimental system can accurately amplify target areas of 2,3,4 exons of HLA-A, B and C genes of the sample without bias and the like, HLA-I type gene sequence information is obtained by NGS sequencing, and the correct HLA type result is obtained by biological information software processing analysis.
The typing results of the sample can play an important reference role in health status and disease analysis, homologous transplantation and the like by combining TRB immune repertoire information.
Example 2
A kit for simultaneously detecting TCR and HLA genotypes comprising the TCR amplification primer and HLA amplification primer of example 1.
Example 3
A system for simultaneously detecting TCR and HLA genotyping, comprising:
a detection device for detecting according to the method described in example 1 to obtain sequencing data;
the analysis device is used for acquiring the sequencing data to perform analysis treatment and obtain TCR and HLA genotyping conditions;
and the output device is used for outputting the genotyping conditions of the TCR and the HLA.
Example 4
And (3) checking TCR and HLA genotyping methods simultaneously.
Samples obtained by whole genome sequencing to obtain known HLA genotypes were tested for typing according to the method of example 1 and compared with known results as shown in the following table.
TABLE 8 verification of comparative experiments
The results show that the method for simultaneously detecting the TCR and HLA genotyping can amplify the TCR and HLA genes in the same reaction system, detect the TCR and the HLA genes by adopting the same sequencing library, and has accurate and reliable genotyping result compared with the independent construction of HLA.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
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Claims (3)
1. A method for simultaneous detection of TCR and HLA genotyping for non-diagnostic purposes, comprising the steps of:
sample extraction: taking a biological sample to be detected, and extracting DNA (deoxyribonucleic acid) in the biological sample;
and (3) PCR amplification: taking the DNA, simultaneously adding a TCR amplification primer and an HLA amplification primer, and simultaneously amplifying a TCR target region and an HLA target region, wherein the HLA target region is the 2,3,4 exon region of an HLA gene;
library construction: taking the amplification products to construct a library;
sequencing on a machine: taking the library, and performing high-throughput sequencing on the machine;
data analysis: processing and analyzing the sequencing data to obtain TCR and HLA genotyping conditions;
in the PCR amplification step, the TCR amplification primer comprises a sequence shown as SEQ ID NO.1-SEQ ID NO. 41; the HLA amplification primer comprises a sequence shown as SEQ ID NO.42-SEQ ID NO. 60;
in the PCR amplification step, the TCR amplification primers were used in the following proportions:
the HLA amplification primers were used in the following proportions:
in the PCR amplification step, the total molar ratio of the TCR amplification primer to the HLA amplification primer is 8-12:1;
in the PCR amplification step, the total concentration of the TCR amplification primer and the HLA amplification primer is 0.3-0.5mM.
2. A kit for simultaneous detection of TCR and HLA genotyping comprising a TCR amplification primer and an HLA amplification primer for simultaneous amplification of a TCR target region and an HLA target region according to claim 1.
3. A system for simultaneously detecting TCR and HLA genotyping, comprising:
detection means for detecting according to the method of claim 1 to obtain sequencing data;
the analysis device is used for acquiring the sequencing data to perform analysis treatment and obtain TCR and HLA genotyping conditions; and the output device is used for outputting the genotyping conditions of the TCR and the HLA.
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| CN103045591A (en) * | 2013-01-05 | 2013-04-17 | 上海荻硕贝肯生物科技有限公司 | HLA gene specific PCR amplification primer, HLA typing method and kit |
| CN105506746A (en) * | 2014-09-22 | 2016-04-20 | 深圳华大基因科技有限公司 | Method for constructing variable region sequencing library, and method for determining variable region nucleic acid sequence |
| EP3060679A1 (en) * | 2013-10-25 | 2016-08-31 | BioNTech AG | Method and kit for determining whether a subject shows an immune response |
| CN112143777A (en) * | 2020-08-18 | 2020-12-29 | 北京臻知医学科技有限责任公司 | Primer group for constructing CDR3 region high-throughput sequencing library of human TCR beta and application thereof |
| CN112322716A (en) * | 2020-11-25 | 2021-02-05 | 深圳泛因医学有限公司 | Specific lymphocyte content analysis method and device based on TCR/BCR high-throughput sequencing |
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Patent Citations (5)
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|---|---|---|---|---|
| CN103045591A (en) * | 2013-01-05 | 2013-04-17 | 上海荻硕贝肯生物科技有限公司 | HLA gene specific PCR amplification primer, HLA typing method and kit |
| EP3060679A1 (en) * | 2013-10-25 | 2016-08-31 | BioNTech AG | Method and kit for determining whether a subject shows an immune response |
| CN105506746A (en) * | 2014-09-22 | 2016-04-20 | 深圳华大基因科技有限公司 | Method for constructing variable region sequencing library, and method for determining variable region nucleic acid sequence |
| CN112143777A (en) * | 2020-08-18 | 2020-12-29 | 北京臻知医学科技有限责任公司 | Primer group for constructing CDR3 region high-throughput sequencing library of human TCR beta and application thereof |
| CN112322716A (en) * | 2020-11-25 | 2021-02-05 | 深圳泛因医学有限公司 | Specific lymphocyte content analysis method and device based on TCR/BCR high-throughput sequencing |
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