Primers for one or more HLA genes

Abstract

The present invention provides one or more primers capable of amplifying or detecting, in a highly comprehensive manner, HLA alleles of HLA-E, HLA-F, or HLA-G gene. More specifically, the present invention provides one or more HLA gene primers selected from the group consisting of: (1) one or more HLA-G gene primers which are (1a) a first primer including a base sequence indicated by SEQ ID NO. 1, and the like and / or (1b) a second primer including a base sequence indicated by SEQ ID NO. 3, and the like; (2) one or more HLA-E gene primers which are (2a) a first primer including a base sequence indicated by SEQ ID NO. 5, and the like, and / or (2b) a second primer including a base sequence indicated by SEQ ID NO. 7, and the like; and (3) one or more HLA-F gene primers which are (3a) a first primer including a base sequence indicated by SEQ ID NO. 9 and the like, and / or (3b) a second primer including a base sequence indicated by SEQ ID NO. 11, and the like.

Description

Technical Field

[0001] The present invention relates to primers for one or more HLA genes, etc.

Background Art

[0002] Human leukocyte antigen (HLA) is an important molecule that controls immune responsiveness and is associated with various disease susceptibilities. HLA is roughly classified into class I molecules and class II molecules. HLA class I molecules function not only as antigen peptide presentation to T cells but also as ligands for immune receptors on natural killer cells, T cells, and myeloid cells.

[0003] There are two groups of HLA class I molecules: classical (class Ia) molecules and non-classical (class Ib) molecules. HLA-A, HLA-B, and HLA-C, which are HLA class Ia molecules, are expressed in almost all human cells. On the other hand, for HLA-E, HLA-F, and HLA-G, which are HLA class Ib molecules, the distribution of tissues in which they are expressed is often limited, and their functions are diverse and not limited to antigen peptide presentation. For example, the HLA-G molecule is mainly expressed in the placenta and presents antigen peptides like HLA class Ia molecules, but the receptor that receives the presentation is the receptor of natural killer cells, and it regulates the function of natural killer cells. Natural killer cells are extremely important in cancer immunotherapy.

[0004] The HLA class I gene cluster has high sequence homology with each other and is arranged in tandem on the short arm of chromosome 6. While very high polymorphism is recognized in the HLA class Ia gene, the polymorphism of the HLA class Ib gene is relatively scarce. On the other hand, since the entire HLA gene region is highly polymorphic, the intergenic region is also rich in polymorphism compared to general genomic regions. Therefore, it is difficult to design PCR primers that can amplify the full length of HLA alleles comprehensively using only the human genome reference sequence (genome assembly version hg38).

[0005] HLA alleles are defined by combinations of polymorphisms in the full length of the gene. Sanger sequencers and next-generation sequencers cannot sequence the entire gene at once because the determinable sequence length is short. In particular, HLA class Ib genes have relatively little polymorphism, so the probability of polymorphic sites being present in the determined sequence is low, making it impossible to phase (phasing) maternal and paternal HLA alleles (Non-Patent Literature 1). Furthermore, there is no established HLA allele typing method for HLA class Ib genes, and no kits are commercially available, so there are few reported cases regarding typing results. Therefore, the HLA allele information for HLA class Ib genes registered in the IMGT / HLA database is limited.

[0006] Nilsson et al. have reported a single primer set capable of amplifying the entire HLA-G gene, including the untranslated region (Non-Patent Document 2).

[0007] Alizadeh et al. have reported a set of long-range PCR primers targeting the HLA-F gene, but the primers are designed for the untranslated region (Non-Patent Literature 3).

[0008] Wang et al. have reported a long-range PCR primer set targeting the HLA-E gene, but it is not possible to amplify the entire HLA-E gene, including the untranslated region (Non-Patent Literature 4). Lucas et al. have also reported sequencing results of the entire HLA-E gene using long-range PCR, but their primers are designed for the untranslated region, and therefore cannot amplify the region including the untranslated region that affects gene expression (Non-Patent Literature 5). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Suzuki S et al.,Front.Immunol.2018 Oct 4;9:2294. [Non-Patent Document 2] Nilsson LL et al.,HLA.2018;92:144-153. [Non-Patent Document 3] Alizadeh M et al.,Hum Immunol.2020;81(5):202-205. [Non-Patent Document 4] Wang SX et al.,HLA.2017;89:327-330. [Non-Patent Document 5] Lucas JAM et al.,HLA.2020;95:561-572. [Overview of the project] [Problems that the invention aims to solve]

[0010] No primers capable of amplifying the entire HLA-E and HLA-F gene, including the untranslated region, have been reported. Regarding the HLA-G gene primers disclosed in the prior art, as shown in the examples, analysis using whole-genome sequencing data revealed that mismatches exist in the primer binding sites in some samples, suggesting a high possibility that PCR amplification may not occur or that uniform amplification may be affected. Furthermore, prior art has analyzed the sequence of HLA class Ib genes using PCR with a pair of primers designed to focus on the coding region of the HLA class Ib gene. However, this method cannot analyze polymorphism in the untranslated region, which is important for gene expression.

[0011] Therefore, an object of the present invention is to provide one or more primers capable of detecting or amplifying HLA alleles of HLA-E, HLA-F, or HLA-G genes with a high degree of comprehensive coverage. Another object of the present invention is to provide one or more primers capable of detecting or amplifying HLA alleles of the above genes with a high degree of comprehensive coverage, and capable of amplifying the entire gene including the untranslated region of the above genes. [Means for solving the problem]

[0012] As a result of diligent research, the inventors have discovered one or more HLA gene primers that can detect or amplify HLA alleles of HLA-E, HLA-F, or HLA-G genes with a high degree of comprehensiveness, and moreover, can amplify the entire gene including the untranslated region of the above genes. In fact, it has been confirmed that the one or more HLA gene primers discovered by the inventors, unlike the prior art, can amplify a large number of HLA allele groups, including HLA alleles that are presumed to be novel, with a high degree of comprehensiveness (Table 6). Based on these findings, the inventors have succeeded in providing one or more HLA gene primers, thereby completing the present invention.

[0013] In other words, the present invention is as follows: [1] One or more HLA gene primers selected from the group consisting of (1) to (3) below: (1) One or more primers for HLA-G genes, A primer comprising one or more HLA-G gene primers, wherein (1a) a first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, and / or (1b) a second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence; (2) One or more primers for HLA-E genes, A primer comprising one or more HLA-E gene primers, wherein (2a) a first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence, and / or (2b) a second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence; and (3) One or more primers for the HLA-F gene, A primer comprising one or more HLA-F gene primers, wherein (3a) a first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence, and / or (3b) a second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence. [2] The one or more HLA gene primers in [1] are an HLA gene amplification primer set selected from the group consisting of (1') to (3') below: (1') A primer set for HLA-G gene amplification, A primer set for HLA-G gene amplification comprising (1a) a first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, and (1b) a second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence; (2') A primer for amplifying the HLA-E gene, A primer set comprising (2a) a first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence, and (2b) a second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence; and (3') A primer for amplifying the HLA-F gene, A primer set for HLA-F gene amplification, comprising (3a) a first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence, and (3b) a second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence. [3] (1a) The first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence is (1a') the first primer containing the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence, (1b) A second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence is (1b') a second primer containing the nucleotide sequence of SEQ ID NO: 4 or its complementary nucleotide sequence. (2a) The first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence is (2a') the first primer containing the nucleotide sequence of SEQ ID NO: 6 or its complementary nucleotide sequence, (2b) A second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence is (2b') a second primer containing the nucleotide sequence of SEQ ID NO: 8 or its complementary nucleotide sequence. (3a) The first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence is the first primer containing the nucleotide sequence of SEQ ID NO: 10 or its complementary nucleotide sequence, (3b) The second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence is the second primer containing the nucleotide sequence of SEQ ID NO: 12 or its complementary nucleotide sequence, a primer for one or more HLA genes of [1] or [2]. [4] A method for detecting HLA genes, including detecting one or more HLA genes in a sample obtained from a human subject using one or more primers for HLA genes of any one of [1] to [3], where the one or more HLA genes are selected from the group consisting of HLA-G gene, HLA-E gene, and HLA-F gene. [5] The method of [4], wherein the detection of one or more HLA genes is performed by amplification of the one or more HLA genes. [6] A detection reagent for HLA genes, comprising one or more primers for HLA genes of any one of [1] to [3]. [7] A detection kit for HLA genes, comprising the following (a) and (b): (a) One or more primers for HLA genes of any one of [1] to [3]; and (b) Polymerase. [Advantages of the Invention]

[0014] According to the present invention, it is possible to highly comprehensively detect or amplify HLA alleles of HLA-E, HLA-F or HLA-G genes. Further, according to the present invention, it is possible to amplify the entire gene length including the untranslated region of HLA-E, HLA-F or HLA-G genes. [Brief Description of the Drawings]

[0015] [Figure 1]Figure 1 shows the results of PCR amplification of HLA-E, HLA-F, and HLA-G genes. L: Molecular weight marker; D: Genomic DNA extracted from a cell line established from an African population (Daudi) used as a template; A: Genomic DNA extracted from a cell line established from a Japanese population (AKIBA) used as a template; L7: Genomic DNA extracted from human peripheral blood mononuclear cells of Hispanic American origin used as a template; N: No genomic DNA used (negative control). [Modes for carrying out the invention]

[0016] The present invention provides one or more HLA gene primers selected from the group consisting of (1) to (3) below. (1) One or more primers for HLA-G genes: (1a) A first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, and / or (1b) A second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence; (2) One or more primers for HLA-E genes: (2a) A first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence, and / or (2b) A second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence; and (3) One or more primers for the HLA-F gene: (3a) A first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence, and / or (3b) A second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence.

[0017] The one or more primers of the present invention are not particularly limited, as long as they can anneal to the target site of an HLA gene selected from the group consisting of HLA-G, HLA-E, and HLA-F genes. For example, any primers including natural nucleic acids or artificial nucleic acids can be used as the one or more primers of the present invention, but DNA primers are preferred from the viewpoint of versatility and cost.

[0018] In one embodiment, one or more primers of the present invention may be primers for a single HLA gene. For example, such one or more primers of the present invention are useful as sequencing primers or targets for RNA-inducible nucleases (e.g., Cas9) because they offer excellent comprehensive coverage of HLA alleles.

[0019] When one or more primers of the present invention are sequencing primers or targets of RNA-inducible nucleases, from the viewpoint of targeting regions within HLA genes, one of the primers is the following primer. (1) Primers for one type of HLA-G gene: (1a) A first primer containing the nucleotide sequence of SEQ ID NO: 1, or (1b) A second primer containing the nucleotide sequence of SEQ ID NO: 3; (2) Primers for one type of HLA-E gene: (2a) A first primer containing the nucleotide sequence of SEQ ID NO: 5, or (2b) A second primer containing the nucleotide sequence of SEQ ID NO: 7; and (3) One type of primer for the HLA-F gene: (3a) A first primer containing the nucleotide sequence of SEQ ID NO: 9, or (3b) A second primer containing the nucleotide sequence of SEQ ID NO: 11.

[0020] Furthermore, if one or more primers of the present invention are sequencing primers or targets of RNA-inducible nucleases, from the viewpoint of targeting untranslated regions upstream of genes that are particularly important for gene expression, one of the primers may be (1a) a first primer containing the nucleotide sequence of SEQ ID NO: 1, (2a) a first primer containing the nucleotide sequence of SEQ ID NO: 5, or (3a) a first primer containing the nucleotide sequence of SEQ ID NO: 9.

[0021] In another embodiment, the one or more primers of the present invention may be a primer set comprising two or more primers. For example, such a primer set is useful as a primer set for gene amplification because it has excellent comprehensiveness of HLA alleles.

[0022] If the present invention provides a primer set containing two or more primers, it may also be the following primer set containing at least one specific primer that is excellent in comprehensive coverage of HLA alleles. (1) A primer set containing two or more HLA-G gene primers: (1a) A primer set comprising a first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, and (1b) a second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence, or A primer set comprising (1a) a first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, or (1b) a second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence, and another primer capable of amplifying the HLA-G gene in combination with the selected primer; (2) A primer set containing two or more HLA-E gene primers: (2a) A primer set comprising a first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence, and (2b) a second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence, or or A primer set comprising (2a) a first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence, or (2b) a second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence, and another primer capable of amplifying the HLA-E gene in combination with the selected primer; or (3) Two or more primers for the HLA-F gene: (3a) A primer set comprising a first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence, and (3b) a second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence, or A primer set comprising (3a) a first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence, or (3b) a second primer selected from the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence, and another primer capable of amplifying the HLA-F gene in combination with the selected primer.

[0023] The number of primers required for gene amplification varies depending on the type of gene amplification method. For example, PCR requires two primers for gene amplification. On the other hand, LAMP (Loop-mediated isothermal amplification) (see, e.g., International Publication No. 00 / 28082) requires four or six primers for gene amplification. Therefore, if amplification by a gene amplification method requiring more than two primers is intended, the primer set may include additional primers.

[0024] Preferably, the one or more primers of the present invention may be the following primer set, which includes two specific primers. (1') Primer set for HLA-G gene amplification: (1a) A primer set comprising a first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, and (1b) a second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence; (2') Primer set for HLA-E gene amplification: (2a) A primer set comprising a first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence, and (2b) a second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence; or (3') Primer set for HLA-F gene amplification: A primer set comprising (3a) a first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence, and (3b) a second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence.

[0025] The primer sets described in (1') to (3') above use two specific primers. These two specific primers are designed to produce large amplification products of 4500 bp or larger (see example). Therefore, the primer sets described in (1') to (3') above are preferably used in gene amplification methods that can easily produce large amplification products. Examples of such gene amplification methods include PCR, SDA (Strand Displacement Amplification), Hybrid Capture, LCR (Ligase Chain Reaction), and Cleavase Invader.

[0026] When the present invention provides a primer set containing one or more primers, it is possible to amplify the HLA alleles of the target gene more comprehensively than with a primer set containing only one specific primer, and moreover, it is possible to amplify the entire gene, including the untranslated region of the gene.

[0027] Primers containing sequence numbers 1, 3, 5, 7, 9, or 11, or their complementary nucleotide sequences, may have additional bases added to their 5' or 3' ends. For example, such bases may be complementary bases to the corresponding bases at the target site. The number of complementary bases is not particularly limited and may be, for example, 1 to 10, preferably 1 to 5. When such bases are added to the primer, annealing to the target site can be more stable, improving detection or amplification efficiency. However, primers containing sequence numbers 1, 3, 5, 7, 9, or 11, or their complementary nucleotide sequences, can anneal to the target site well on their own, so additional bases do not need to be added to their 5' or 3' ends. Also, the primers may be chemically modified (e.g., modified with amino groups) and may have linkers added.

[0028] In certain embodiments, the first and second primers, which have additional bases added to their 5' or 3' ends, may be as follows: (1a) The first primer containing the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence may be (1a') the first primer containing the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence. (1b) The second primer containing the nucleotide sequence of SEQ ID NO: 3 or its complementary nucleotide sequence may be (1b') the second primer containing the nucleotide sequence of SEQ ID NO: 4 or its complementary nucleotide sequence. (2a) The first primer containing the nucleotide sequence of SEQ ID NO: 5 or its complementary nucleotide sequence may be (2a') the first primer containing the nucleotide sequence of SEQ ID NO: 6 or its complementary nucleotide sequence. (2b) The second primer containing the nucleotide sequence of SEQ ID NO: 7 or its complementary nucleotide sequence may be (2b') the second primer containing the nucleotide sequence of SEQ ID NO: 8 or its complementary nucleotide sequence. (3a) The first primer containing the nucleotide sequence of SEQ ID NO: 9 or its complementary nucleotide sequence may be (3a') the first primer containing the nucleotide sequence of SEQ ID NO: 10 or its complementary nucleotide sequence. (3b) The second primer containing the nucleotide sequence of SEQ ID NO: 11 or its complementary nucleotide sequence may be (3b') the second primer containing the nucleotide sequence of SEQ ID NO: 12 or its complementary nucleotide sequence.

[0029] The following is a summary of the nucleotide sequences of sequence numbers 1-12 (see Table 5 for details). [Table 1]

[0030] Primers containing sequence numbers 2, 4, 6, 8, 10, or 12, or their complementary nucleotide sequences, may have additional bases added to their 5' or 3' ends. For example, such bases may be complementary bases to the bases at the target site. The number of complementary bases is not particularly limited and may be, for example, 1 to 10, preferably 1 to 5. When such bases are added to the primer, annealing to the target site can be more stable, improving detection or amplification efficiency. However, primers containing sequence numbers 2, 4, 6, 8, 10, or 12, or their complementary nucleotide sequences, can anneal to the target site very well on their own, so additional bases do not need to be added to their 5' or 3' ends. Also, the primers may be chemically modified (e.g., modified with amino groups) and may have linkers added.

[0031] Primers containing sequence numbers 1-12, or their complementary nucleotide sequences, may have a functional moiety (e.g., a non-complementary nucleotide sequence such as a barcode sequence) added to their 5' end to enable identification. Examples of such functional moieties include Universal Sequences (PacBio) and Adapter Overhang Nucleotide Sequences (Illumina).

[0032] The primers used in the present invention, as described above, can be designed to have a specific number of bases (nucleotide length). Such a number of bases may be, for example, 15 or more, preferably 16 or more, more preferably 17 or more, even more preferably 18 or more, and particularly preferably 20 or more. Such a number of bases may also be, for example, 50 or less, preferably 45 or less, more preferably 40 or less, even more preferably 35 or less, and particularly preferably 30 or less. More specifically, such a number of bases may be, for example, 15 to 50, preferably 16 to 45, more preferably 17 to 40, even more preferably 18 to 35, and particularly preferably 20 to 30.

[0033] The present invention also provides a method for detecting HLA genes. The method of the present invention includes detecting one or more HLA genes selected from the group consisting of HLA-G genes, HLA-E genes, and HLA-F genes in a sample obtained from a human subject using one or more primers of the present invention.

[0034] Any sample containing the target of one or more primers of the present invention can be used as a sample obtained from a human subject. Therefore, the sample obtained from a human subject is not particularly limited as long as it contains the genome and / or a transcript of the target HLA gene, and examples include samples taken from humans and samples prepared from samples taken from humans (e.g., cell samples). Preferably, a genome-containing sample is used as the sample. As a genome-containing sample, a minimally invasive sample is preferred. Examples of such samples include hair, saliva, blood (e.g., whole blood, plasma, or serum), and mucous membranes (e.g., oral mucosa, nasal mucosa).

[0035] For the detection of HLA genes in a sample, genomic DNA or HLA gene transcripts may be extracted from the sample. Such extraction can be performed by any method. The extracted genomic DNA or HLA gene transcripts can be subjected to an appropriate gene amplification method (e.g., PCR or LAMP) depending on the size of the intended amplification product. Alternatively, the sample may be subjected directly to a gene amplification method (e.g., direct PCR).

[0036] The method of the present invention is useful, for example, for HLA testing for transplantation, selection of cancer drugs / therapies (e.g., cancer vaccines), or assessment of disease risk.

[0037] The present invention also provides an HLA gene detection reagent comprising one or more primers of the present invention. The reagent of the present invention allows for the convenient execution of the method of the present invention.

[0038] The reagent of the present invention may contain one or more primers of the present invention in the form of a powder (e.g., lyophilized) or a solution. The solution is preferably an aqueous solution. Examples of aqueous solutions include water (e.g., sterile distilled water) and buffer solutions. Examples of buffer solutions include TE (Tris-EDTA) buffer, hydrochloric acid-potassium chloride buffer, glycine-hydrochloric acid buffer, citrate buffer, acetate buffer, citrate-phosphate buffer, phosphate buffer, Tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer, carbonic acid-bicarbonate buffer, borate buffer, and tartaric acid buffer. When the reagent of the present invention is a solution containing one or more primers of the present invention, the concentration of the primer in the solution varies depending on factors such as the application of the primer and the dilution ratio when using the primer, but may be, for example, 0.1 to 100 mM, preferably 1 to 10 mM. The solution may contain other components such as stabilizers.

[0039] The present invention also provides an HLA gene detection kit comprising (a) one or more primers of the present invention, and (b) a polymerase. The kit of the present invention allows for the convenient execution of the method of the present invention.

[0040] As the polymerase, an appropriate polymerase can be used depending on the type of gene amplification method. For example, for PCR, the use of a heat-stable polymerase is preferable, and for LAMP, the use of a strand-displacement polymerase is preferable. DNA polymerase is preferred as the polymerase.

[0041] The kit of the present invention may include, in addition to (a) and (b), further components. Such components include a deoxynucleoside triphosphate (dNTP) mixture, a reaction buffer, a molecular weight marker, and a control (e.g., a standard of amplified products of various HLA alleles). If the kit of the present invention includes further components, each component may be provided in a form isolated from the others, for example, in different containers (e.g., tubes), or in a pre-mixed form (e.g., PreMix).

[0042] In certain embodiments, the detection may be performed in real time (e.g., real-time PCR). In this case, examples of methods enabling real-time detection include the intercalator method and the fluorescent substance-labeled probe method. Therefore, when real-time detection is intended, the kit of the present invention may further contain a fluorescent substance or a fluorescent substance-labeled probe as an additional component. Examples of the fluorescent substance include fluorescent substances used in the intercalator method (e.g., SYBR (registered trademark) Green I). Examples of the fluorescent substance-labeled probe include a probe in which a fluorescent substance is bound to one of the 5'-end or 3'-end and a quencher is bound to the other of the 5'-end or 3'-end (e.g., TaqMan (registered trademark) probe).

Examples

[0043] The present invention will be described in detail by the following examples, but the present invention is not limited to the following examples.

[0044] The primer design procedure and evaluation for performing gene-specific PCR on three HLA class Ib genes (HLA-E, HLA-F, HLA-G) will be described.

[0045] <Primer Design> By running the PCR primer design tool Primer3 on the regions stored in the human genomic reference sequence (genome assembly version hg38) and Alternate sequences (chr6_GL000250v2_alt, chr6_GL000251v2_alt, chr6_GL000252v2_alt, chr6_GL000253v2_alt, chr6_GL000254v2_alt, chr6_GL000²55v2_alt, chr6_GL000256v2_alt, chr6_GL383533v1_alt), one set of primers for each gene was designed (HLA-G_v1, HLA-E_v1, HLA-F_v1).

[0046] <Confirmation of HLA Allele Coverage> We confirmed in silico, using publicly available whole-genome sequencing (WGS) data, whether the designed primer set could comprehensively amplify HLA alleles via PCR.

[0047] We downloaded WGS data from 145 publicly available HipSci Resources from the European Nucleotide Archive (ENA, https: / / www.ebi.ac.uk / ena, study PRJEB15299) and created a FASTQ file containing only reads that may originate from HLA genes or their vicinity using BWA 0.7.17 and samtools 1.10.

[0048] HLA allele calling was performed on the FASTQ files of each created sample using HLAHD 1.2.1 (Tables 2, 3, and 4).

[0049] [Table 2]

[0050] [Table 3]

[0051] [Table 4]

[0052] For each sample's FASTQ file, consensus sequences of the HLA class Ib gene and its neighboring sequences were created using freebayes 1.3.1, whatshap 1.0, and bcftools 1.10.2, and then correlated with the HLA allele information for each sample.

[0053] The binding site that serves as the core sequence of the designed primers was evaluated using Bowtie2 2.3.5.1 to determine if it exists in the consensus sequences of all 145 samples in the HipSci Resource, and it was confirmed that it exists in the consensus sequences.

[0054] <Comparison of HLA Allele Coverage of HLA-G Gene Primers> Regarding the known HLA-G gene primers (Non-Patent Document 2), the coverage for the consensus sequences of 145 samples in the HipSci Resource was evaluated using Bowtie2 2.3.5.1. As a result, among 145 samples (290 alleles), mismatches were found in 14 alleles, suggesting that PCR amplification may not be observed or that it is highly likely to affect uniform amplification. In contrast, for both the forward primer and reverse primer of the designed primer set HLA-G_v1, no mismatches were found in all 290 alleles, confirming that the allele coverage is very high.

[0055] <Confirmation of Specific PCR Amplification by Experiment> To experimentally confirm whether the primer sets shown in Table 5 can specifically amplify by PCR, PCR reactions were performed using genomic DNA extracted from cell lines established from African populations (Daudi: D), cell lines established from Japanese populations (AKIBA: A), and human peripheral blood mononuclear cells (L7) from Hispanic Americans as templates, with each HLA class Ib gene-specific primer set. Nuclease-free Water (N) was used as a non-template control.

[0056]

Table 5

[0057] For the PCR reaction, a total of 25 μL of solution was prepared using PrimeSTAR GXL Premix (Takara Bio Inc.): 12.5 μL of PrimeSTAR GXL Premix (2X), 2.5 μL of forward primer (2 μM), 2.5 μL of reverse primer (2 μM), 5 μL of nuclease-free water, and 2.5 μL of genomic DNA solution (5 ng). This solution was incubated at 94°C for 1 minute, followed by two steps: 98°C for 10 seconds and 68°C for 8 minutes. This process was repeated 30 times. A Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific Inc.) was used for this PCR.

[0058] Next, after the PCR reaction, the PCR amplification product was purified using Sera-Mag Select (Cytiva) and eluted with 25 μL of Buffer EB (QIAGEN).

[0059] The purified PCR amplification product was subjected to electrophoresis using the Genomic DNA ScreenTape System (Agilent Technologies, Inc.) to confirm the status of PCR amplification (Figure 1).

[0060] <Sequencing using a long-read sequencer> The PCR amplification products were sequenced using the Sequel system (Pacific Biosciences of California, Inc.).

[0061] PCR amplification was performed using the primer sets listed in Table 5. The SMRTbell Express Template Prep Kit 2.0 was used, and the instrument settings, reaction conditions, and reagent addition amounts basically followed the Procedure & Checklist Part Number 101-791-700 version 02 provided by PacBio.

[0062] HLA allele typing was performed on the consensus sequences obtained using the Sequel system, with the IMGT / HLA database as the reference sequence.

[0063] We performed analyses using genomic DNA extracted from cell lines established from African populations (Daudi:D), cell lines established from Japanese populations (AKIBA:A), and six human peripheral blood mononuclear cells from Hispanic Americans (L7, L222, L255, L275, L340, L365) as templates.

[0064] As a result, it was confirmed that the primer set selected in this study can highly comprehensively amplify a large number of HLA allele groups, including those presumed to be novel (Table 6).

[0065] [Table 6]

[0066] <Identifying the core sequence> To identify the core sequences for forward or reverse primers, we shortened the 5' and 3' ends of the designed primers by one base each and checked for perfect match against the human genome reference sequence (genome assembly version hg38). This allowed us to identify core sequences that uniquely matched the upstream and downstream of the target gene (sequences shown in bold and underlined in Table 5). For these core sequences, we confirmed that the binding sites of all 145 primers in the HipSci Resource uniquely and perfectly matched the consensus sequences of the target genes.

Claims

1. A primer set for HLA-G gene amplification, which is (1-1) or (1-2) below: (1-1) A primer set comprising (1a) a first primer containing the nucleotide sequence of SEQ ID NO: 1, and (1b) a second primer containing the nucleotide sequence of SEQ ID NO: 3; or (1-2) A primer set comprising (1c) a first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1, and (1d) a second primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO:

3.

2. (1a) The first primer containing the nucleotide sequence of SEQ ID NO: 1 is (1a') the first primer containing the nucleotide sequence of SEQ ID NO: 2, (1b) The second primer containing the nucleotide sequence of SEQ ID NO: 3 is (1b') the second primer containing the nucleotide sequence of SEQ ID NO: 4, (1c) The first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1 is (1c') the first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2, and The HLA-G gene amplification primer set according to claim 1, wherein the second primer containing the complementary nucleotide sequence of the nucleotide sequence of (1d) SEQ ID NO: 3 is the second primer containing the complementary nucleotide sequence of the nucleotide sequence of (1d') SEQ ID NO:

4.

3. A primer set for HLA-E gene amplification, which is (2-1) or (2-2) below: (2-1) A primer set comprising (2a) a first primer containing the nucleotide sequence of SEQ ID NO: 5, and (2b) a second primer containing the nucleotide sequence of SEQ ID NO: 7; or (2-2) A primer set comprising (2c) a first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 5, and (2d) a second primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO:

7.

4. (2a) The first primer containing the nucleotide sequence of SEQ ID NO: 5 is (2a') the first primer containing the nucleotide sequence of SEQ ID NO: 6 (2b) The second primer containing the nucleotide sequence of SEQ ID NO: 7 is (2b') the second primer containing the nucleotide sequence of SEQ ID NO:

8. (2c) The first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 5 is (2c') the first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 6, and The HLA-E gene amplification primer set according to claim 3, wherein the second primer containing the complementary nucleotide sequence of the nucleotide sequence of (2d) SEQ ID NO: 7 is the second primer containing the complementary nucleotide sequence of the nucleotide sequence of (2d') SEQ ID NO:

8.

5. A primer set for HLA-F gene amplification, which is (3-1) or (3-2) below: (3-1) A primer set comprising (3a) a first primer containing the nucleotide sequence of SEQ ID NO: 9, and (3b) a second primer containing the nucleotide sequence of SEQ ID NO: 11; or (3-2) A primer set comprising (3c) a first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 9, and (3d) a second primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO:

11.

6. (3a) The first primer containing the nucleotide sequence of SEQ ID NO: 9 is (3a') the first primer containing the nucleotide sequence of SEQ ID NO: 10, (3b) The second primer containing the nucleotide sequence of SEQ ID NO: 11 is (3b') the second primer containing the nucleotide sequence of SEQ ID NO: 12, (3c) The first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 9 is (3c') the first primer containing the complementary nucleotide sequence of the nucleotide sequence of SEQ ID NO: 10, and The HLA-F gene amplification primer set according to claim 5, wherein the second primer containing the complementary nucleotide sequence of the nucleotide sequence of (3d) SEQ ID NO: 11 is the second primer containing the complementary nucleotide sequence of the nucleotide sequence of (3d') SEQ ID NO:

12.

7. A method for detecting the HLA gene, This method includes detecting one or more HLA genes in a sample obtained from a human subject using one or more primer sets described in any one of claims 1 to 6, A method for selecting one or more HLA genes from a group consisting of HLA-G genes, HLA-E genes, and HLA-F genes.

8. The method according to claim 7, wherein the detection of one or more HLA genes is performed by amplification of one or more HLA genes.

9. A reagent for detecting HLA genes, comprising one or more primer sets according to any one of claims 1 to 6.

10. HLA gene detection kits including (a) and (b) below: (a) One or more primer sets according to any one of claims 1 to 6; and (b) Polymerase.

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