Kit for multiplex SNP detection using fragment analysis and multiplex SNP detection method

Abstract

The present invention relates to a kit for multiplex SNP detection and a multiplex SNP detection method. According to the present invention, up to tens of haplotypes of sequence polymorphism sites such as a plurality of SNPs or alleles can be rapidly and accurately detected in high multiplex through a CE method after performing a single PCR amplification reaction on a plurality of spaced-apart nucleotide sequence elements using primer pairs fluorescently labeled in one direction.

Description

SNP multiplexing kit and SNP multiplexing method using fragment analysis

[0001] This patent application claims priority to Korean Patent Application No. 10-2024-0129892 filed with the Korean Intellectual Property Office on September 25, 2024, the disclosures of said patent application are incorporated herein by reference.

[0002] The present invention relates to a kit for SNP multiplex testing using fragment analysis and a method for SNP multiplex testing.

[0003]

[0004] Polymerase Chain Reaction (PCR), commonly used in the field of molecular diagnostics, is a technique that amplifies DNA into millions of identical copies using a unique heat-resistant polymerase (DNA copying enzyme) and a pair of oligonucleotide primers with sequences complementary to the target gene region. In particular, Multiplex Real-Time PCR is a method that adds more than one pair of primers and a fluorescence-labeled probe to the PCR reaction to monitor the fluorescent signal accumulated during a single PCR amplification process in real time and amplify one or more DNA fragments. While this method offers advantages such as speed, high sensitivity, and low cost for simultaneously identifying multiple gene regions, it has disadvantages such as cross-reactivity between primer sets and the limited number of fluorescence-labeled probes that can be used for multiplex analysis.

[0005] Representative analysis methods capable of multiplexing include NGS (next-generation sequencing) and microarray methods. The NGS method involves dividing the genome into countless fragments and combining their respective nucleotide sequences to decode the genome. By overcoming the limitations of conventional DNA sequencing (Sanger sequencing), it has significantly reduced the time and cost of DNA sequencing analysis and improved sequencing data production capabilities; however, it requires a large volume of samples and involves complex experimental procedures. Furthermore, the quality of genomic information decoded by NGS is degraded due to factors such as noise signals.

[0006] Microarray methods can be used to simultaneously measure the expression of many genes and genotype multiple regions of the genome. This method analyzes data by hybridizing two complementary DNA sequences (or strands), detecting fluorescence when DNA fragments attached to the surface are exposed to a fluorescently labeled target sequence, and comparing the fluorescence intensities. Since this method utilizes chips equipped with numerous SNP probes, it can process 100,000 to 500,000 SNPs simultaneously in a single reaction. In the absence of existing NGS data, designing microarray chips is difficult, and designing them to include newly discovered gene targets is costly and time-consuming.

[0007] In the commonly used electrophoresis method, target genes from DNA fragments amplified by PCR are analyzed by separating them by size using gel electrophoresis with an agarose gel. While this method has the advantage of analyzing multiple samples at a low cost, the range of detectable targets is limited.

[0008] Another method is capillary electrophoresis (CE) utilizing polyacrylamide components. This method allows for precise separation of DNA fragment sizes and enables fluorescence detection, making it suitable for automated data analysis. Methods for analyzing DNA fragments using CE can be broadly classified into two categories: Short tandem repeats (STRs) and Single nucleotide polymorphism (SNP) genotyping.

[0009] First, short tandem repeats (STRs) analysis is a method for analyzing 1 to 6 repeating nucleotide sequence elements that make up a significant portion of the human genome. It analyzes products amplified by PCR using primer pairs fluorescence-labeled in one direction and is primarily applied in forensic evidence, paternity testing, and cancer genomics research. However, it has the disadvantage that the number of STRs that can be analyzed at once is limited, and it cannot identify nucleotide sequence variations within the STR repeat unit region.

[0010] Next, SNP genotyping analysis analyzes the genotypes of SNPs that occur at a frequency of approximately 1 bp per 1,000 base pairs (bp) in the human genome. Most commonly, a fluorescently labeled ddNTP (dideoxynucleotide terminator) is used to stop the PCR reaction, converting each base to a specific color for base sequence analysis. This method is primarily applied to human physiological research, such as disease genetics and pharmacogenetics, which links base sequence variations to phenotypic changes, thereby elucidating the molecular basis of diseases. A representative example is the SNaPshot method, which analyzes the genotype of the PCR interruption product using fluorescently labeled ddNTPs via the CE method. While this method offers high accuracy and multi-analysis capabilities allowing the analysis of up to 10 gene targets, it has the disadvantages of being time-consuming due to high costs and the need for manual interpretation of genotypes.

[0011] Therefore, there is an urgent need for the development of highly specific primer designs that enable the simultaneous analysis of more SNP genotypes with a single PCR amplification reaction.

[0012]

[0013] The inventors have made diligent research efforts to develop a method for analyzing a large number of SNPs at once. As a result, the present invention was completed by identifying that multiple SNPs can be analyzed at once by designing primers for amplifying separated SNP sequences on a single nucleic acid such that the lengths of each fragment to be amplified differ, labeling the ends of the forward or reverse primers with fluorescence, and performing capillary electrophoresis.

[0014] Therefore, the objective of the present invention is to provide a kit for SNP multiplex testing using fragment analysis and a method for SNP multiplex testing.

[0015]

[0016] According to one aspect of the present invention, the present invention provides a kit for SNP multiplex testing.

[0017] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0018] The nucleotides constituting the nucleic acid molecule may be isolated from nature or produced by chemical synthesis. However, they may preferably be isolated from psychrophilic bacteria, and more preferably from Photobacterium rayognathii.

[0019] In one embodiment of the present invention, the gene to be analyzed in the SNP multiplex test kit contains two or more single nucleotide polymorphisms (SNPs) within the gene.

[0020] In one embodiment of the present invention, Wilson's disease, which is the subject of analysis of the present invention, is known to be caused by a mutation in the ATP7B gene, and mutations at six positions (R778L, A874V, T1029I, L1083F, G1186S, and N1270S) of this gene are known to be gene mutations that cause about 66.4% of the disease.

[0021] In one embodiment of the present invention, the subject of analysis of the present invention may be a single nucleotide polymorphism (SNP) of one or more genes involved in vitamin metabolism, and the analysis may be utilized for the purpose of individual nutrient metabolism efficiency and health management.

[0022] In one embodiment of the present invention, the SNP multiplex test kit comprises one or more sets of SNP multiplex test primers, each comprising a plurality of primer pairs that detect single nucleotide polymorphisms (SNPs) at different locations of a gene.

[0023] The term “primer” as used herein means an oligonucleotide that can act as an initiator of synthesis under conditions in which the synthesis of a primer extension product complementary to a nucleic acid strand (template) is induced, that is, in the presence of a polymer such as a nucleotide and DNA polymerase, and at a suitable temperature and pH.

[0024] As used herein, the term “probe” means a single-stranded nucleic acid molecule comprising a site or sites substantially complementary to a target nucleic acid sequence. The “target nucleic acid,” “target nucleic acid sequence,” or “target sequence” means a nucleic acid sequence to be detected and is annealed or hybridized with a primer or probe under hybridization, annealing, or amplification conditions.

[0025] More specifically, the probe and primer are single-stranded deoxyribonucleotide molecules. The probe or primer used in the present invention may include naturally occurring dNMP (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides. Additionally, the probe or primer may include ribonucleotides.

[0026] The primer must be long enough to prime the synthesis of the extension product in the presence of the polymer. The exact length of the primer will be determined by a number of factors, including, for example, temperature, application, and the source of the primer.

[0027] The terms “annealing” or “priming” as used in this specification mean the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, said apposition causes a polymerase to polymerize the nucleotide to form a nucleic acid molecule complementary to the template nucleic acid or a part thereof.

[0028] The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0029] As used herein, the term “hybridization” means the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization may occur between two nucleic acid strands that are completely matched or substantially matched with some mismatch. The complementarity for hybridization may depend on hybridization conditions, particularly temperature. As used herein, the terms “annealing” and “hybridization” are not different and are used interchangeably.

[0030] In one embodiment of the present invention, the amplification of the gene is carried out by PCR (polymerase chain reaction).

[0031] The above Polymerase Chain Reaction (PCR) is the most well-known nucleic acid amplification method, and many variations and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR were developed by modifying the traditional PCR procedure to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (IPCR), vectorette PCR, TAIL-PCR (thermal asymmetric interlaced PCR), and multiplex PCR have been developed for specific applications. For more details on PCR, see McPherson, MJ, and Moller, SG PCR. It is described in BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY (2000), and its teachings are incorporated herein by reference.

[0032] In a specific embodiment of the present invention, each set of primers for multiple SNP testing comprises: i) a wild-type SNP detection primer subset including a primer pair for detecting wild-type SNPs; and ii) a mutant-type SNP detection primer subset including a primer pair for detecting mutant-type SNPs located at the same gene location as the same wild-type SNP.

[0033] In one embodiment of the present invention, the i) wild type SNP detection primer subset and the ii) mutant type SNP detection primer subset are each characterized by gene amplification in different tubes.

[0034] In a specific embodiment of the present invention, the kit of the present invention enables further multiplex analysis because the primer set configuration distinguishes between wild type and mutant type tubes and performs PCR amplification separately.

[0035] The above terms “multiple detection,” “multiplex detection,” or “multiplexing detection” refer to the simultaneous detection of multiple target nucleic acid sequences in a reaction vessel (e.g., a reaction tube).

[0036] As used herein, the term "multiplex PCR" refers to the simultaneous amplification of multiple targets by a polymerase chain reaction in a reaction vessel.

[0037] Various DNA polymerases may be used in the above polymerase chain reaction, including the 'Klenow' fragment of E. coli DNA polymerase I, heat-stable DNA polymerases, and bacteriophage T7 DNA polymerases. Specifically, the polymerase is a heat-stable DNA polymerase that can be obtained from various bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).

[0038] According to one embodiment of the present invention, the kit of the present invention comprises Taq DNA polymerase or Pfu DNA polymerase.

[0039] According to another embodiment of the present invention, the kit of the present invention includes Taq DNA polymerase.

[0040] The kit of the present invention may additionally include dUTP (deoxyuridine triphosphate) and UDG (Uracil DNA Glycosilase) to block carryover or crossover contamination caused by polymerase chain reaction products. UDG recognizes and cuts uracil contained in the DNA template strand of the previous amplification product, and as the cut DNA template strand loses its function as a template strand, the amplification reaction does not occur. By using dUTP and UDG, the present invention fundamentally blocks the generation of amplification products caused by contamination of the previous amplification product, thereby excluding false positive results caused by laboratory contamination of the previous amplification product and improving the accuracy of the test.

[0041] When carrying out a polymerization reaction, it is desirable to provide the components necessary for the reaction in excess in the reaction vessel. The excess of the components necessary for the amplification reaction refers to an amount such that the amplification reaction is not substantially limited by the concentration of the components. Mg 2+ It is desirable to provide cofactors such as dATP, dCTP, dGTP, and dTTP to the reaction mixture in such a way that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changes in conditions, such as the addition of reactants.

[0042] In the present invention, annealing is performed under strict conditions that enable specific binding between the target nucleotide sequence and the primer. The strict conditions for annealing are sequence-dependent and vary depending on surrounding environmental variables.

[0043] In one embodiment of the present invention, the wild type SNP detection primer subset and the mutant type SNP detection primer subset are designed such that the size of the amplification target products of the primer pair detecting wild type SNPs and the primer pair detecting mutant type SNPs, which are located at the same corresponding gene location, are equal to each other.

[0044] In one embodiment of the present invention, for N1270S, an SNP associated with the onset of Wilson's disease, the size of the amplification target product for wild type and mutant type is 676 bp, for T1029I, the size of the amplification target product for wild type and mutant type is 547 bp, for R778L, the size of the amplification target product for wild type and mutant type is 471 bp, for A874V, the size of the amplification target product for wild type and mutant type is 342 bp, for G1186S, the size of the amplification target product for wild type and mutant type is 264 bp, and for L1083F, the size of the amplification target product for wild type and mutant type is 198 bp, but this is merely an exemplary design and is not necessarily limited thereto.

[0045] In another embodiment, an individual's vitamin metabolism ability is evaluated based on genes related to the metabolism of vitamins A, B, C, D, E, K, etc., and associated SNPs, thereby enabling the presentation of personalized nutritional design or a guide for consuming health functional foods. To this end, in one embodiment of the present invention, variations in vitamin metabolism-related genes, including the 22 types of SNPs listed in Table 1 below, are subject to analysis. However, this is merely an exemplary design and is not necessarily limited thereto.

[0046] Example of Vitamin Metabolism-Related Gene and SNP Information No. Gene SNP (rs No.) Related Vitamin Amplification Product Size (bp)1KIAA2013rs2639453B9138bp2PKD1L2rs6420424A166bp3GCrs2282679D222bp4MTHFRrs1801133B9288bp5SLC5A6rs1395B5344bp6FUT6rs3760776B123 59bp7LINC02356rs10774624B3375bp8ADCYAP1R1rs2267739B6392bp9SLC23A1rs6596473C425bp10HNF4Ars1800961B4581bp11SLC23A1rs33972313C475bp12 CYP4F2rs2108622E&K501bp13GCKRrs1260326B4443bp14NONE(NBPF3-ALPL)rs1697421B6640bp15CUBNrs1801222B12653bp16SCARB1rs11057830E668bp17SL C22A3rs7769879B3742bp18TTRrs1667255A839bp19SLC17A1rs35875210B5882bp20CTNNA2rs4852146K892bp21ZPR1rs964184E&K963bp22GCrs7041D1,016bp

[0047]

[0048] When primers are designed as above, the presence of wild-type SNPs and mutant-type SNPs can be easily confirmed simply by checking whether the target site is amplified.

[0049] In one embodiment of the present invention, the size of the product to be amplified by each primer pair is 50 to 1200 base pairs (bp), but is not limited thereto.

[0050] In a specific embodiment of the present invention, the size of the product to be amplified must be within the range of 50 to 1200 bp so that when capillary electrophoresis is performed, it is easy to determine whether amplification has occurred by detecting the size of each amplified fragment.

[0051] In one embodiment of the present invention, a fluorescent label is attached to one end of the forward primer or the reverse primer of the primer pair.

[0052] In one embodiment of the present invention, the one end may be a 5' end or a 3' end.

[0053] In one embodiment of the present invention, the fluorescent label may be any known in the art, and examples thereof are as follows:

[0054] 5-FAM TM , 6-FAM TM , TET TM , VIC, JOE TM , HEX TM , LIZ, NED TM , TAMRA TM , ROX TM , ATTO™425, Alexa Fluor ® 488, ATTO TM 488, Rhodamine Green TM -X, Yakima Yellow ® , Alexa Fluor ® 532, SUN, ATTO TM 532, MAX, TYE TM 563, Cy3 TM , Alexa Fluor ® 546, ATTO TM 550, 5-TAMRA TM , TAMRA TM , ATTO TM 565, Rhodamine Red TM , ATTO TM Rho101, TEX 615, Alexa Fluor ® 594, Texas Red ® -X, Texas Red, ATTO TM 590, Lightcycler ® 640, ATTO TM 647N, TYETM 665, Cy5 TM , Alexa Fluor ® 647, Alexa Fluor ® 660, 5' IRDye ® 700, TYE 705, Cy5.5 TM , ATTO TM 700, Dy 750, Alexa Fluor ® 750, 5' IRDye ® 800CW, 5' IRDye ® 800, Cy5.5, Cy2 TM , YOPRO TM -1, YOYO TM -1, Calcein, FITC, FluorX TM , Alexa TM , rhodamine 110, Oregon Green TM 500, Oregon Green TM 488, RiboGreen TM , Rhodamine Green TM , Rhodamine 123, Magnesium Green TM , Calcium Green TM , TO-PRO TM -1, TOTO1, BODIPY530 / 550, Dil, BODIPY TMR, BODIPY558 / 568, BODIPY564 / 570, TRITC, Magnesium Orange TM , Phycoerythrin R&B, Rhodamine Phalloidin, Calcium Orange TM , Pyronin Y, RhodamineB, Cy3.5 TM , Calcium Crimson TM , Nile Red, YO-PRO TM -3, YOYO TM -3, R-phycocyanin, CPhycocyanin, TO-PRO TM-3, TOTO3, DiD DilC(5), Thiadicarbocyanine, BiosearchBlue, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL FluorRed 610, CAL Fluor Red 635, Fluorescein, Fluorescein-C3, Pulsar 650, Quasar 570, Quasar 670, SFC-V, Chamel560, SFC-N, SFC574, Chamel610, SFC620, SFC647, Chamel670, SFC670, Chamel705 and Quasar 705.

[0055] In a specific embodiment of the present invention, the fluorescent label may be selected from the group consisting of 5-FAM, 6-FAM, ATTO-550, SUN, VIC, NED, ROX, Cy3, TET, HEX, JOE, TMR, Chamel 610, SFC-N, and PET, but is not limited thereto.

[0056] In one embodiment of the present invention, a pair of primers within a primer set is attached with the same or different types of fluorescent labels.

[0057] In one embodiment of the present invention, as shown in FIG. 6, FAM fluorescence was attached to the 5' of a forward or reverse primer to amplify all six targets (N1270S: 676 bp, T1029I: 547 bp, R778L: 471 bp, A874V: 342 bp, G1186S: 264 bp, L1083F: 198 bp), and it was confirmed that FAM fluorescence was measured in all six types of targets.

[0058] In one embodiment of the present invention, the size of the amplification target product of each primer pair included in the one primer set differs by 1 bp or more. For example, 1 to 50 bp or more, 1 to 40 bp or more, 1 to 30 bp or more, 1 to 20 bp or more, 1 to 10 bp or more, 3 to 50 bp or more, 3 to 40 bp or more, 3 to 30 bp or more, 3 to 20 bp or more, 3 to 10 bp or more, 5 to 50 bp or more, 5 to 40 bp or more, 5 to 30 bp or more, 5 to 20 bp or more, 5 to 10 bp or more, 7 to 50 bp or more, 7 to 40 bp or more, 7 to 30 bp or more, 7 to 20 bp or more, 7 to 10 bp or more, 10 to 50 bp or more, 10 to 40 bp or more, 10 to 30 bp or more, 10 to 20 bp There may be a difference of 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, or 50 bp or more, but is not limited thereto.

[0059] Since the amplification target product according to the present invention can determine whether a mutation is detected from the size of the fragment separated through gel electrophoresis or capillary electrophoresis, there must be a difference of at least 1 bp or more to easily determine whether a mutation is detected.

[0060] In one embodiment of the present invention, the positions within the gene of the amplification target products of each primer pair included in the one primer set do not overlap each other.

[0061] The primer set included in the kit of the present invention comprises primer pairs capable of detecting a plurality of SNPs, and each primer pair amplifies the target product by multiplex PCR within a single tube. Therefore, if the in-genetic locations of the target products of each primer pair performing the PCR reaction within a single tube overlap with each other, the amplification efficiency may be reduced due to interference. Accordingly, it is desirable that the in-genetic locations of the target products of each primer pair included in the single primer set do not overlap with each other.

[0062] In one embodiment of the present invention, the present invention is designed to detect SNP genotypes of up to 1,200 bp amplicons with high multiplex (tens of) in four fluorescent channels (FAM, VIC, NED, PET) using a primer pair in which a fluorescent label is attached to the 5' end of a unidirectional primer. Therefore, PCR products can be designed in various sizes up to 1,200 bp, and SNPs can be detected by attaching different fluorescent labels to products of similar sizes, so high multiplex can be achieved by using four fluorescent labels.

[0063] According to another aspect of the present invention, the present invention provides a method for testing multiple SNPs.

[0064] In one embodiment of the present invention, the SNP multiplex testing method comprises the following steps:

[0065] (a) adding a nucleic acid sample isolated from a patient to different PCR amplification tubes, each comprising i) a subset of wild-type SNP detection primers including a primer pair for detecting wild-type SNPs, and ii) a subset of mutant-type SNP detection primers including a primer pair for detecting mutant-type SNPs located at the same gene location as the wild-type SNP, and performing a PCR reaction; and

[0066] (b) a step of detecting the PCR reaction product amplified by i) a wild type SNP detection primer subset, and ii) a mutant type SNP detection primer subset.

[0067] In one embodiment of the present invention, the wild type SNP detection primer subset and the mutant type SNP detection primer subset are designed such that the size of the amplification target products of the primer pair detecting wild type SNPs and the primer pair detecting mutant type SNPs, which are located at the same corresponding gene location, are equal to each other.

[0068] In addition, in one embodiment of the present invention, the SNP detection primer subset comprises two or more primer pairs.

[0069] Accordingly, the PCR reaction of the present invention can be performed through multiplex PCR using a primer set comprising two or more primer pairs.

[0070] In one embodiment of the present invention, the PCR reaction of step (a) may be performed using additional primer subsets that detect SNPs at gene locations different from i) wild type SNP detection primer subsets and ii) mutant type SNP detection primer subsets.

[0071] In one embodiment of the present invention, by using a primer subset that detects a target different from the above primer subset, it is possible to detect a larger number of SNPs at once.

[0072] In one embodiment of the present invention, the detection of the PCR reaction product is performed through gel electrophoresis or capillary electrophoresis.

[0073] In one embodiment of the present invention, a fluorescent label is attached to one end of the forward primer or the reverse primer of the primer pair.

[0074] In one embodiment of the present invention, the size of the amplification target product of each primer pair included in the one primer set differs by 1 bp or more.

[0075] In another embodiment of the present invention, the gene locations of the amplification target products of each primer pair included in the one primer set do not overlap each other.

[0076] Since the SNP multiplex testing method according to one embodiment of the present invention is performed using the SNP multiplex testing kit according to one embodiment of the present invention described above, any content overlapping with that mentioned in relation to the SNP multiplex testing kit described above applies equally.

[0077]

[0078] The present invention provides a kit for SNP multiplex testing and a method for SNP multiplex testing. According to the present invention, after a single PCR amplification reaction of multiple nucleotide sequence elements located apart from each other using a pair of primers fluorescence-labeled in one direction, up to tens of nucleotide sequence polymorphism sites such as multiple SNPs or haplotypes of alleles can be rapidly and accurately detected with high multiplex through the CE method.

[0079]

[0080] Figures 1 and 2 show photographs of the electrophoresis results of the amplification products following the Wilson's disease Multiplex PCR (6 targets; N1270S, T1029I, R778L, A874V, G1186S, L1083F) reaction of a total of three clinical samples (HET 1, HET 3, HET 4) (Figure 1: wild type, Figure 2: mutant type).

[0081] Figures 3 and 4 show photographs of the electrophoresis results of the amplified products following the Multiplex PCR (22 target + 1 Exogenous internal control) reaction for vitamin metabolism gene testing of a total of 4 clinical samples (Sample #1, Sample #9, Sample #13, Sample #4) (Figure 3: wild type, Figure 4: mutant type).

[0082] Figures 5 to 7 show the results of a Wilson's disease mono PCR reaction using a clinical sample (HET 4). The PCR reaction was performed by labeling the 5' end of the forward primer of the R778L target with FAM, ATTO-550, SUN, ROX, and Cy3 fluorescence, and the 5' end of the forward primer of the T10291I target with HEX and ROX fluorescence, respectively. The results showed the identification of target bands (Figures 5 and 6: 471 bp, Figure 7: 547 bp) in the Wild / Mutant mixture.

[0083] Figures 8 and 9 show the results of the CE experiment after confirming the electrophoresis results of the Wilson's disease multiplex PCR amplification products of clinical sample HET 4. In the wild-type mixture, FAM fluorescence was detected at 6 targets (N1270S: 676 bp, T1029I: 547 bp, R778L: 471 bp, A874V: 342 bp, G1186S: 264 bp, L1083F: 198 bp) (Figure 8), and in the mutant-type mixture, FAM fluorescence was detected only at the N1270S and R778L target sizes (Figure 9).

[0084] Figure 10 shows the CE test results after confirming the electrophoresis results of the multiplex PCR amplification products of the vitamin metabolism gene test of clinical sample Sample #1. In the wild-type mixture, FAM fluorescence was measured in 16 out of 22 targets (rs2639453, rs2282679, rs1801133, rs3760776, rs2267739, rs6596473, rs33972313, rs2108622, rs1800961, rs1697421, rs11057830, rs7769879, rs35875210, rs4852146, rs964184, rs7041), while Chamel610 fluorescence was measured in the exogenous internal control, and in the mutant-type PCR mixture, a total of 12 targets FAM fluorescence was measured in (rs2639453, rs6420424, rs1395, rs3760776, rs10774624, rs1260326, rs2108622, rs1801222, rs1667255, rs4852146, rs964184, rs7041), and SFC-N fluorescence was measured in the exogenous internal control.

[0085] Figures 11 to 17 show the results of a mono PCR reaction conducted using clinical sample HET 4 with FAM, ATTO-550, SUN, ROX, and Cy3 fluorescence labeled on the 5' end of the forward primer targeting R778L to verify the applicability of various fluorescent dyes in Wilson's disease experiments (Figures 11 to 15), and the results of a CE experiment after a mono PCR amplification reaction with HEX and ROX fluorescence labeled on the 5' end of the forward primer targeting T1029I (Figures 16 and 17), in which each target fluorescence was detected in all fluorescences.

[0086] Figure 18 shows the expected image when a total of 50 SNPs are targeted. It is expected that if a primer set is designed using a total of 4 fluorescent channels (FAM, VIC, NED, PET), the SNPs of 50 target genes can be identified within a maximum of 1,000 base pairs as shown in the figure.

[0087]

[0088] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0089]

[0090] Examples

[0091]

[0092] Throughout this specification, “%” used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0093]

[0094] Experimental Methods and Materials

[0095] 1. Experimental materials

[0096] 1-1. Clinical Sample Information

[0097] This technology verification experiment consisted of two experiments (Wilson's disease, vitamin test), and a total of seven clinical samples (or nucleic acids) were used for verification.

[0098] 1-1-1. Wilson's disease

[0099] A total of three clinical samples (or nucleic acids) were used in the Wilson's disease experiment. These clinical samples included two samples (HET1, HET4) in which N1270S and R778L mutations were confirmed through the Wilson disease screening test (Allele Specific PCR) among the Seegene Medical Foundation's molecular genetic tests, and one sample (HET3) in which the L1083F mutation was confirmed (Table 2).

[0100] Wilson's disease clinical sample informationTargetSampleATP7B geneN1270ST1029IR778LA874VG1186SL1083FHET1HeteroWildHeteroWildWildWildHET3WildWildWildWildWildHeteroHET4HeteroWildHeteroWildWildWild

[0101]

[0102] 1-1-2. Genetic testing related to vitamin metabolism

[0103] A total of four clinical samples (or nucleic acids) were used for the vitamin metabolism gene testing experiment. These four clinical samples (Sample#1, Sample#9, Sample#13, Sample#4) were identified after analyzing genes related to the metabolism of vitamins A, B, C, D, E, and K, as well as 22 associated SNPs, using Sanger Sequencing (Table 3).

[0104]

[0105] 비타민 대사 유전자 검사 임상 샘플 정보VitaminTarget geners no.SNPSampleSample#1Sample#9Sample#13Sample#4ATTRrs1667255A>CMutantHeteroWildHeteroPKD1L2rs6420424G>AMutantMutantHeteroWildB3LINC02356rs10774624G>AMutantMutantMutantMutantSLC22A3rs7769879G>CWildMutantHeteroMutantB4GCKRrs1260326T>CMutantWildMutantMutantHNF4rs1800961C>TWildWildWildWildB5SLC5A6rs1395G>AMutantMutantHeteroMutantSLC17A1rs35875210A>GWildHeteroWildWildB6NONE(NBPF3-ALPL)rs1697421C>TWildHeteroMutantHeteroADCYAP1R1rs2267739C>GWildWildWildHeteroB9MTHFRrs1801133C>TWildHeteroHeteroHeteroKIAA2013rs2639453T>CHeteroHeteroHeteroMutantB12FUT6rs3760776G>AHeteroWildHeteroWildCUBNrs1801222A>GMutantHeteroMutantMutantCSLC23A1rs33972313C>TWildWildWildWildSLC23A1rs6596473G>CWildMutantMutantHeteroDGCrs7041A>CHeteroWildWildHeteroGCrs2282679T>GWildWildWildHeteroESCARB1rs11057830G>AWildWildWildHeteroE&KZPR1rs964184G>CHeteroMutantHeteroWildCYP4F2rs2108622C>THeteroWildHeteroHeteroKCTNNA2rs4852146C>THeteroHeteroHeteroHetero

[0106]

[0107] 1-2. Oligo Information

[0108] 1-2-1. Wilson's disease

[0109] Wilson's disease is caused by mutations in the ATP7B gene, and in Korean patients, six point mutations account for approximately 66.4% (R778L 39.4%, A874V 10.1%, T1029I 3.3%, L1083F 4.5%, G1186S 0.8%, N1270S 6.3%). In the Wilson's disease experiment to validate this technology, two types of PCR mixtures (Wild type, Mutant type) were used, and six pairs of forward and reverse primers were added to each mixture.

[0110] The amplification product sizes for each target region are N1270S: 676 base pairs, T1029I: 547 base pairs, R778L: 471 base pairs, A874V: 342 base pairs, G1186S: 264 base pairs, L1083F: 198 base pairs.

[0111] For the dye verification experiment, six types of dyes (FAM, SUN, HEX, ATTO-550, Cy3, ROX) were labeled at the 5' end, and the fluorescence signal was confirmed through a mono PCR reaction using a primer set targeting R778L and T1029I (Table 4).

[0112] Wilson's disease primer informationGeneNo.TargetTypeTowardPrimer Name5' labeledFluorescenceProduct Size (bp)ATP7B1N1270S CommonFN1270S-F1FAM676WildRN1270S-WT-R5-MutantRN1270S-Mut-R5-2T1029I CommonFT1029I-F1FAM,HEX,ROX 547WildRT1029I-WT-R5-MutantRT1029I-Mut-R8-3R778LCommonFR778L-F1FAM,ATTO-550,SUN,ROX,Cy3471WildRR778L-WT-R2- MutantRR778L-Mut-R3-4A874VCommon RA874V-R3FAM342WildFA874V-WT-F6-MutantFA874V-Mut-F6-5G1186SCommon RG1186S-R1FAM2 64WildFG1186S-WT-F4-MutantFG1186S-WT-F4-6L1083FCommonFL1083F-F1FAM198WildRL1083F-WT-R5-MutantRL1083F-Mut-R5-

[0113] Abbreviation.

[0114] F: Forward, R: Reverse, bp: Base pair

[0115] Primers for the T1029I and R778L target genes were labeled with various fluorescence for fluorescent dye validation experiments.

[0116]

[0117] 1-2-2. Vitamin Metabolism Gene Test

[0118] In the vitamin metabolism gene test experiment for verifying this technology, two types of PCR Mixtures (Wild type, Mutant type) were used, and 22 pairs of forward and reverse primers and 1 pair of internal control forward and reverse primers were added to each Mixture.

[0119] 각 타겟 부위별 증폭 산물 사이즈는 (1) Vitamin A_rs1667255: 839 base pairs, (2) Vitamin A_rs6420424 166 base pairs (3) Vitamin B3_rs10774624: 375 base pairs, (4) Vitamin B3_rs7769879: 742 base pairs (5) Vitamin B4_rs1800961: 443 base pairs, (6) Vitamin B4_ rs1260326: 581 base pairs (7) Vitamin B5_rs1395: 344 base pairs, (8) Vitamin B5_ rs35875210: 882 base pairs, (9) Vitamin B6_rs1697421: 640 base pairs, (10) Vitamin B6_rs2267739: 392 base pairs, (11) Vitamin B9_rs1801133: 288 base pairs, (12) Vitamin B9_rs2639453: 138 base pairs, (13) Vitamin B12_rs3760776: 359 base pairs, (14) Vitamin B12_rs1801222: 653 base pairs, (15) Vitamin C_rs33972313: 475 base pairs, (16) Vitamin C_rs6596473: 425 base pairs, (17) Vitamin D_rs7041: 1,016 base pairs, (18) Vitamin D_rs2282679: 222 base pairs, (19) Vitamin E_rs11057830: 668 base pairs, (20) Vitamin E&K_rs964184: 963 base pairs, (21) Vitamin E&K_rs2108622: 501 base pairs, (22) Vitamin K_rs4852146: 892 base pairs (23) Internal control: 520 base pairs 이다.

[0120] In this experiment, three types of dyes (FAM, SFC-N, Chamel610) were labeled at the 5' ends of forward or reverse primers. Fluorescence signals were confirmed through a multiplex PCR reaction using a fluorescent primer set of FAM for the vitamin target and SFC-N or Chamel610 for the internal control target (Table 5).

[0121]

[0122] Abbreviation.

[0123] F: Forward, R: Reverse, bp: Base pair, IC: Exogenous internal control

[0124]

[0125] 1-3. Types of Fluorescent Dyes

[0126] A total of five fluorescence wavelengths were measured using a G5 dye set on a 3500xL genetic analyzer (ThermoFisher) (a 3730xL genetic analyzer is also compatible). Among them, the Orange wavelength was used as a size standard with GeneScan. TM 1200 LIZ TM dye Size Standard (Applied Biosystems TM Using (Catalog No. 4379950), it was possible to analyze up to a total maximum size of 1,200 base pairs. For the remaining four fluorescences, alternative fluorescent dyes of the same wavelength from other manufacturers (SUN, HEX, Cy3, ATTO-550, ROX, SFC-N, Chamel610) can be used in addition to the dyes recommended by the manufacturer (ThermoFisher) (6-FAM, VIC, NED, PET), and these fluorescences were confirmed in this experiment (Table 6).

[0127] Genetic Analyzer Fluorescent Dye Information Genetic Analyzer (ThermoFisher) Available Dye Fluorescence* DyeSetMatrix StandardColor DyeEx. Max(nm) Em. Max(nm) Manufacturer DyeEx. Max(nm) Em. Max(nm)G5DS-33●Blue6-FAM494522IDT6-FAM494522●GreenVIC538554IDTSUN538554HEX538555●YellowNED546575 IDTCy3550564ATTO-550560575SFCSFC-N547572●RedPET558595IDTROX588608SFCChamel610588610●OrangeGS1200 Liz(Size standard)638655Thermo-FisherGS1200 Liz(Size standard)638655

[0128] Abbreviation.

[0129] Ex: Excitation wavelength in nanometers, Em: Emission wavelength in nanometers

[0130] *(Alternative) Fluorescence can be added

[0131]

[0132] 2. Experimental Method

[0133] 2-1. PCR Amplification and Electrophoresis

[0134] First, the PCR mastermix was prepared as shown in Tables 7 and 8 below, then lightly mixed or vortexed about 5 times, and then lightly centrifuged.

[0135] (A) Wilson's diseaseNo.PCR Mastermix1 Test volume (μL)1PCRMixtureWild type5Mutant type22X Multiplex master mix (4 unit, w / dye)10Total15

[0136] (B) Vitamin Metabolism Gene Test No. PCR Mastermix 1 Test volume (μL) 1 PCR Mixture Wild type 5 Mutant type 2 EM 4 5 EM 4 Buffer 5 Total 15

[0137]

[0138] Next, 15 µl of PCR mastermix was dispensed into PCR tubes. 5 µl of the sample nucleic acid was added to the tubes (total nucleic acid amount (A): 25 ng, (B): 100 ng). The lids were closed and the PCR tubes were gently centrifuged. PCR was performed according to the following program.

[0139] (A) Wilson's diseaseSegmentNo. of cyclesTemperature(℃)Duration119415 min2339430 sec6530 sec721 min317210min

[0140] (B) Vitamin Metabolism Gene Test Segment No. of cycles Temperature(°C) Duration 1 19 4 15 min 2 3 5 9 4 30 sec 6 5 30 sec 7 2 1 min 30 sec 3 1 7 2 10 min

[0141]

[0142] After the PCR reaction was completed, 5 µL of the amplified DNA fragments were transferred to each well of an agarose gel and run in an electrophoresis apparatus at 280°C for 30 minutes. After the electrophoresis was completed, the DNA fragment bands were checked and their sizes analyzed.

[0143]

[0144] 2-2. Sample Denaturation

[0145] A cocktail was prepared as shown in Table 11 below.

[0146] Ratio1200 LIZvolume (μL)Hi-Divolume (μL)Totalvolume (μL)1:1811819

[0147]

[0148] The PCR product, whose amplification results were confirmed by electrophoresis, was diluted 1 / 10 with RNase-free water. The amplification product of the present invention can be used at different dilution ratios depending on the characteristics of the item.

[0149] As shown in Table 12 below, the diluted PCR product and cocktail (1200Liz+HiDi) were mixed.

[0150] Ratio*1 / 10 PCR productCocktail(1200Liz+HiDi)Totalvolume (μL)1:1911920

[0151] *Based on manufacturer's recommended manufacturing ratio

[0152] In addition, denaturation of the mixed mixture was performed according to the following program.

[0153] SegmentNo. of cyclesTemperature(℃)Duration11953 min2143 min314∞

[0154]

[0155] 2-3. Capillary electrophoresis

[0156] After sample denaturation was completed, the size of the DNA fragments was separated by capillary electrophoresis using a genetic analyzer (3500 / 3500xL / 3500xL Dx Genetic Analyzer (3500 Series instrument)).

[0157] Capillary Electrophoresis Protocol Information 3500xL Genetic Analyzer* 3730xL DNA Analyzer Capillary length 5036 Run Module name** Long Fragment Analysis Assay xLGS1200LIZ 36 POP7

[0158] This verification experiment was conducted using only the 3500xL Dx Genetic Analyzer.

[0159] Follow the Run protocol provided by the manufacturer.

[0160]

[0161] Experimental results

[0162]

[0163] 1. Electrophoresis Results

[0164] 1-1. Multiplex PCR (Wilson's disease, 6 targets)

[0165] Figure 1 shows the electrophoresis results of the amplification products from the Multiplex PCR (6 targets; N1270S, T1029I, R778L, A874V, G1186S, L1083F) reaction of a total of 3 clinical samples (HET 1, HET 3, HET 4).

[0166] The 5' end of all forward or reverse primers of the two types of PCR Mixture (wild, mutant) is labeled with 6-FAM fluorescence.

[0167] As shown in Figure 1, first, as a result of PCR amplification of the wild-type PCR mixture, all three clinical samples (HET 1, HET 3, HET 4) showed the detection of six target bands (N1270S: 676 bp, T1029I: 547 bp, R778L: 471 bp, A874V: 342 bp, G1186S: 264 bp, L1083F: 198 bp). The reason all six target bands were detected is that wild bases were also detected because they were heterozygote samples.

[0168] In addition, as shown in Figure 2, in the Mutant type PCR mixture, detections were found only in the N1270S and R778L target bands in clinical sample HET 1, L1083F in HET 3, and N1270S and R778L target bands in HET 4, respectively. This result indicates that PCR-amplified bands were found only in the same target regions as the actual reported test results.

[0169]

[0170] 1-2. Multiplex PCR (Vitamin Metabolism Gene Test, 22 Targets + Exogenous Internal Control)

[0171] The electrophoresis results of the amplification products from the Multiplex PCR (22 targets + Internal control) reaction of a total of 4 clinical samples (Sample #1, Sample #9, Sample #13, Sample #4) are shown in Figures 3 and 4.

[0172] The 5' end of all forward or reverse primers of the two types of PCR Mixtures (wild, mutant) is labeled with 6-FAM fluorescence, the 5' end of the reverse primer of the internal control of the Wild PCR Mixture is labeled with Chamel610 fluorescence, and the 5' end of the reverse primer of the internal control of the Mutant PCR Mixture is labeled with SFC-N fluorescence.

[0173] As shown in Figures 3 and 4, based on the size markers, the PCR amplification results of the PCR mixture (wild, mutant) show rs2639453 and rs6420424 in the 100–200 bp range; rs2282679 and rs1801133 in the 200–300 bp range; rs1395, rs3760776, rs10774624, and rs2267739 in the 300–400 bp range; rs6596473, rs1260326, and rs33972313 in the 400–500 bp range; rs2108622 and rs1800961 in the 500–600 bp range; and rs1697421 in the exogenous internal control 600–700 bp range. Amplification products of targets rs1801222, rs11057830, rs7769879 in the 700–800 bp interval, rs1667255, rs35875210, and rs4852146 in the 800–900 bp interval, rs964184 in the 900–1,000 bp interval, and rs7041 in the 1,000–1,100 bp interval were observed. Examining the results by sample, the presence of amplification varied in each interval, which is interpreted as being due to genetic differences (SNP variations) between samples. Additionally, it was confirmed that among the Negative Controls, only the Exogenous internal control showed amplification.

[0174] Therefore, the electrophoresis results confirmed that amplified products were formed in each segment, and targets within the same segment were not individually distinguished.

[0175]

[0176] 1-3. Dye verification (Wilson's disease, mono PCR)

[0177] Next, the results of the mono PCR reaction using one clinical sample (HET 4) for the dye verification experiment are shown in Figures 5 to 7.

[0178] First, as shown in Figures 5 and 6, when PCR reactions were performed by labeling the 5' end of the forward primer of the R778L target with FAM, ATTO-550, SUN, ROX, and Cy3 fluorescence, respectively, it was confirmed that the target band (Size: 471 bp) was amplified in the Wild / Mutant mixture.

[0179] In addition, as shown in Figure 7, when PCR was performed by labeling the 5' end of the forward primer of the T1029I target with HEX and ROX fluorescence, it was confirmed that the target band (Size: 547 bp) was amplified only in the wild mixture (since the HET4 sample has a wild type T1029 target).

[0180]

[0181] 2. Capillary electrophoresis results

[0182] 2-1. Multiplex PCR (Wilson's disease, 6 targets)

[0183] After confirming the electrophoresis results of the multiplex PCR amplification products of clinical sample HET 4, the CE test results are shown in Figure 8.

[0184] As shown in Figure 8, FAM fluorescence was measured in all 6 targets (N1270S: 676 bp, T1029I: 547 bp, R778L: 471 bp, A874V: 342 bp, G1186S: 264 bp, L1083F: 198 bp) in the CE results of the wild-type PCR mixture.

[0185] As shown in Figure 9, the Mutant type PCR mixture was detected only in N1270S and R778L target sizes and fluorescence.

[0186] The size, height (intensity), and area of ​​the fluorescence measured by the CE method can be seen in Figures 8 and 9.

[0187]

[0188] 2-2. Multiplex PCR (Vitamin Metabolism Gene Test, 22 Targets + Exogenous Internal Control)

[0189] After confirming the electrophoresis results of the multiplex PCR amplification product of clinical sample Sample #1, the CE test results are shown in Figure 10.

[0190] As shown in Figure 10, the CE results of the wild-type PCR mixture showed that FAM fluorescence was measured in 16 out of 22 targets (rs2639453, rs2282679, rs1801133, rs3760776, rs2267739, rs6596473, rs33972313, rs2108622, rs1800961, rs1697421, rs11057830, rs7769879, rs35875210, rs4852146, rs964184, rs7041) and Chamel610 fluorescence was measured in the exogenous internal control.

[0191] As shown in Figure 10, FAM fluorescence was detected in a total of 12 targets (rs2639453, rs6420424, rs1395, rs3760776, rs10774624, rs1260326, rs2108622, rs1801222, rs1667255, rs4852146, rs964184, rs7041) of the Mutant type PCR mixture, and SFC-N fluorescence was detected in the Exogenous internal control. The size, height (intensity), and area of ​​the fluorescence measured by the CE method can be seen in Table 15.

[0192]

[0193]

[0194] 2-3. Dye verification (Wilson's disease, mono PCR)

[0195] As a result of conducting a mono PCR reaction using clinical sample HET 4 with the R778L target by labeling the 5' end of the forward primer with FAM, ATTO-550, SUN, ROX, and Cy3 fluorescence, each target fluorescence was detected in all fluorescences (FAM, ATTO-550, SUN, ROX, Cy3) (Figs. 11 to 15).

[0196] In addition, HEX and ROX fluorescence were labeled at the 5' end of the forward primer of the T1029I target, and as a result of the CE experiment after the mono PCR amplification reaction, each target fluorescence was detected in all fluorescences (HEX, ROX) (Figs. 16 and 17).

[0197] Therefore, from the above results, it was confirmed through Dye verification experiments that various fluorescent materials of four wavelength ranges (excluding size standard LIZ) can be used in this technology.

Claims

1. An SNP multiplexing kit comprising one or more sets of primer sets for SNP multiplexing, each comprising a plurality of primer pairs for detecting single nucleotide polymorphisms (SNPs) at different locations of a gene, wherein A SNP multiplex test kit, wherein each of the above SNP multiplex test primer sets comprises: i) a wild-type SNP detection primer subset including a primer pair for detecting wild-type SNPs; and ii) a mutant-type SNP detection primer subset including a primer pair for detecting mutant-type SNPs existing at the same gene location as the same wild-type SNP.

2. An SNP multiplex test kit according to claim 1, wherein the i) wild type SNP detection primer subset and ii) mutant type SNP detection primer subset are each gene amplified in different tubes.

3. An SNP multiplex test kit according to claim 1, wherein the wild type SNP detection primer subset and the mutant type SNP detection primer subset are designed such that the size of the amplification target products of the primer pair detecting wild type SNPs and the primer pair detecting mutant type SNPs, which are located at the same corresponding gene location, are equal to each other.

4. An SNP multiplex test kit according to claim 1, wherein the size of the amplification target product of each primer pair is 50 to 1200 base pairs (bp).

5. An SNP multiplex test kit according to claim 1, wherein one end of the forward primer or reverse primer of the primer pair is labeled with a fluorescent label.

6. An SNP multiplex test kit according to claim 5, wherein the fluorescent label is selected from the group consisting of 5-FAM, 6-FAM, ATTO-550, SUN, VIC, NED, ROX, Cy3, TET, HEX, JOE, TMR, Chamel610, SFC-N, and PET.

7. An SNP multiplex test kit according to claim 5, wherein the primer pairs within a single primer set are attached with the same or different types of fluorescent labels.

8. An SNP multiplex test kit according to claim 1, wherein the size of the amplification target product of each primer pair included in the single primer set differs by 1 bp or more.

9. An SNP multiplex test kit according to claim 1, wherein the in-gene locations of the amplification target products of each primer pair included in the single primer set do not overlap each other.

10. SNP multiplex testing method including the following steps: (a) adding a nucleic acid sample isolated from a patient to different PCR amplification tubes, each comprising i) a subset of wild-type SNP detection primers including a primer pair for detecting wild-type SNPs, and ii) a subset of mutant-type SNP detection primers including a primer pair for detecting mutant-type SNPs located at the same gene location as the wild-type SNP, and performing a PCR reaction; and (b) a step of detecting the PCR reaction product amplified by i) a wild type SNP detection primer subset, and ii) a mutant type SNP detection primer subset.

11. An SNP multiplex test kit according to claim 10, wherein the wild type SNP detection primer subset and the mutant type SNP detection primer subset are designed such that the size of the amplification target products of the primer pair detecting wild type SNPs and the primer pair detecting mutant type SNPs, which are located at the same corresponding gene location, are equal to each other.

12. An SNP multiplexing method according to claim 10, wherein the SNP detection primer subset comprises two or more primer pairs.

13. An SNP multiplexing method according to claim 10, wherein the PCR reaction is performed through multiplex PCR.

14. An SNP multiplexing method according to claim 10, wherein the PCR reaction of step (a) is further performed using i) a wild-type SNP detection primer subset and ii) a primer subset that detects SNPs at gene locations different from the mutant-type SNP detection primer subset.

15. An SNP multiplex test method according to claim 10, wherein the detection of the PCR reaction product is performed through gel electrophoresis or capillary electrophoresis.

16. An SNP multiplexing method according to claim 10, wherein one end of the forward primer or reverse primer of the primer pair is labeled with a fluorescent label.

17. An SNP multiplexing method according to claim 10, wherein the size of the amplification target products of each primer pair included in the above-mentioned primer set differs by 1 bp or more.

18. An SNP multiplexing method according to claim 10, wherein the in-gene locations of the amplification target products of each primer pair included in the above-mentioned primer set do not overlap each other.

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