Kit for detecting expression typing and expression quantity of human HLA-I / II type genes

Abstract

The utility model discloses a kit for detecting human HLA-I / II type gene expression typing and expression quantity, which comprises a kit body and a flip type kit cover rotationally connected with the kit body, a paper support is arranged in the kit body, and the paper support divides the interior of the kit body into an upper layer and a lower layer; more than twenty reagent tube holes are formed in the paper support and are divided into three areas, one area is a reverse transcription reagent area used for containing reverse transcription reagent tubes for cDNA synthesis, and the other area is a reverse transcription reagent area used for containing reverse transcription reagent tubes for cDNA synthesis; the second region is an enrichment reagent region and is used for accommodating HLA enrichment amplification related reagent tubes, namely an HLA Target Mix1 reagent tube for capturing HLA-I / II type genes, an HLA Target Mix2 reagent tube for capturing HLA-I / II type genes and a PCR (Polymerase Chain Reaction) reagent for capturing HLA-I / II type genes; and the third region is a library building reagent region and is used for containing a reagent for constructing the DNA library after targeted capture. According to the kit and the use method thereof, the immune state of cells can be evaluated more truly; the method is simple, effective and stable in process.

Description

Technical Field

[0001] This utility model relates to a kit for detecting the expression genotype and expression level of human HLA class I / II genes, belonging to the field of kits. Background Technology

[0002] Sequencing has become a routine experimental technique. The significant advantages of recent generation sequencing technologies include: massively parallel signature sequencing (MPSS), high-throughput sequencing, polymerase cloning (Polony Sequencing), 454 pyrosequencing, Illumina (Solexa) Sequencing, ABI SOLiD Sequencing, Ion semiconductor sequencing, and DNA nanoball sequencing.

[0003] The Major Histocompatibility Complex (MHC) is a collective term for a group of genes encoding major histocompatibility antigens in animals. In 1958, Dausset et al. discovered that different specific leukocyte antibodies existed in the serum of patients who had received multiple blood transfusions, multiparous women, and volunteers immunized with allogeneic leukocytes. These antibodies were used to identify many different specific leukocyte antigens, termed Human Leukocyte Antigens (HLA). Through pedigree and population genetic analysis, it was found that the human MHC is located on chromosome 6 and is called the HLA complex. MHC can be divided into two categories: classical MHC and non-classical MHC. Classical MHC includes MHC I, MHC II, and MHC III genes, encoding MHC I, MHC II, and MHC III molecules, respectively.

[0004] Current research on MHC primarily focuses on class I and class II bulk MHC. However, a review of relevant kits reveals only those capable of detecting specific portions of MHC, such as exons 2-4 of class I A gene, exons 2-4 of class I B gene, and exon 2 of the DRB1 gene, as described in patent CN101892317B; and the full lengths of class I A, B, and C genes, as described in patent CN105039332B. Extensive literature indicates significant polymorphism in HLA class I genes, showing polymorphism in exons 1-7; class II genes are concentrated in exons 2-4 of six genes: DRB1, DRA1, DPA1, DPB1, DQA1, and DQB1. Currently, no kits capable of detecting such a broad range of bulk MHC transcripts have been found. Utility Model Content

[0005] The purpose of this invention is to provide a kit for detecting the expression genotype and expression level of human HLA class I / II genes.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by this utility model is as follows:

[0007] A kit for detecting the expression genotyping and expression level of human HLA class I / II genes includes a box body and a flip-top lid rotatably connected to the box body. The box body contains a paper tray that divides the box body into upper and lower layers. The paper tray has a reverse transcription reagent area, an enrichment reagent area, and a library construction reagent area. The reverse transcription reagent area has more than 4 test tube wells, the enrichment reagent area has more than 6 test tube wells, and the library construction reagent area has more than 17 test tube wells. The reverse transcription reagent section contains wells for reverse transcription of cDNA, including reverse transcriptase tubes, TSO primer tubes, Poly d(T) primer tubes, and reverse transcription buffer tubes. The enrichment reagent section contains wells for HLA-I / II gene capture: HLA Target Mix 1 and HLA Target Mix 2, 2X Multiplex PCR Master Mix, deionized ultrapure water, and purified magnetic beads. The library preparation reagent section contains wells for fragmentation and end-repair enzymes and buffers, 1X TE buffer, ligation adapters, ligase, ligation buffer, and 2X PCR Mix, Universal Primer, Sample Index Primer, deionized ultrapure water, and purified magnetic beads. All tubes are capped.

[0008] All the test tubes listed above are not overlapped; each test tube corresponds to one test tube. The following are listed: Reverse Transcriptase Test Tube (containing reverse transcriptase), TSO Primer Test Tube (containing TSO primers), Poly d(T) Primer Test Tube (containing Poly d(T) primers), Reverse Transcription Buffer Test Tube (containing reverse transcription buffer), HLA Target Mix 1 Test Tube (containing HLA Target Mix 1 for HLA-I / II gene capture), HLA Target Mix 2 Test Tube (containing HLA Target Mix 2 for HLA-I / II gene capture), 2X Multiplex PCR Master Mix Test Tube (containing 2X Multiplex PCR Master Mix), Fragmentation and End Repair Enzyme Test Tube (containing fragmentation and end repair enzymes), Fragmentation and End Repair Buffer Test Tube (containing fragmentation and end repair buffer), 1X TE Buffer Test Tube (containing 1X TE buffer). Buffer test tube; ligation adapter test tube refers to a test tube containing a ligation adapter; ligase test tube refers to a test tube containing ligase; ligation buffer test tube refers to a test tube containing a ligation buffer; 2X PCR Mix test tube refers to a test tube containing 2X PCR Mix; Universal Primer test tube refers to a test tube containing a Universal Primer; Sample Index Primer test tube refers to a test tube containing a Sample Index Primer; Deionized ultrapure water test tube refers to a test tube containing deionized ultrapure water; Purified magnetic bead test tube refers to a test tube containing purified magnetic beads.

[0009] To save materials, reduce volume, and meet daily needs, all test tubes are 1.5mL tubes.

[0010] To save materials, reduce size, and facilitate use, the reagent kit has a cubic structure with a length of 20±1cm, a width of 15±1cm, and a height of 12±1cm.

[0011] To simplify the structure, save costs, and ensure the stability of the device, the paper tray includes a support surface and support feet arranged around the bottom of the support surface. The support feet are located on the bottom surface of the box. The reverse transcription reagent area, enrichment reagent area, and library preparation reagent area are located on the support surface.

[0012] In this application, terms such as "top," "bottom," and "upper" all refer to the relative positions of the device during normal use.

[0013] As another improvement of this application, the paper tray includes an upper support surface, a lower support surface, and a support ring. The support ring connects the upper and lower support surfaces into a single structure. The lower support surface is a sponge layer and contacts the bottom surface of the inner casing. The reverse transcription reagent area, enrichment reagent area, and library preparation reagent area are located on the upper support surface. This provides better protection for each test tube.

[0014] For ease of use and better protection of reagents, the paper tray has 27-30 test tube wells. The inner surface of each well has a first elastic layer, and all wells have a diameter of 9mm. Any extra wells are for future use.

[0015] For ease of differentiation and operation, preferably, except for ionized ultrapure water and purified magnetic beads, all reagent tubes in the reverse transcription reagent area have blue caps, all reagent tubes in the enrichment reagent area have yellow caps, all reagent tubes in the library construction reagent area have green caps, the deionized ultrapure water test tubes have colorless caps on the top, and the purified magnetic bead test tubes have red caps on the top.

[0016] Further optimization involves including one tube of reverse transcriptase, one tube of TSO primers, one tube of Poly d(T) primers, and one tube of reverse transcription buffer in the reverse transcription reagent area. All tubes are equipped with caps.

[0017] Further optimization involves the following wells in the enrichment reagent area: one HLATarget Mix 1 tube for HLA-I / II gene capture, one HLA Target Mix 2 tube for HLA-I / II gene capture, one 2X Multiplex PCR Master Mix tube, two deionized ultrapure water tubes, and one purified magnetic bead tube. All tubes are equipped with caps.

[0018] Further optimization involves the following wells in the library preparation reagent area: one tube for fragmentation and end-repair enzyme, one tube for fragmentation and end-repair buffer, one tube for 1X TE buffer, one tube for ligation adapter, one tube for ligase, one tube for ligation buffer, one tube for 2X PCR Mix, one tube for Universal Primer, six tubes for Sample Index Primer, two tubes for deionized ultrapure water, and one tube for purified magnetic beads. All tubes are equipped with caps.

[0019] Any technologies not mentioned in this utility model are based on existing technologies.

[0020] This kit for detecting HLA class I / II gene expression typing and levels can more accurately assess the immune status of cells; the method is simple, effective, and the procedure is stable. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the kit for detecting human HLA class I / II gene expression typing and expression level according to this invention;

[0022] Figure 2 This is a schematic diagram of the paper tray structure of the kit for detecting human HLA class I / II gene expression typing and expression level according to this utility model;

[0023] In the diagram: 1. Box body; 2. Box lid; 3. Paper tray; 3-1. Reverse transcriptase tube; 3-2. TSO primer tube; 3-3. Poly d(T) primer tube; 3-4. Reverse transcription buffer tube; 3-5. HLATarget Mix 1 tube for HLA-I / II gene capture; 3-6. HLA Target Mix 2 tube for HLA-I / II gene capture; 3-7. 2X Multiplex PCR Master Mix tube; 3-8. Deionized ultrapure water tube; 3-9. Purification magnetic bead tube; 3-10. Fragmentation and end-repair enzyme tube; 3-11. Fragmentation and end-repair buffer tube; 3-12. 1X TE Buffer tube; 3-13. Ligator tube; 3-14. Ligase tube; 3-15. Ligation buffer tube; 3-16. 2X PCR Mix for library amplification. Test tubes; 3-17, Universal Primer test tube; 3-18, Sample Index Primer test tube; 3-19, Deionized Ultrapure Water test tube; 3-20, Purified Magnetic Beads test tube.

[0024] Figure 3 Electrophoresis image of total RNA extracted from frozen peripheral blood PBMC samples from patients;

[0025] Figure 4 Peak shape after HLA purification by second targeted enrichment magnetic beads;

[0026] Figure 5 HLA next-generation sequencing library peak shape diagram;

[0027] Figure 6 Statistical chart of HLA class I / II gene expression typing detection results using different library construction methods for the same sample;

[0028] Figure 7 Statistical graph of expression genotyping and expression levels of HLA class I / II genes constructed using different methods for the same sample;

[0029] Figure 8Comparison of typing results after enrichment and library construction of intact RNA and degraded RNA from the same sample. Detailed Implementation

[0030] To better understand this utility model, the following embodiments further illustrate the content of this utility model, but the content of this utility model is not limited to the following embodiments.

[0031] Example 1

[0032] As shown in the figure, a kit for detecting the expression genotyping and expression level of human HLA class I / II genes is presented. The kit has a cubic structure, with a length of 20±1cm, a width of 15±1cm, and a height of 12±1cm. The kit includes a box body and a flip-top lid rotatably connected to the box body. Inside the box body is a paper tray that divides the box body into upper and lower layers. The paper tray includes a support surface and support feet arranged around the bottom perimeter of the support surface, which are located on the bottom surface of the box body. The support surface has a reverse transcription reagent area, an enrichment reagent area, and a library construction reagent area. The reverse transcription reagent area has more than 4 test tube wells, the enrichment reagent area has more than 6 test tube wells, and the library construction reagent area has more than 17 test tube wells. The inner surface of the test tube wells is provided with a second elastic layer, and the pore diameter of each test tube well is 9mm.

[0033] The reverse transcription reagent section contains one tube of reverse transcriptase, one tube of TSO primers, one tube of Poly d(T) primers, and one tube of reverse transcription buffer. The enrichment reagent section contains one tube of HLA Target Mix 1 for HLA-I / II gene capture, one tube of HLA Target Mix 2 for HLA-I / II gene capture, one tube of 2X Multiplex PCR Master Mix, two tubes of deionized ultrapure water, and one tube of purified magnetic beads. The library preparation reagent section contains one tube of fragmentation and end-repair enzyme, one tube of fragmentation and end-repair buffer, one tube of 1X TE buffer, one tube of ligation adapter, one tube of ligase, one tube of ligation buffer, and one tube of 2X PCR Mix, one Universal Primer, six Sample Index Primers, two tubes of deionized ultrapure water, and one tube of purified magnetic beads required for library amplification.

[0034] All test tubes are equipped with caps. The remaining wells are reserved for future kit upgrades and to accommodate additional reagent wells. All test tubes are 1.5 mL. Different colors are used to distinguish different reagents on the caps for user convenience. Except for the ultrapure water and purified magnetic beads, all reverse transcription reagent tubes have blue caps, all enrichment reagent tubes have yellow caps, and all library preparation reagent tubes have green caps. The deionized ultrapure water tubes have colorless caps, and the purified magnetic bead tubes have red caps.

[0035] Example 2

[0036] The paper tray is basically the same as in Example 1, except that: the paper tray includes an upper support surface, a lower support surface and a support ring, the support ring connects the upper support surface and the lower support surface into a whole structure, the lower support surface is a sponge layer, the lower support surface is in contact with the bottom surface of the synchrotron, and the reverse transcription reagent area, the enrichment reagent area and the library preparation reagent area are located on the upper support surface.

[0037] Primer information for this utility model

[0038] Table 1. Primer information required for the first targeted enrichment

[0039]

[0040] Table 2. Primer information required for the second targeted enrichment

[0041]

[0042] TSO primers consist of a known oligo and one or more bases at its 3' end for template conversion. The known oligo is usually a sequence commonly used by those skilled in the art, such as the Illumina sequencing platform Read1 sequence: 5'-CTACACGACGCTCTTCCGATCT-3'. The one or more bases at the 3' end for template conversion are usually 3-5 Gs in the form of deoxyribonucleic acid or 3-5 Gs in the form of ribonucleic acid. Between the known oligo and the bases at the 3' end for template conversion, a base sequence of any length, such as UMI, can also be added.

[0043] Poly d(T) primers consist of the Oligo d(T)10-29 sequence and a known sequence at its 5' end. This known sequence is usually a sequence commonly used by those skilled in the art, such as the Illumina sequencing platform Read2 sequence: 5'-AAGCAGTGGTATCAACGCAGAGTAC-3'.

[0044] Application Example 1. Obtaining Total Human HLA RNA

[0045] Total RNA was obtained from samples using the QIAGEN® RNeasy Mini Kit. Sample sources could include fresh human blood, fresh tissue blocks obtained surgically or by puncture, human cell lines cultured in the laboratory, or even frozen peripheral blood PBMCs. The obtained total RNA was tested for integrity using Agilent Tapestation Screen Tape and Reagents, and its concentration and purity were determined using Nanodrop. An electrophoresis image of total RNA extracted from a patient's frozen peripheral blood PBMC sample is shown below. Figure 3 As shown, the detection concentration was 42 ng / ul, and the purity was OD260 / 280=2.0, indicating that the RNA quality was good and the integrity was very high.

[0046] Application Example 2. Obtaining the first strand of cDNA via reverse transcription

[0047] Reverse transcription was performed using TransScript® Uni Reverse Transcriptase (catalog number: AU101) according to the instructions. During reverse transcription, a universal sequence was added to the 5' end of the cDNA via a template switch. The reverse transcriptase is preferably a template switch-enabled reverse transcriptase, all of which achieve the desired effect of this invention. More preferably, TransScript® IV Reverse Transcriptase, TransScript® II Reverse Transcriptase, Vazyme HiScript® Reverse Transcriptase, Vazyme HiScript® II Reverse Transcriptase, Vazyme HiScript® III Reverse Transcriptase, Vazyme M-MLV(H-) Reverse Transcriptase, ABclonal® ABScript II Reverse Transcriptase, ABclonal® ABScript III Reverse Transcriptase, Yeasen Hifair® V Reverse Transcriptase, or Invitrogen SuperScript® IV Reverse Transcriptase are used.

[0048] Application Example 3. For RNA samples with good integrity, a universal 5' upstream primer and Target Mix downstream primers are used to target and amplify HLA class I / II genes.

[0049] 1. First targeted enrichment of HLA-I / II

[0050] The first targeted enrichment was performed using the Hieff® Multiplex PCR Kit. Following the instructions, the PCR components required for the first enrichment, the reverse transcribed first-strand cDNA, the 5' universal upstream primer, and the downstream primer combination from HLA-I / II Target Mix 1 were added to the reaction tubes respectively. The first targeted enrichment was performed on the PCR instrument under the conditions shown in Table 3 below.

[0051] Table 3. Conditions for the first targeted enrichment PCR reaction

[0052]

[0053] 2. Purification of products after the first targeted enrichment of HLA-I / II

[0054] The reaction product was purified using 0.8X Agencourt AMPure XP Beads according to the instructions to remove excess oligos and PCR components (such as salt ions) to obtain a template for the second targeted enrichment.

[0055] 3. Second targeted enrichment of HLA-I / II

[0056] Add the purified first-enriched products to reaction tubes and perform a second targeted enrichment using the Hieff® Multiplex PCR Kit under the conditions shown in Table 2 below on a PCR instrument, following the manufacturer's instructions. For the second targeted enrichment, use the same 5' universal upstream primer and the downstream primer combination from HLA-I / II Target Mix 2. Perform the second targeted enrichment under the conditions shown in Table 4 below on a PCR instrument.

[0057] Table 4. Conditions for the second targeted enrichment PCR reaction

[0058]

[0059] 4. Purification of the product from both ends after the second targeted enrichment of HLA-I / II

[0060] The reaction product was purified using 0.5X and 0.8X Agencourt AMPure XP Beads according to the instructions, and fragments of the desired length were screened out.

[0061] Enriched product analysis: The peak chromatogram of the enriched product was detected using Agilent Tapestation, as shown in the schematic diagram. Figure 4As shown, the Qubit concentration was detected. The enriched product can be stored at 4°C for 72 hours or at -20°C for one week.

[0062] Application Example 4: For RNA samples with poor integrity (degraded), the upstream and downstream primer combinations in Target Mix are used to target and amplify HLA class I / II genes.

[0063] 1. First targeted enrichment of HLA-I / II

[0064] The first targeted enrichment was performed using the Hieff® Multiplex PCR Kit. Following the instructions, the PCR components required for the first enrichment, the reverse transcribed first-strand cDNA, and the upstream and downstream primer combinations from HLA-I / II Target Mix 1 were added to the reaction tubes, and the first targeted enrichment was performed on the PCR instrument under the conditions shown in Table 5 below.

[0065] Table 5. Conditions for the first targeted enrichment PCR reaction

[0066]

[0067] 2. Purification of products after the first targeted enrichment of HLA-I / II

[0068] The reaction product was purified using 0.8X Agencourt AMPure XP Beads according to the instructions to remove excess oligos and PCR components (such as salt ions) to obtain a template for the second targeted enrichment.

[0069] 3. Second targeted enrichment of HLA-I / II

[0070] Add the purified first enrichment products to reaction tubes and perform a second targeted enrichment on the PCR instrument according to the conditions in Table 2 below, using the Hieff® Multiplex PCR Kit. The second targeted enrichment uses the upstream and downstream primer combination in HLA-I / II Target Mix 2 and is performed on the PCR instrument according to the conditions in Table 6 below.

[0071] Table 6. Conditions for the second targeted enrichment PCR reaction

[0072]

[0073] 4. Purification of the product after the second targeted enrichment of HLA-I / II: The reaction product was purified using 0.5X and 0.8X AgencourtAMPure XP Beads according to the instructions, and the desired fragment length was selected.

[0074] Enriched product analysis: The peak chromatogram of the enriched product was detected using Agilent Tapestation, and the Qubit concentration was also determined. The enriched product can be stored at 4°C for 72 hours or at -20°C for one week.

[0075] Next-generation sequencing library construction and quality control for Illumina platform

[0076] Sequencing library preparation and purification

[0077] Using library construction kits suitable for the Illumina next-generation sequencing platform, such as the VAHTS Universal Plus DNALibrary Prep Kit for Illumina V2, ABclonal FS DNA Lib Prep Kit for Illumina V2, or other equivalent kits or library construction module combinations, construct next-generation sequencing libraries from the products obtained by targeted capture according to their instructions. Specific steps include...

[0078] 1. Use a disruption enzyme to break down enriched DNA under appropriate conditions, perform end repair, and add an A to the 3' end.

[0079] 2. Purify or screen fragment lengths using an appropriate concentration of Agencourt AMPure XP Beads. The concentration of magnetic beads should be selected according to the instructions based on the desired fragment size.

[0080] 3. Use ligase to ligate the adapters.

[0081] 4. Use an appropriate concentration of Agencourt AMPure XP Beads to purify the ligation product or screen for fragment length. The concentration of magnetic beads should be selected according to the instructions based on the desired fragment size.

[0082] 5. Sequencing adapters and sample indexes were added to both ends of the fragment by PCR, while the next-generation sequencing library was amplified.

[0083] 6. Use an appropriate concentration of Agencourt AMPure XP Beads to sort the reaction products by fragment length. The concentration of magnetic beads should be selected according to the instructions based on the expected fragment size to obtain the sequencing library.

[0084] After obtaining the next-generation sequencing library, qPCR is required for quantification. The library concentration is detected using Qubit, and the product fragment size is detected using Agilent Tapestation Screen Tape and Reagents. Figure 5).

[0085] Application Example 5: Comparative Analysis of HLA Results Obtained by Different Methods

[0086] 1. Preprocessing of raw FASTQ data from the computer

[0087] Based on previous bioinformatics analysis experience, the raw FastQ data were initially quality controlled using software such as FastQC, FastP, Trimmatic, and Bowtie2 to remove low-quality data and adapters, and aligned to the reference genome. The quality of the aligned data was then further controlled.

[0088] 2. HLA typing analysis

[0089] Using software such as HLA-VBseq, a matrix was constructed, and HLA typing was performed, with the typing detection results analyzed. The results are as follows: Figure 6 As can be seen from the figure, compared with the GEX library construction method of the 10X Genomics platform, the method of this patent has a higher detection rate for HLA typing of the same sample.

[0090] 3. Comparative analysis of HLA results obtained by different methods

[0091] HLA typing and expression levels obtained by different methods were compared and analyzed end-to-end. The same sample was constructed using both the GEX library construction method on the 10X Genomics platform and the library construction method of this patent. The comparison results are shown below. Figure 7 As shown in the figure, the number of read counts detected for each HLA type in the second-generation sequencing libraries constructed by the method of this patent and the 10X method are basically the same. Using another sample, second-generation sequencing libraries were constructed according to the manufacturer's instructions using both highly intact extracted RNA and degraded RNA. The detected HLA types were consistent, and the number of read counts corresponding to each HLA type was also basically the same. Figure 8 This demonstrates that the method of this patent effectively solves the problem of poor quality of extracted RNA, caused by easy degradation of the extracted RNA or poor quality of the extracted RNA due to sample quality issues (such as long-term cryopreservation), which affects HLA capture and library construction.

[0092] This invention relates to a kit for detecting the expression genotyping and expression levels of human HLA-I / II genes. The use of this kit allows for more targeted sequencing libraries, fully revealing cellular HLA polymorphism. Based on the transcriptome level of HLA-I / II cDNA, it effectively displays the HLA ensemble state during cellular expression, rather than the intracellular HLA state at the DNA level. HLA genotyping during transcription is essential to accurately reflect cellular immune status. The kit comprehensively includes genotyping of all nine HLA-I and HLA-II genes, providing a large and comprehensive sample size for a more accurate assessment of cellular immune status.

Claims

1. A kit for detecting HLA-I / II class gene expression typing and expression quantity of human, characterized in that: The kit comprises a box body and a flip cover connected with the box body, a paper support is arranged in the box body, and the box body is divided into two layers by the paper support; the paper support is provided with a reverse transcription reagent area, an enrichment reagent area and a library building reagent area; the reverse transcription reagent area is provided with four or more test tube holes, the enrichment reagent area is provided with six or more test tube holes, and the library building reagent area is provided with seventeen or more test tube holes; the reverse transcription reagent area is provided with a reverse transcription enzyme test tube, a TSO primer test tube, a Poly d(T) primer test tube and a reverse transcription buffer test tube; the test tube holes of the enrichment reagent area are provided with an HLA Target Mix1 test tube for HLA-I / II class gene capture, an HLA Target Mix2 test tube for HLA-I / II class gene capture, a 2X Multiplex PCR Master Mix test tube, a deionized ultrapure water test tube and a purification magnetic bead test tube; the test tube holes of the library building reagent area are provided with a fragmentation and end repair enzyme test tube, a fragmentation and end repair buffer test tube, a 1X TE buffer test tube, a linker test tube, a ligase test tube, a ligation buffer test tube, a 2X PCR Mix test tube required for library amplification, a Universal Primer test tube, a Sample Index Primer test tube, a deionized ultrapure water test tube and a purification magnetic bead test tube, and all the test tubes are provided with test tube covers.

2. The kit for detecting human HLA-I / II class gene expression typing and expression amount according to claim 1, characterized in that: All the test tubes are 1.5ml test tubes.

3. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized in that: The kit has a cubic structure, and the length, width and height thereof are 20±1cm, 15±1cm and 12±1cm respectively.

4. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized by: The paper support comprises a support surface and support feet arranged along the bottom of the support surface, and the support feet are arranged on the inner bottom surface of the kit body; the reverse transcription reagent area, the enrichment reagent area and the library building reagent area are arranged on the support surface.

5. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized by: The paper support comprises an upper support surface, a lower support surface and a support ring, the support ring connects the upper support surface and the lower support surface into an integrated structure, the lower support surface is a sponge layer, the lower support surface is in contact with the inner bottom surface of the kit body, and the reverse transcription reagent area, the enrichment reagent area and the library building reagent area are arranged on the upper support surface.

6. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, wherein: The paper support is provided with 27-30 test tube holes, and the diameters of all the test tube holes are 9mm.

7. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized by: Except the deionized ultrapure water test tube and the purification magnetic bead test tube, all the test tubes in the reverse transcription reagent area are provided with blue caps, all the test tubes in the enrichment reagent area are provided with yellow caps, and all the test tubes in the library building reagent area are provided with green caps; the test tube cover of the deionized ultrapure water test tube is provided with a colorless cap at the top, and the test tube cover of the purification magnetic bead test tube is provided with a red cap at the top.

8. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized by: The reverse transcription reagent area is provided with one reverse transcription enzyme test tube, one TSO primer test tube, one Poly d(T) primer test tube and one reverse transcription buffer test tube, and all the test tubes are provided with test tube covers.

9. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized by: The tube hole in the enrichment reagent area is provided with 1 tube of HLA Target Mix1 tube for HLA-I / II class gene capture, 1 tube of HLA Target Mix2 tube for HLA-I / II class gene capture, 1 tube of 2X Multiplex PCR Master Mix tube, 2 tubes of deionized ultrapure water tube and 1 tube of purified magnetic bead tube, and all the tubes are provided with a tube cap.

10. The kit for detecting the expression pattern and the expression amount of human HLA-I / II class genes according to claim 1 or 2, characterized by: The tube hole in the library building reagent area is provided with 1 tube of fragmentation and end repair enzyme tube and 1 tube of fragmentation and end repair buffer tube, 1 tube of 1X TE buffer tube, 1 tube of adapter ligation tube, 1 tube of ligase tube, 1 tube of ligation buffer tube, 1 tube of 2X PCR Mix tube required for library amplification, 1 tube of Universal Primer tube, 6 tubes of Sample Index Primer tube, 2 tubes of deionized ultrapure water tube and 1 tube of purified magnetic bead tube, and all the tubes are provided with a tube cap.

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