WO2014157825A1 - Method and kit for testing hla alleles using speed multiplex pcr - Google Patents

Method and kit for testing hla alleles using speed multiplex pcr Download PDF

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Publication number
WO2014157825A1
WO2014157825A1 PCT/KR2014/001097 KR2014001097W WO2014157825A1 WO 2014157825 A1 WO2014157825 A1 WO 2014157825A1 KR 2014001097 W KR2014001097 W KR 2014001097W WO 2014157825 A1 WO2014157825 A1 WO 2014157825A1
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Prior art keywords
seq
hla
primer
nos
drb1
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PCT/KR2014/001097
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French (fr)
Korean (ko)
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장욱진
최희백
유미경
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(주)진스랩
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Priority claimed from KR1020130033330A external-priority patent/KR101320622B1/en
Application filed by (주)진스랩 filed Critical (주)진스랩
Publication of WO2014157825A1 publication Critical patent/WO2014157825A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method and kit for simultaneously testing HLA-A, B, and DR alleles using real-time multiplex polymerase chain reaction, which specifically binds to HLA alleles in DNA extracted from a sample.
  • kit for classifying HLA alleles and the type of HLA alleles with fluorescence values which can be detected using a number of primers and fluorescent probes capable of detecting amplified products amplified by these primers, It is about.
  • HLA Human Leukocyte Antigen
  • the HLA molecule is responsible for delivering foreign antigens to CD8 + T cells and CD4 + T cells.
  • the type of HLA molecule was analyzed by serological method. However, as the polymerase chain reaction was developed, the type analysis was performed at the DNA level. As a result of HLA typing at the DNA level, a large number of new HLA alleles have been identified. Currently, 2,013 types of HLA-A, 2,605 types of HLA-B, 1,551 types of HLA-C, and 1,260 types of HLA-DRB. As of July 2012.
  • HLA assays include serotyping by antibody-antigen reactions and molecular typing (DNA typing) to determine HLA gene sequence differences.
  • the serological method has a relatively low complexity of the test method and the reliability of the results compared to the molecular biological method is currently used a lot of molecular biological methods worldwide.
  • PCR-SSP sequence specific primers
  • PCR-SSOP sequence specific oligonucleotide probes
  • PCR-SBT sequence based analysis
  • PCR-SSP method similar to the present technique has a disadvantage in that a large number of tubes are used for HLA test because only one single base can be seen in one tube. There was also a demand for it.
  • An object of the present invention is to specifically amplify a site with a polymorphism of two genes (HLA-A and HLA-B, HLA-B and HLA-DRB) in one tube in detecting a site with a polymorphism in an HLA gene.
  • the present invention provides a method and kit for performing multiplex polymerase chain reaction and detecting the presence or absence of amplification by using a fluorescence probe.
  • the present invention provides a primer set capable of specifically amplifying HLA alleles for two or more genes selected from the group consisting of HLA-A, HLA-B and HLA-DRB by real-time PCR, and It provides a HLA allele type kit comprising a set of probes that can probe the product amplified by the real-time PCR.
  • the present invention is the step of performing a real time PCR on the DNA extracted from the sample using the detection set for the HLA allele of the present invention; And identifying the HLA allele by identifying the real-time PCR result.
  • HLA allele type selection kit using the real-time polymerase chain reaction of the present invention is a general primer specific for HLA alleles instead of fluorescent primers to amplify the genes in multiplex and detect only the amplification of each type gene
  • the use of fluorescent probes allows the identification of HLA alleles through real-time polymerase chain reactions without electrophoresis, and is more economical than HLA test kits (96 wells per person) and test methods (general electrophoresis) currently available on the market.
  • HLA test kits 96 wells per person
  • test methods general electrophoresis
  • a single test kit can simultaneously test patients and donors for organ transplantation, and thus have excellent technical convenience and economic efficiency. As a result, it can be easily used for type analysis of genes in which several single base mutations exist in a gene.
  • 1A to 1E show analysis tables for distinguishing HLA-A, B, and DR allele types.
  • Figure 2 shows the result of the gene amplification after HLA-A, B, DR allele types.
  • Figure 3 shows the CT value after gene amplification by HLA-A, B, DR allele type.
  • the present invention provides a set of primers capable of specifically amplifying HLA alleles for two or more genes selected from the group consisting of HLA-A, HLA-B and HLA-DRB by real-time PCR, and amplified by the real-time PCR.
  • a HLA allele typing kit comprising a set of probes capable of probing a product.
  • the multiplex HLA allele type set which can detect the HLA allele of the present invention by real-time polymerase chain reaction, has primers capable of specifically binding to and amplifying alleles of the HLA-A, B, and DRB genes, respectively.
  • HLA alleles can be tested through real-time polymerase chain reaction using fluorescently labeled probes that can hybridize to amplification products for each HLA gene and identify the amplification products.
  • the HLA alleles detectable through the detection set of the present invention are HLA-A, HLA-B, HLA-DRB genes.
  • the real-time polymerase chain reaction described in the present invention uses a device that integrates a thermal cycler and a spectral fluorescence photometer, and analyzes the amount of DNA to be obtained by monitoring the generation process of PCR amplification products in real time. Means the way.
  • the real-time PCR method does not require electrophoresis for the identification of PCR amplification products, and is an excellent method for rapid and quantitative comparison of the amount of amplification products in a region where amplification occurs exponentially.
  • the primer is capable of specifically amplifying HLA alleles by type.
  • the primers may be constructed to have a length of 17 to 22 nt based on a position indicating diversity of the HLA alleles.
  • the primers are HLA-A alleles via conventional PCR, for example HLA-A * 01, A * 02, A * 03, A * 11, A * 23, A * 24, A * 25, A * 26 , A * 29, A * 30, A * 31, A * 32, A * 33, A * 34, A * 36, A * 43, A * 66, A * 68, A * 69, A * 74, A * 80 and the like, or HLA-B alleles, for example B * 07, B * 08, B * 13, B * 14, B * 15 (B62), B * 15 (63), B * 15 (70 ), B * 15 (71), B * 15 (72), B * 15 (75), B * 15 (76), B * 15 (77), B * 15, B * 18, B * 27, B * 35, B * 37, B * 38, B * 39, B * 40, B * 40 (B60), B * 40 (B61), B * 41, B * 42, B * 44, B * 45, B * 46, B * 47, B * 48, B *
  • the primers were used to simultaneously amplify HLA-A and HLA-B, HLA-B and HLA-DRB genes through multiplex real-time polymerase chain reaction.
  • the amplification products that can be amplified through the primer in consideration of the conditions that do not occur non-specific reaction is 91 to 274bp, wherein the Tm value of all the primers was 58 to 65.
  • HLA-A, B and DRB alleles that can be specifically amplified by each primer are as follows.
  • HLA alleles A * 02, A * 24, A * 25
  • the fluorescence probe may select a region without polymorphism in the HLA gene so as to detect all of the products amplified by the primers, and may operate in a length of 27 to 33 nt.
  • SEQ ID NOs: 151, 152, 153 It may have the sequence described in 154, 155, 156, 157, 158, 159, 160, 161 or 162.
  • the presence or absence of amplification can be detected through a fluorescent probe having a sequence set forth in SEQ ID NO: 151, 152, 153, 154 or 155.
  • the presence or absence of amplification can be detected through a fluorescent probe having a sequence set forth in SEQ ID NO: 156, 157, 158, 159 or 160.
  • the presence or absence of amplification can be detected through a fluorescent probe having a sequence set forth in SEQ ID NO: 161 or 162.
  • the Tm value of the fluorescent probe may be 60 to 65 °C.
  • the fluorescent probe is labeled with fluorescent labeling factors at the 5 'and 3' ends of the probe, and each probe is labeled with a different fluorescent labeling factor according to the type of detection set of the HLA allele.
  • Fluorescent labeling factors differ in excitation and emission wavelengths depending on the type, and the method of use is also different. Therefore, the fluorescent labeling factors used together in one PCR reaction should be selected and used separately in consideration of this. Colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
  • the fluorescent labeling factor is labeled on a probe included in the HLA allele test set according to the present invention in a conventional manner, and the labeling method is an interchelating method, a TaqMan TM probe method and a molecular beacon (Molecualr beacon) methods are available.
  • the labeling method is an interchelating method, a TaqMan TM probe method and a molecular beacon (Molecualr beacon) methods are available.
  • TaqMan TM probe Method for adding TaqMan TM probe method was 5 used in this invention in a raised the reaction solution of PCR oligonucleotide (TaqMan probe) modified with "a terminal fluorescent marker (FAM, etc.) to the 3 'end a quencher substance (TAMRA etc.)
  • FAM terminal fluorescent marker
  • TAMRA quencher substance
  • the probe 5 'end is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3'
  • the terminus may be labeled with one fluorescence inhibitor (Quencher) selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and molecular grove binding non-fluorescence quencher (MGBNFQ).
  • the detection set of the present invention may further include a primer for amplifying the internal positive control gene to increase the reliability of the test and to be included in the entire PCR tube used in the experiment process to check the status of the template and the PCR conditions.
  • a primer for amplifying the internal positive control gene to increase the reliability of the test and to be included in the entire PCR tube used in the experiment process to check the status of the template and the PCR conditions.
  • -globin, human -actin, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), or Homo sapiens adenomatous polyposis coli (APC) gene can be used.
  • GPDH glyceraldehydes-3-phosphate dehydrogenase
  • APC Homo sapiens adenomatous polyposis coli
  • a primer pair consisting of the sequences set forth in SEQ ID NOs: 149 and 150 capable of amplifying the Homo sapiens adenomatous polyposi
  • the detection set of the present invention may further include a fluorescent probe for detecting the presence or absence of the amplification of the positive control gene, preferably those having the sequence set forth in SEQ ID NO: 163.
  • a detection set comprising primers and fluorescent probes for the HLA-A, B, DRB1 alleles are as follows.
  • the types of positive control primer pairs (SEQ ID NOs: 149 and 150) and fluorescence probes (SEQ ID NO: 163) are the same in all detection sets.
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 151, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 155, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 155, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 155, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probe SEQ ID NO: 152
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 154, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 153, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 155, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 155, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probe SEQ ID NO: 152
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 155, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 154, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 152, 158
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probe SEQ ID NO: 152
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 162, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescent probes SEQ ID NOs: 161, 159
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probes SEQ ID NOs: 161, 156
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probe SEQ ID NO: 157
  • Positive control primer pairs SEQ ID NOs: 149 and 150
  • HLA fluorescence probe SEQ ID NO: 157
  • the detection set of the present invention can perform multiplex real-time PCR using at least two or more primers and fluorescent probes specific to the type of HLA allele, using one or more combinations thereof simultaneously.
  • the invention also relates to an HLA allele type selection kit comprising the detection set of the invention.
  • HLA alleles that can be typed through the HLA allele type selection kit of the present invention are HLA-A, HLA-B, HLA-DRB1 alleles.
  • the primer pair and the fluorescence probe may be packaged in strips or microplates and packaged by methods known in the art.
  • the kit of the present invention may further include one or more selected from the group consisting of Taq polymerase, a reaction buffer including MgCl 2 , dNTP, and a stabilizer, in addition to those known in the art.
  • Other reagents such as a master mix for real-time PCR.
  • the major histocompatibility antigen complex can be distinguished at the gene level, it can be widely used for more accurate histocompatibility determination.
  • the present invention also provides
  • the first step is to prepare a primer for amplifying the HLA allele, and construct a primer having a length of 17 to 24 nt based on a position indicating the diversity of the HLA allele.
  • the primer is SEQ ID NO: 41 to which can selectively amplify the HLA-DRB1 allele using two species selected from the sequence set forth in SEQ ID NO: 1 to 40 to selectively amplify the HLA-A allele
  • Two kinds selected from the sequences set forth in 77 are used, and two or more kinds selected from the sequences set forth in SEQ ID NOS: 78 to 148 capable of selectively amplifying the HLA-B allele are used.
  • the primers include a primer pair consisting of the sequences set forth in SEQ ID NOs: 149 and 150 for amplification of the internal positive control genes to increase the reliability of the test and to be included in the entire PCR tube used during the experiment to check the status of the template and the PCR conditions. It may further include.
  • a fluorescent probe capable of amplifying the primers described above, and detecting the amplified product is classified into five types of HLA-A, five types of HLA-B, and HLA- for each HLA gene. Two types of DRB1 exist.
  • the constructed fluorescence probe selects a region without polymorphism in the HLA gene and constructs it with a length of 27 to 33 nt.
  • it may be a sequence described in SEQ ID NOs: 151, 152, 153, 154, and 155 capable of detecting the amplification of the HLA-A allele. It may also be the sequence set forth in SEQ ID NO: 156, 157, 158, 159, 160 capable of detecting the amplification of the HLA-B allele. It may also be the sequence set forth in SEQ ID NO: 161 or 162 capable of detecting the amplification of the HLA-DRB1 allele.
  • the method may further include a fluorescent probe having a sequence set forth in SEQ ID NO: 163 for detecting amplification of a positive control gene.
  • the third step is the step of amplifying the primer through real-time PCR and the step of real-time measuring the presence or absence of amplification when amplified by a fluorescent probe.
  • single or multiplex real-time PCR reaction conditions can take conventional conditions.
  • single real-time PCR and multiplex real-time PCR reaction may be performed under the same conditions. For example, initial denaturation is performed at 95 ° C. for 10 minutes and then denatured in one step (denaturation, 25 seconds at 95 ° C.). ), Annealing (45 seconds at 70 ° C.) and extension (30 seconds at 72 ° C.), five times, denaturation (25 seconds at 95 ° C.), annealing, at 70 ° C. 45 seconds) and extension (30 seconds at 72 ° C.) may be carried out 30 times to achieve a total of 35 times. Fluorescence generated by the fluorescence probe is measured in the extension step of the real time PCR.
  • the HLA allele typing method of the present invention can be carried out using conventional real-time PCR methods and apparatus.
  • the real-time PCR method is a method of detecting and quantifying fluorescence performed in real time every cycle of PCR based on the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). This method distinguishes specific amplification products from non-specific amplification products and makes it easy to obtain analysis results in an automated manner.
  • FRET Fluorescence Resonance Energy Transfer
  • the real-time PCR devices that can be used in the present invention include, but are not limited to, AB Real-time PCR devices 7900, 7500, 7300, LightCycler 480 from Roche, Mx3000p from Stratagene, and Chromo 4 from BioRad.
  • the laser of the real-time PCR device senses the fluorescent labeling factor labeled on the fluorescent probe of the amplified PCR product to implement the peak as shown in FIG. 2.
  • the results can be automatically analyzed by executing a program embedded in the device without the electrophoresis process.
  • KoRASTM Kozen Biotech
  • the analyzed result can be represented by the O / X type or the Ct type, so that even the unskilled person can easily know the analyzed result.
  • the detection set for the HLA allele of the present invention and the HLA allele typing method using the same not only significantly shorten the PCR process for identifying the existing HLA allele, but also can quickly check the result in real time. have.
  • This method can be distinguished by laser sensitization of the presence of small concentrations of DNA fragments or less than 100 bp PCR products that were difficult to discern by electrophoresis. There is no concern about contamination due to contamination, and all the test processes are operated as an automatic system until the results are analyzed and the test is completed. Therefore, there is a low probability of error or error of the tester. This is very useful in minimizing the inspection time and labor required.
  • Primer 2 AGCCCCGCTTCATCGCA (SEQ ID NO: 2)
  • Primer 3 TGGCTGCGACGTGGGGT (SEQ ID NO: 3)
  • Primer 4 GTTCTCACACCATCCAGATA (SEQ ID NO: 4)
  • Primer 5 CACACCCTCCAGATGATGTT (SEQ ID NO: 5)
  • Precursor 6 CCCATGAGGTATTTCTACACC (SEQ ID NO: 6)
  • Precursor 7 GGAGTATTGGGACCGGAAC (SEQ ID NO: 7)
  • Precursor 8 CCACTCCATGAGGTATTTCAC (SEQ ID NO: 8)
  • Primer 9 CACAGACTGACCGAGTGG (SEQ ID NO: 9)
  • Primer 10 CCGTGTCCCGGCCCGGCAGT (SEQ ID NO: 10)
  • Primer 11 CCACTCCATGAGGTATTTCTT (SEQ ID NO: 11)
  • Primer 12 CGGGTACCAGCAGGACGCT (SEQ ID NO: 12)
  • Precursor 13 CCCACTCCATGAGGTATTTCA (SEQ ID NO: 13)
  • Precursor 14 CCTCCGCGGGTACCGG (SEQ ID NO: 14)
  • Precursor 15 TCCTCCGCGGGTACCGGC (SEQ ID NO: 15)
  • Precursor 16 CTCCATGAGGTATTTCTACTCC (SEQ ID NO: 16)
  • Primer 17 GCGACGTGGGGTCGGACT (SEQ ID NO: 17)
  • Primer 18 ACGCAGTTCGTGCGGTTT (SEQ ID NO: 18)
  • Precursor 19 GAGCCCCGCTTCATCGCA (SEQ ID NO: 19)
  • Primer 20 CACACCCTCCAGATGATGTT (SEQ ID NO: 20)
  • Precursor 21 TCTGTGAGTGGGCCTTCAT (SEQ ID NO: 21)
  • Primer 22 CACTTTCCGTGTCTCCCC (SEQ ID NO: 22)
  • Primer 23 CCACTCCACGCACGTGCCA (SEQ ID NO: 23)
  • Promoter 24 GCCCTCCAGGTAGGCTCTCT (SEQ ID NO: 24)
  • Precursor 25 CCTCCAGGTAGGCTCTCTG (SEQ ID NO: 25)
  • Precursor 26 GCGCGATCCGCAGGCTCT (SEQ ID NO: 26)
  • Precursor 27 CCGGGGTCACTCACCGT (SEQ ID NO: 27)
  • Precursor 28 CTTCACATTCCGTGTCTGCA (SEQ ID NO: 28)
  • Precursor 29 GTCTGTGAGTGGGCCTTCAC (SEQ ID NO: 29)
  • Precursor 30 CTTCACATTCCGTGTGTTCCG (SEQ ID NO: 30)
  • Precursor 31 CTCCGCCTCATGGGCCGT (SEQ ID NO: 31)
  • Primer 32 GGTCCCAATACTCAGGCCT (SEQ ID NO: 32)
  • Primer 33 GAGCCACTCCACGCACGT (SEQ ID NO: 33)
  • Precursor 34 AGGTATCTGCGGAGCCCG (SEQ ID NO: 34)
  • Precursor 35 CCTTCATATTCCGTGTCTGCA (SEQ ID NO: 35)
  • Precursor 36 CCTCCAGGTAGGCTCTCAA (SEQ ID NO: 36)
  • Precursor 37 GCAGGGTCCCCAGGTCCA (SEQ ID NO: 37)
  • Precursor 38 TCCCCAGGTTCGCTCGGTT (SEQ ID NO: 38)
  • Precursor 39 TCTCAACTGCTCCGCCACAC (SEQ ID NO: 39)
  • Precursor 40 CACTCGGTCAGTCTGTGAC (SEQ ID NO: 40)
  • Precursor 41 CTTGTGGCAGCTTAAGTTTGAA (SEQ ID NO: 41)
  • Precursor 42 CCTGTGGCAGCCTAAGAGG (SEQ ID NO: 42)
  • Primer 43 GTTTCTTGGAGTACTCTACGTC (SEQ ID NO: 43)
  • Primer 44 GTTTCTTGGAGCAGGTTAAACA (SEQ ID NO: 44)
  • Precursor 45 CCTGTGGCAGGGTAAGTATA (SEQ ID NO: 45)
  • Precursor 46 AGTACTCTACGGGTGAGTGTT (SEQ ID NO: 46)
  • Precursor 47 GACGGAGCGGGTGCGGTA (SEQ ID NO: 47)
  • Primer 48 GCGGTTGCTGGAAAGACGCG (SEQ ID NO: 48)
  • Precursor 49 GTTTCTTGGAGTACTCTACGTC (SEQ ID NO: 49)
  • Precursor 50 GGTTCCTGGACAGATACTTCC (SEQ ID NO: 50)
  • Primer 51 TACTCTACGTCTGAGTGTCAA (SEQ ID NO: 51)
  • Precursor 52 ACAGCACGTTTCTTGGAGCT (SEQ ID NO: 52)
  • Primer 53 GAGCGAGTGTGGAACCTGAT (SEQ ID NO: 53)
  • Precursor 54 GTTTCTTGCAGCAGGATAAGTA (SEQ ID NO: 54)
  • Precursor 55 CCGCCTCTGCTCCAGGAG (SEQ ID NO: 55)
  • Precursor 56 TCCACCGCGGCCCGCGC (SEQ ID NO: 56)
  • Promoter 57 TAGGTGTCCACCGCGGCG (SEQ ID NO: 57)
  • Primer 58 GCAGTAGTTGTCCACCCGGC (SEQ ID NO: 58)
  • Precursor 59 CGCACGTACTCCTCTTGGTG (SEQ ID NO: 59)
  • Precursor 60 CCCGTAGTTGTGTCTGCACAC (SEQ ID NO: 60)
  • Precursor 61 TGCAGTAGGTGTCCACCAG (SEQ ID NO: 61)
  • Precursor 62 ACCCCGTAGTTGTGTCTGCACAC (SEQ ID NO: 62)
  • Primer 63 GCTGCACTGTGAAGCTCTCAC (SEQ ID NO: 63)
  • Primer 64 CTGGCTGTTCCAGTACTCCT (SEQ ID NO: 64)
  • Primer 65 CTGTTCCAGGACTCGGCGA (SEQ ID NO: 65)
  • Primer 66 CCCGCTCGTCTTCCAGGAT (SEQ ID NO: 66)
  • Precursor 67 CCCGCCTGTCTTCCAGGAA (SEQ ID NO: 67)
  • Primer 68 CTGGCTGTTCCAGTACTCGG (SEQ ID NO: 68)
  • Precursor 69 CACCTCGGCCCGCCTCC (SEQ ID NO: 69)
  • Primer 70 CACCGCGGCCCGCCTCT (SEQ ID NO: 70)
  • Precursor 71 CTGTTCCAGTGCTCCGCAG (SEQ ID NO: 71)
  • Primer 72 GCTYACCTCGCCKCTGCAC (SEQ ID NO: 72)
  • Precursor 73 TCTGCAGTAGGTGTCCACCAG (SEQ ID NO: 73)
  • Precursor 74 GCTGCACTGTGAAGCTCTCCA (SEQ ID NO: 74)
  • Primer 75 GCAGTAATTGTCCACCCGGC (SEQ ID NO: 75)
  • Precursor 76 CTGCACTGTGAAGCTCTCCA (SEQ ID NO: 76)
  • Primer 77 CCGCTGCACTGTGAAGCTCT (SEQ ID NO: 77)
  • Primer 78 GGTCTCACACCCTCCAGT (SEQ ID NO: 78)
  • Primer 79 GCCTCCTCCGCGGGCATA (SEQ ID NO: 79)
  • Primer 80 CCCACTCCATGAGGTATTTCGA (SEQ ID NO: 80)
  • Primer 81 CGCGAGTCCGAGGACGGA (SEQ ID NO: 81)
  • Primer 82 GCTACGTGGACGACACGCT (SEQ ID NO: 82)
  • Primer 83 GGTCTCACACTTGGCAGAG (SEQ ID NO: 83)
  • Primer 84 CACACCATCCAGAGGATGTC (SEQ ID NO: 84)
  • Primer 85 TCCGCGGGTATGACCAGGA (SEQ ID NO: 85)
  • Primer 86 GCTGCGACCTGGGGCCC (SEQ ID NO: 86)
  • Primer 87 CCGCGGGTACCACCAGGA (SEQ ID NO: 87)
  • Primer 88 CCCACTCCATGAGGTATTTCC (SEQ ID NO: 88)
  • Primer 89 GTCTCACACCCTCCAGAGC (SEQ ID NO: 89)
  • Precursor 90 GGAGCCCCGCTTCATTG (SEQ ID NO: 90)
  • Primer 91 GAGGTATTTCYACACCGCCA (SEQ ID NO: 91)
  • Primer 92 GAGGTATTTCTACACCGCCA (SEQ ID NO: 92)
  • Primer 93 GTCTCACACCCTCCAGAGC (SEQ ID NO: 93)
  • Primer 94 AGATCACCCAGCGCAAGTG (SEQ ID NO: 94)
  • Primer 95 CATGAGGTATTTCCACACCT (SEQ ID NO: 95)
  • Primer 96 CTACGTGGACGACACGCA (SEQ ID NO: 96)
  • Primer 97 CGCGAGTCCGAGGATGGC (SEQ ID NO: 97)
  • Precursor 98 GCCGCGAGTCCGAGAGA (SEQ ID NO: 98)
  • Precursor 100 ATGAGGTATTTCTACACCTCCG (SEQ ID NO: 100)
  • Precursor 101 GCCGCGAGTCCGAGGAC (SEQ ID NO: 101)
  • Primer 102 ACGCCGCGAGTCCGACAGG (SEQ ID NO: 102)
  • Precursor 103 GCCGCGAGTCCGAGAGA (SEQ ID NO: 103)
  • Precursor 104 GGGGAGCCCCGCTTCATCT (SEQ ID NO: 104)
  • Primer 105 GCTACGTGGACGACACGCT (SEQ ID NO: 105)
  • Precursor 106 CCGCGGGCATGACCAGTC (SEQ ID NO: 106)
  • Primer 107 CTCAGTGGGCTACGTGGACGG (SEQ ID NO: 107)
  • Primer 108 TCCGCGGGCATAACCAGTTA (SEQ ID NO: 108)
  • Primer 109 AGTACGCCTACGACGGCAAA (SEQ ID NO: 109)
  • Primer 110 GGCCAG GGTCTCACACTTGG (SEQ ID NO: 110)
  • Precursor 111 CCGCGGGCATAACCAGTTC (SEQ ID NO: 111)
  • Primer 112 G GGTCTCACATCATCCAGGT (SEQ ID NO: 112)
  • Primer 113 GACGACACGCAGTTCGTGA (SEQ ID NO: 113)
  • Primer 114 GACGACACGCTGTTCGTGA (SEQ ID NO: 114)
  • Precursor 115 GACGACACCCAGTTCGTGA (SEQ ID NO: 115)
  • Primer 116 GACGGCACCCAGTTCGTGA (SEQ ID NO: 116)
  • Primer 118 CTAGCGCGCTCCAGCTTGT (SEQ ID NO: 118)
  • Primer 119 CAGTCTGTGTGTTGGTCTTGA (SEQ ID NO: 119)
  • Primer 120 GGTCAGTCTGTGCCTTGGC (SEQ ID NO: 120)
  • Primer 121 TCTGCGCGGAGGCCTTCAT (SEQ ID NO: 121)
  • Primer 122 CCTCCAGGTAGGCTCTGTCC (SEQ ID NO: 122)
  • Primer 123 CCAGGTATCTGCGGAGCGA (SEQ ID NO: 123)
  • Primer 124 CCTCCAGGTAGGCTCTGTCA (SEQ ID NO: 124)
  • Primer 125 GGAGCCACTCCACGCACAG (SEQ ID NO: 125)
  • Primer 126 CTTGTAGTAGCGGAGCGCGA (SEQ ID NO: 126)
  • Primer 127 CGCTGTCGAACCTCACGAACA (SEQ ID NO: 127)
  • Primer 128 GAGCAGGGTCCGCAGGTC (SEQ ID NO: 128)
  • Primer 129 CCTCCAACTTGCGCTGGGA (SEQ ID NO: 129)
  • Primer 130 CTCCTTCCTCGGACTCGT (SEQ ID NO: 130)
  • Primer 131 AGTCTGTGTGTTGGTCTTGC (SEQ ID NO: 131)
  • Primer 132 CTGTGCCTGGCGCTTGTACT (SEQ ID NO: 132)
  • Primer 133 TGTGCCTGGGCCTTGTA (SEQ ID NO: 133)
  • Primer 134 GGTCTTGDAGATCTGTGTGTTC (SEQ ID NO: 134)
  • Primer 135 TGGTCTTGGAGATCTGTGTCTC (SEQ ID NO: 135)
  • Primer 136 TGGTTGTAGTAGCGGAGCGC (SEQ ID NO: 136)
  • Primer 137 AGTCTGTGTGTTGGTCTTGG (SEQ ID NO: 137)
  • Precursor 138 GTCTGTGTGTTGGTCTTGTAG (SEQ ID NO: 138)
  • Primer 139 TGGTCTTGGAGATCTGTGTCTC (SEQ ID NO: 139)
  • Primer 140 GTTCCGCAGGCTCTCTCGGTA (SEQ ID NO: 140)
  • Primer 141 GTTGTAGTAGCGGAGCGCGG (SEQ ID NO: 141)
  • Primer 142 TTGTAGTAGCCGCGCAGGT (SEQ ID NO: 142)
  • Primer 143 GGAGCCACTCCACGCACGT (SEQ ID NO: 143)
  • Primer 144 CCCAATACTCCGGCCCCTCT (SEQ ID NO: 144)
  • Primer 145 CACGTGCCCTCCAGGTAGGT (SEQ ID NO: 145)
  • Primer 146 GGAGCCACTCCACGCACAG (SEQ ID NO: 146)
  • Primer 148 CGCTCTGGTTGTAGTAGCC (SEQ ID NO: 148)
  • HLA-A fluorescence probe 1 FAM 5'-CGT GGA TAG AGC AGG AGG GGC CGG AGT 3 'BHQ1 (SEQ ID NO: 151)
  • HLA-A fluorescence probe 2 FAM 5'-CGA CAC GCA GTT CGT GCG GTT CGA CAG C 3 'BHQ1 (SEQ ID NO: 152)
  • HLA-A Fluorescent Probe 3 FAM 5'-CCC TGC GCG GCT ACT ACA ACC AGA GC 3 'BHQ1 (SEQ ID NO: 153)
  • HLA-A fluorescence probe 4 FAM 5'-TGA AGG CCC ACT CAC AGA CTG ACC GA 3 'BHQ1 (SEQ ID NO: 154)
  • HLA-A Fluorescent Probe 5 FAM 5'-AGG ACC TGC GCT CTT GGA CCG CGG CGG AC 3 'BHQ1 (SEQ ID NO: 155)
  • HLA-B Fluorescent Probe 1 Cy5 5'-CAA RGA TTA CAT CGC CCT GAA CGA GGA CC 3 'BHQ1 (SEQ ID NO: 156)
  • HLA-B fluorescence probe 2 Cy5 5'-AGA GCA RGA GGG GCC GGA RTA TTG GGA C 3 'BHQ1 (SEQ ID NO: 157)
  • HLA-B Fluorescence Probe 3 Cy5 5'-TGG AGT GGC TCC GCA GAT ACC TGG AGA ACG 3 'BHQ1 (SEQ ID NO: 158)
  • HLA-B fluorescent probe 4 Cy5 5'-CGT GAG GTT CGA CAG CGA CGC CGC GAG TCC 3 'BHQ1 (SEQ ID NO: 159)
  • HLA-B fluorescent probe 5 Cy5 5'-CCC GGC CGC GGG GAG CCC CGC TTC ATC 3 'BHQ1 (SEQ ID NO: 160)
  • HLA-DRB1 Fluorescent Probe 1 FAM 5'-CGC TTC GAC AGC GAC GTG GGG GAG TWC CG 3 'BHQ1 (SEQ ID NO: 161)
  • HLA-DRB1 fluorescent probe 2 FAM 5'-TCA TTT CTT CAA CGG GAC GGA GCG GGT GC 3 'BHQ1 (SEQ ID NO: 162)
  • Positive control standard gene fluorescent probe 2 Cyanine 3 5'-CCA CAC AGA ACT AAC CTC CAA CCA ACA ATC AGC 3'BHQ2 (SEQ ID NO: 163).
  • DNA isolated from blood was analyzed for specific amplification pairs capable of amplifying 2 mM dATP, dGTP, dCTP, dTTP and HLA-A, B, and DRB1 alleles, primer pairs for amplifying positive control standard genes, and for measuring amplification.
  • annealing (45 seconds at 70 ° C.) and extension (30 seconds at 72 ° C.) are performed five times
  • Annealing (45 seconds at 70 ° C.) and extension (30 seconds at 72 ° C.) were performed 30 times in total
  • real-time amplification was measured at 30 seconds at 72 ° C. in 2 steps. .
  • the amplification was measured at 490 nm for FAM, 532 nm for Cyanine 3, and 670 nm for cyanine 5.
  • the concentration of the specific primer used was carried out at 1.0 M and the positive control group was used at 0.5 M.
  • the polymerase chain reaction buffer used was Tris base 67 mM, ammonium sulphate 16.6 mM Tween 0.1%, and MgCl 2 0.2 mM.
  • the dNTP concentration used was 0.2 mM and Taq polymerase (GLS) was used.
  • the real time PCR instrument used was a product of ABI 7500.
  • the number of the tube whose starting point was measured is A1 (HLA-A CT value: 23.383, IC CT value: 25.039), A3 (HLA-A CT value: 22.186, IC CT value: 27.259), A10 (HLA-B CT value: 22.092, IC CT value: 22.092), B3 (HLA-A CT value: 20.735, IC CT value: 24.040), B4 (HLA-A CT value: 22.013, HLA-B CT value: 28.033 , IC CT value: 26.093), C4 (HLA-DR CT value: 23.597, IC CT value: 25.301), C10 (HLA-DR CT value: 24.170, IC CT value: 25.495), D7 (HLA-B CT value: 23.106, IC CT value: 26.308), D8 (HLA-DR CT value: 21.199, IC CT value: 26.180), D9 (HLA-B CT value: 22.568,
  • CT value was measured between 20 and 28 only in IC gene.
  • Table 1 shows that the A1, A3, B3, and B4 tubes were amplified in the HLA-A gene to be identified as HLA-A * 01, HLA-A * 03, and in the HLA-B gene, A10, B4, and D7.
  • D9, D11 tubes are amplified to HLA-B * 44, HLA-B * 57, HLA-Bw4 positive
  • C4, C10, C12, D8, D9 tubes are amplified to HLA-DRB1 * 07, HLA-DRB1 * 13, HLA-DRB3 positive and HLA-DRB4 positive were found.

Abstract

The present invention relates a method and a kit capable of simultaneously testing HLA-A, -B, and -DR alleles using a real-time multiplex polymerase chain reaction and, more specifically, to a method and a kit for HLA allele typing through a fluorescence value which is shown by using primers capable of specifically binding to HLA alleles for DNA extracted from a sample and a fluorescent probe capable of detecting amplification products amplified by the primers.

Description

스피드 멀티플렉스 PCR법을 이용한 HLA 대립유전자 검사방법 및 검사키트HLA allele test method and test kit using speed multiplex PCR method
본 발명은 실시간 멀티플렉스 중합효소연쇄반응을 이용하여 HLA-A, B, DR 대립유전자를 동시에 검사할 수 있는 방법 및 키트에 관한 것으로 시료에서 추출한 DNA를 대상으로 HLA 대립유전자에 특이적으로 결합할 수 있는 시발체들과 이들 시발체에 의하여 증폭된 증폭산물을 탐지할 수 있는 형광탐침자들을 이용하여 나타나는 형광 값을 가지고 HLA 대립유전자의 형별을 구분하는 방법 및 HLA 대립유전자의 형별을 구분하기 위한 키트에 관한 것이다.The present invention relates to a method and kit for simultaneously testing HLA-A, B, and DR alleles using real-time multiplex polymerase chain reaction, which specifically binds to HLA alleles in DNA extracted from a sample. In the kit for classifying HLA alleles and the type of HLA alleles with fluorescence values, which can be detected using a number of primers and fluorescent probes capable of detecting amplified products amplified by these primers, It is about.
사람의 백혈구 항원 (Human Leukocyte Antigen; 이하 HLA라 함)은 사람에서 발견되는 가장 다형성을 지닌 유전적 시스템으로 장기 및 조혈모세포이식의 성공에 있어서 주요한 인자로 알려져 있다. HLA 분자는 외래항원을 CD8+ T세포와 CD4+ T세포에 전달해주는 역할을 하고 있다. HLA 분자의 형별은 혈청학적 방법을 이용하여 형별을 분석하였으나 중합효소연쇄반응이 개발되면서 DNA 수준에서의 형별분석이 이루어지게 되었다. 이러한 DNA 수준의 HLA 형별검사가 이루어지면서 많은 수의 새로운 HLA 대립유전자가 밝혀지게 되어 현재 HLA-A는 2,013종류, HLA-B는 2,605종류, HLA-C는 1,551종류, HLA-DRB은 1,260종류가 밝혀져 있다 (2012년 7월 기준).Human Leukocyte Antigen (HLA) is the most polymorphic genetic system found in humans and is known as a major factor in the success of organ and hematopoietic stem cell transplantation. The HLA molecule is responsible for delivering foreign antigens to CD8 + T cells and CD4 + T cells. The type of HLA molecule was analyzed by serological method. However, as the polymerase chain reaction was developed, the type analysis was performed at the DNA level. As a result of HLA typing at the DNA level, a large number of new HLA alleles have been identified. Currently, 2,013 types of HLA-A, 2,605 types of HLA-B, 1,551 types of HLA-C, and 1,260 types of HLA-DRB. As of July 2012.
HLA 검사 방법으로는 항체-항원 반응에 의한 혈청학적 방법(Serotyping)과 HLA 유전자 염기서열 차이를 판별하는 분자생물학적 방법(DNA typing)이 있다. 혈청학적 방법은 검사 방법의 복잡함과 결과의 신뢰도가 분자생물학적 방법보다 상대적으로 낮아 현재 전 세계적으로 분자생물학적 방법을 많이 사용하고 있다.HLA assays include serotyping by antibody-antigen reactions and molecular typing (DNA typing) to determine HLA gene sequence differences. The serological method has a relatively low complexity of the test method and the reliability of the results compared to the molecular biological method is currently used a lot of molecular biological methods worldwide.
분자생물학적 방법으로는 크게 세 종류의 방법을 사용하는 데 첫 번째로 중합효소연쇄반응을 실시한 후 그 결과를 전기영동으로 확인하여 분석하는 PCR-SSP(sequence specific primers)방법, 두 번째로 중합효소연쇄반응을 실시한 후 탐침자를 이용하여 그 결과를 분석하는 PCR-SSOP(sequence specific oligonucleotide probes)방법, 세 번째로 중합효소연쇄반응을 실시한 후 염기서열분석에 의해 그 결과를 분석하는 PCR-SBT(sequence based typing)방법이 있고 이들 방법을 이용한 제품들이 개발되어 판매되고 있다. 그러나 종래의 방법은 사용자가 중합효소 연쇄반응을 기본적으로 실시하고 이후 다른 여러 반응 즉 혼성화반응(hybridization)과 전기영동을 하여야 HLA 검사를 할 수 있는 불편함과 시간적인 소모가 요구되었다.As molecular biology methods, three kinds of methods are used. First, PCR-SSP (sequence specific primers) method, which conducts polymerase chain reaction, and confirms and analyzes the result by electrophoresis, and second, polymerase chain PCR-SSOP (sequence specific oligonucleotide probes) method that analyzes the results using a probe after the reaction, and PCR-SBT (sequence based analysis) to analyze the results by sequencing after polymerase chain reaction. typing) and products using these methods are being developed and sold. However, the conventional method requires the user to perform the polymerase chain reaction basically and then perform several other reactions, that is, hybridization and electrophoresis, so that inconvenience and time consumption for HLA test are required.
따라서, 중합효소연쇄반응만으로 HLA 검사를 할 수 있는 실시간 중합효소 연쇄반응에 대한 방법이 계속적으로 요구되고 있다. 본 기술과 유사한 PCR-SSP법은 한 튜브에서 하나의 단일염기만을 볼 수 있는 방법으로 HLA 검사를 하는데 많은 수의 튜브를 이용하여야 하는 단점을 가지고 있어 한 튜브에서 여러 유전자를 증폭하는 multiplex PCR법에 대한 요구 또한 계속적으로 요구되었다.Therefore, there is a continuous need for a method for real-time polymerase chain reaction that can perform HLA test only by polymerase chain reaction. The PCR-SSP method similar to the present technique has a disadvantage in that a large number of tubes are used for HLA test because only one single base can be seen in one tube. There was also a demand for it.
본 발명의 목적은 HLA유전자에 다형성이 있는 부위를 검출함에 있어서 하나의 튜브에서 두 유전자 (HLA-A와 HLA-B, HLA-B와 HLA-DRB)의 다형성이 있는 부위를 특이적으로 증폭할 수 있는 멀티플렉스 중합효소연쇄반응을 실시하고 증폭 유무를 형광탐침자를 이용하여 검출하여 HLA 대립유전자 형별을 할 수 있는 방법 및 키트를 제공하는 것이다.An object of the present invention is to specifically amplify a site with a polymorphism of two genes (HLA-A and HLA-B, HLA-B and HLA-DRB) in one tube in detecting a site with a polymorphism in an HLA gene. The present invention provides a method and kit for performing multiplex polymerase chain reaction and detecting the presence or absence of amplification by using a fluorescence probe.
상기 목적을 달성하기 위하여, 본 발명은 HLA-A, HLA-B 및 HLA-DRB로 이루어진 군에서 선택된 2 이상의 유전자에 대한 HLA 대립유전자를 실시간 PCR에 의해 특이적으로 증폭할 수 있는 시발체 세트, 및 상기 실시간 PCR에 의해 증폭된 산물을 탐침 할 수 있는 탐침자 세트를 포함하는 HLA 대립유전자 형별 키트를 제공한다. 또한 본 발명은 또한 시료에서 추출한 DNA를 대상으로 본 발명의 HLA 대립유전자용 검출세트를 이용하여 실시간 중합효소연쇄반응(real time PCR)을 실시하는 단계; 및 상기 실시간 PCR 결과를 확인하여 HLA 대립유전자를 형별하는 단계를 포함하는 HLA 대립유전자의 형별 방법을 제공한다.In order to achieve the above object, the present invention provides a primer set capable of specifically amplifying HLA alleles for two or more genes selected from the group consisting of HLA-A, HLA-B and HLA-DRB by real-time PCR, and It provides a HLA allele type kit comprising a set of probes that can probe the product amplified by the real-time PCR. In another aspect, the present invention is the step of performing a real time PCR on the DNA extracted from the sample using the detection set for the HLA allele of the present invention; And identifying the HLA allele by identifying the real-time PCR result.
본 발명의 실시간 중합효소연쇄반응을 이용한 HLA 대립유전자 형별 선별 키트는 형광시발체 대신 HLA 대립유전자에 특이적인 일반 시발체로 상기 유전자를 멀티플렉스로 증폭하고, 각각의 종류 유전자의 증폭 유무만을 검출할 수 있는 형광탐침자를 사용함으로써 전기영동 없이 실시간 중합효소연쇄반응을 통해 HLA 대립유전자를 형별 가능하며, 또한 현재 상품으로 나와 있는 HLA 검사키트(96 well/1인) 및 검사방법 (일반 전기영동법)보다 더 경제적이고 효율성이 높은 키트(96 well/2인)와 검사방법 (Real-time multiplex PCR)을 사용함으로써, 하나의 검사 키트로 장기이식 희망 환자와 공여자를 동시에 검사할 수 있어 기술적 편의성과 경제성이 우수한 시스템으로 한 유전자에 여러 단일염기변이가 존재하는 유전자의 형별 분석 시 용이하게 사용될 수 있다.HLA allele type selection kit using the real-time polymerase chain reaction of the present invention is a general primer specific for HLA alleles instead of fluorescent primers to amplify the genes in multiplex and detect only the amplification of each type gene The use of fluorescent probes allows the identification of HLA alleles through real-time polymerase chain reactions without electrophoresis, and is more economical than HLA test kits (96 wells per person) and test methods (general electrophoresis) currently available on the market. By using an efficient and highly efficient kit (96 well / 2 people) and a test method (Real-time multiplex PCR), a single test kit can simultaneously test patients and donors for organ transplantation, and thus have excellent technical convenience and economic efficiency. As a result, it can be easily used for type analysis of genes in which several single base mutations exist in a gene.
도 1a 내지 도 1e는 HLA-A, B, DR 대립유전자 형별을 구분하기 위한 분석표를 나타낸 것이다.1A to 1E show analysis tables for distinguishing HLA-A, B, and DR allele types.
도 2는 HLA-A, B, DR 대립유전자 형별을 유전자 증폭 후의 결과그림을 나타낸 것이다.Figure 2 shows the result of the gene amplification after HLA-A, B, DR allele types.
도 3은 HLA-A, B, DR 대립유전자 형별 유전자 증폭 후 CT값을 나타낸 것이다.Figure 3 shows the CT value after gene amplification by HLA-A, B, DR allele type.
이하, 본 발명의 구성을 구체적으로 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.
본 발명은 HLA-A, HLA-B 및 HLA-DRB로 이루어진 군에서 선택된 2 이상의 유전자에 대한 HLA 대립유전자를 실시간 PCR에 의해 특이적으로 증폭할 수 있는 시발체 세트, 및 상기 실시간 PCR에 의해 증폭된 산물을 탐침 할 수 있는 탐침자 세트를 포함하는 HLA 대립유전자 형별 키트에 관한 것이다.The present invention provides a set of primers capable of specifically amplifying HLA alleles for two or more genes selected from the group consisting of HLA-A, HLA-B and HLA-DRB by real-time PCR, and amplified by the real-time PCR. A HLA allele typing kit comprising a set of probes capable of probing a product.
본 발명의 HLA 대립유전자를 실시간 중합효소연쇄반응으로 검출 할 수 있는 멀티플렉스 HLA 대립유전자형별 세트는 HLA-A, B, DRB유전자의 대립유전자에 각각 특이적으로 결합하여 증폭할 수 있는 시발체들과 각 HLA 유전자 별로 증폭산물에 혼성 가능하여 증폭산물을 확인할 수 있는 형광 표지된 탐침자를 이용하여 실시간 중합효소연쇄반응을 통해 HLA 대립유전자를 검사할 수 있다.The multiplex HLA allele type set, which can detect the HLA allele of the present invention by real-time polymerase chain reaction, has primers capable of specifically binding to and amplifying alleles of the HLA-A, B, and DRB genes, respectively. HLA alleles can be tested through real-time polymerase chain reaction using fluorescently labeled probes that can hybridize to amplification products for each HLA gene and identify the amplification products.
본 발명의 검출세트를 통해 검출할 수 있는 HLA 대립유전자는 HLA-A, HLA-B, HLA-DRB유전자이다.The HLA alleles detectable through the detection set of the present invention are HLA-A, HLA-B, HLA-DRB genes.
본 발명에서 언급한 실시간 중합효소연쇄반응법은 열순환기(thermal cycler)와 분광 형광 광도계가 일체화된 장치를 이용하여, 실시간으로 PCR 증폭산물의 생성과정을 모니터링하여 얻고자 하는 DNA의 양을 분석하는 방법을 의미한다. 실시간 PCR 방법은 PCR 증폭산물의 확인을 위한 전기영동이 필요 없으며, 증폭이 지수 함수적으로 일어나는 영역에서 증폭 산물의 양을 비교하여 더욱 정확한 정량이 가능한, 신속성과 정량성이 뛰어난 방법이다.The real-time polymerase chain reaction described in the present invention uses a device that integrates a thermal cycler and a spectral fluorescence photometer, and analyzes the amount of DNA to be obtained by monitoring the generation process of PCR amplification products in real time. Means the way. The real-time PCR method does not require electrophoresis for the identification of PCR amplification products, and is an excellent method for rapid and quantitative comparison of the amount of amplification products in a region where amplification occurs exponentially.
상기 시발체는 HLA 대립유전자를 종류별로 특이적으로 증폭시킬 수 있는 것으로, 일 구체예에 따르면, HLA 대립유전자의 다양성을 나타내는 위치를 기점으로 하여 17 내지 22 nt의 길이로 작재할 수 있다.The primer is capable of specifically amplifying HLA alleles by type. According to one embodiment, the primers may be constructed to have a length of 17 to 22 nt based on a position indicating diversity of the HLA alleles.
상기 시발체는 통상의 PCR을 통해 HLA-A 대립유전자, 예를 들어 HLA-A*01, A*02, A*03, A*11, A*23, A*24, A*25, A*26, A*29, A*30, A*31, A*32, A*33, A*34, A*36, A*43, A*66, A*68, A*69, A*74, A*80 등, 또는 HLA-B 대립유전자, 예를 들어, B*07, B*08, B*13, B*14, B*15(B62), B*15(63), B*15(70), B*15(71), B*15(72), B*15(75), B*15(76), B*15(77), B*15, B*18, B*27, B*35, B*37, B*38, B*39, B*40, B*40(B60), B*40(B61), B*41, B*42, B*44, B*45, B*46, B*47, B*48, B*49, B*50, B*51, B*52, B*53, B*54, B*55, B*56, B*57, B*58, B*59, B*67, B*78, B*81, B*82, B*83, Bw4, Bw6등, 또는 HLA-DRB 대립유전자, 예를 들어, DRB1*01, DRB1*15, DRB1*16, DRB1*03, DRB1*04, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB3, DRB4, DRB5 등을 증폭시킬 수 있다.The primers are HLA-A alleles via conventional PCR, for example HLA-A * 01, A * 02, A * 03, A * 11, A * 23, A * 24, A * 25, A * 26 , A * 29, A * 30, A * 31, A * 32, A * 33, A * 34, A * 36, A * 43, A * 66, A * 68, A * 69, A * 74, A * 80 and the like, or HLA-B alleles, for example B * 07, B * 08, B * 13, B * 14, B * 15 (B62), B * 15 (63), B * 15 (70 ), B * 15 (71), B * 15 (72), B * 15 (75), B * 15 (76), B * 15 (77), B * 15, B * 18, B * 27, B * 35, B * 37, B * 38, B * 39, B * 40, B * 40 (B60), B * 40 (B61), B * 41, B * 42, B * 44, B * 45, B * 46, B * 47, B * 48, B * 49, B * 50, B * 51, B * 52, B * 53, B * 54, B * 55, B * 56, B * 57, B * 58 , B * 59, B * 67, B * 78, B * 81, B * 82, B * 83, Bw4, Bw6, etc., or HLA-DRB alleles, for example DRB1 * 01, DRB1 * 15, DRB1 * 16, DRB1 * 03, DRB1 * 04, DRB1 * 11, DRB1 * 12, DRB1 * 13, DRB1 * 14, DRB1 * 07, DRB1 * 08, DRB1 * 09, DRB1 * 10, DRB3, DRB4, DRB5, etc. Can be amplified.
본 발명에서는 상기 시발체들을 이용하여 멀티플렉스 실시간 중합효소연쇄반응을 통해 HLA-A와 HLA-B, HLA-B와 HLA-DRB 유전자를 동시에 증폭시키는데 사용하였다.In the present invention, the primers were used to simultaneously amplify HLA-A and HLA-B, HLA-B and HLA-DRB genes through multiplex real-time polymerase chain reaction.
일 구체예에 따르면, 비특이적 반응이 일어나지 않는 조건을 고려하여 상기 시발체를 통해 증폭될 수 있는 증폭산물의 크기는 91 내지 274bp이고, 이때 모든 시발체의 Tm 값은 58 내지 65 이었다.According to one embodiment, the amplification products that can be amplified through the primer in consideration of the conditions that do not occur non-specific reaction is 91 to 274bp, wherein the Tm value of all the primers was 58 to 65.
상기 각 시발체가 특이적으로 증폭할 수 있는 HLA-A, B, DRB 대립유전자의 종류는 다음과 같다.The types of HLA-A, B and DRB alleles that can be specifically amplified by each primer are as follows.
1)One)
- 시발체 쌍 : 서열번호 1, 21, 78, 117-Primer pair: SEQ ID NO: 1, 21, 78, 117
- 검출 HLA 대립유전자 : A*01, A*36, B*07, B*40(B60), B*40, B*42, B*48, B*81Detection HLA alleles: A * 01, A * 36, B * 07, B * 40 (B60), B * 40, B * 42, B * 48, B * 81
2)2)
- 시발체 쌍 : 서열번호 2, 22, 79, 118-Primer pair: SEQ ID NO: 2, 22, 79, 118
- 검출 HLA 대립유전자 : A*02, A*03, A*30, B*07, B*08, B*35, B*37, B*41, B*42, B*44, B*78Detection HLA alleles: A * 02, A * 03, A * 30, B * 07, B * 08, B * 35, B * 37, B * 41, B * 42, B * 44, B * 78
3)3)
- 시발체 쌍 : 서열번호 3, 23, 80, 119-Primer pair: SEQ ID NO: 3, 23, 80, 119
- 검출 HLA 대립유전자 : A*02, A*03, A*32, B*08, B*15, B*51Detection HLA allele: A * 02, A * 03, A * 32, B * 08, B * 15, B * 51
4)4)
- 시발체 쌍 : 서열번호 4, 24, 81, 119-Primer pair: SEQ ID NO: 4, 24, 81, 119
- 검출 HLA 대립유전자 : A*01, A*03, A*11, B*08, B*18, B*35, B*37, B*38, B*44, B*51, B*53, B*78Detection HLA alleles: A * 01, A * 03, A * 11, B * 08, B * 18, B * 35, B * 37, B * 38, B * 44, B * 51, B * 53, B * 78
5)5)
- 시발체 쌍 : 서열번호 5, 25, 82, 120-Primer pair: SEQ ID NOs: 5, 25, 82, 120
- 검출 HLA 대립유전자 : A*24, A*33, B*27, B*44Detection HLA allele: A * 24, A * 33, B * 27, B * 44
6)6)
- 시발체 쌍 : 서열번호 6, 26-Primer pair: SEQ ID NO: 6, 26
- 검출 HLA 대립유전자 : A*02,A*24, A*25Detected HLA alleles: A * 02, A * 24, A * 25
7)7)
- 시발체 쌍 : 서열번호 7, 27, 81, 121-Primer pair: SEQ ID NO: 7, 27, 81, 121
- 검출 HLA 대립유전자 : A*01, A*11, A*25, A*66, A*26, A*33, A*34, B*58Detection HLA alleles: A * 01, A * 11, A * 25, A * 66, A * 26, A * 33, A * 34, B * 58
8)8)
- 시발체 쌍 : 서열번호 8, 28, 83, 122-Primer pair: SEQ ID NOs: 8, 28, 83, 122
- 검출 HLA 대립유전자 : A*29, B*41, B*44, B*45Detection HLA allele: A * 29, B * 41, B * 44, B * 45
9)9)
- 시발체 쌍 : 서열번호 9, 27, 84, 122-Primer pair: SEQ ID NOs: 9, 27, 84, 122
- 검출 HLA 대립유전자 : A*01, A*03, A*11, A*25, A*66, A*26, A*34, A*43, B*08, B*37Detection HLA alleles: A * 01, A * 03, A * 11, A * 25, A * 66, A * 26, A * 34, A * 43, B * 08, B * 37
10)10)
- 시발체 쌍 : 서열번호 10, 29, 85, 123-Primer pair: SEQ ID NOs: 10, 29, 85, 123
- 검출 HLA 대립유전자 : A*30, B*44, B*83Detection HLA allele: A * 30, B * 44, B * 83
11)11)
- 시발체 쌍 : 서열번호 11, 26, 86, 124-Primer pair: SEQ ID NOs: 11, 26, 86, 124
- 검출 HLA 대립유전자 : A*02, A*25, A*32, B*35, B*41, B*44, B*45, B*53, B*82Detection HLA alleles: A * 02, A * 25, A * 32, B * 35, B * 41, B * 44, B * 45, B * 53, B * 82
12)12)
- 시발체 쌍 : 서열번호 8, 30, 87, 125-Primer pair: SEQ ID NOs: 8, 30, 87, 125
- 검출 HLA 대립유전자 : A*33, B*15(B63), B*15, B*27, B*51, B*57Detection HLA alleles: A * 33, B * 15 (B63), B * 15, B * 27, B * 51, B * 57
13)13)
- 시발체 쌍 : 서열번호 12, 31, 88, 126-Primer pair: SEQ ID NOs: 12, 31, 88, 126
- 검출 HLA 대립유전자 : A*02, A*25, A*66, A*26, A*34, A*43, B*18, B*27, B*40, B*44, B*49, B*51, B*57Detection HLA alleles: A * 02, A * 25, A * 66, A * 26, A * 34, A * 43, B * 18, B * 27, B * 40, B * 44, B * 49, B * 51, B * 57
14)14)
- 시발체 쌍 : 서열번호 13, 32, 89, 122-Primer pair: SEQ ID NO: 13, 32, 89, 122
- 검출 HLA 대립유전자 : A*29, A*31, B*07, B*08, B*35, B*37, B*41, B*42, B*44, B*78Detection HLA alleles: A * 29, A * 31, B * 07, B * 08, B * 35, B * 37, B * 41, B * 42, B * 44, B * 78
15)15)
- 시발체 쌍 : 서열번호 14, 33, 90, 126-Primer pair: SEQ ID NOs: 14, 33, 90, 126
- 검출 HLA 대립유전자 : A*01, A*02, A*03, A*11, A*24, A*29, A*30, A*32, A*33, A*34, A*36, A*68, B*44, B*49, B*51, B*52, B*58Detection HLA alleles: A * 01, A * 02, A * 03, A * 11, A * 24, A * 29, A * 30, A * 32, A * 33, A * 34, A * 36, A * 68, B * 44, B * 49, B * 51, B * 52, B * 58
16)16)
- 시발체 쌍 : 서열번호 15, 34, 91, 127-Primer pairs: SEQ ID NOs: 15, 34, 91, 127
- 검출 HLA 대립유전자 : A*01, A*03, A*11, A*24, A*29, A*80, B*08, B*15, B*35, B*40, B*40(60), B*44, B*41, B*44, B*45, B*47, B*49, B*50, B*57Detection HLA alleles: A * 01, A * 03, A * 11, A * 24, A * 29, A * 80, B * 08, B * 15, B * 35, B * 40, B * 40 ( 60), B * 44, B * 41, B * 44, B * 45, B * 47, B * 49, B * 50, B * 57
17)17)
- 시발체 쌍 : 서열번호 16, 35-Primer pair: SEQ ID NOs: 16, 35
- 검출 HLA 대립유전자 : A*11, A*43Detection HLA allele: A * 11, A * 43
18)18)
- 시발체 쌍 : 서열번호 17, 36, 92, 128-Primer pair: SEQ ID NOs: 17, 36, 92, 128
- 검출 HLA 대립유전자 : A*02, A*69, B*15, B*40, B*47, B*53Detection HLA allele: A * 02, A * 69, B * 15, B * 40, B * 47, B * 53
19)19)
- 시발체 쌍 : 서열번호 18, 37, 93, 129-Primer pair: SEQ ID NOs: 18, 37, 93, 129
- 검출 HLA 대립유전자 : A*29, A*74, B*27, B*40, B*40(B60), B*48, B*81Detection HLA alleles: A * 29, A * 74, B * 27, B * 40, B * 40 (B60), B * 48, B * 81
20)20)
- 시발체 쌍 : 서열번호 19, 38, 94, 118-Primer pair: SEQ ID NOs: 19, 38, 94, 118
- 검출 HLA 대립유전자 : A*80, B*07, B*15, B*27, B*35, B*37, B*40, B*40(60), B*42, B*44, B*48, B*53, B*55Detection HLA alleles: A * 80, B * 07, B * 15, B * 27, B * 35, B * 37, B * 40, B * 40 (60), B * 42, B * 44, B * 48, B * 53, B * 55
21)21)
- 시발체 쌍 : 서열번호 20, 39, 95, 130-Primer pair: SEQ ID NO: 20, 39, 95, 130
- 검출 HLA 대립유전자 : A*23, A*24, A*29, A*31, B*13, B*18, B*40, B*40(60), B*40(61), B*44Detection HLA alleles: A * 23, A * 24, A * 29, A * 31, B * 13, B * 18, B * 40, B * 40 (60), B * 40 (61), B * 44
22)22)
- 시발체 쌍 : 서열번호 6, 40-Primer pair: SEQ ID NO: 6, 40
- 검출 HLA 대립유전자 : A*11, A*34, A*66, A*68, A*69Detection HLA alleles: A * 11, A * 34, A * 66, A * 68, A * 69
23)23)
- 시발체 쌍 : 서열번호 41, 55, 96, 131-Primer pair: SEQ ID NO: 41, 55, 96, 131
- 검출 HLA 대립유전자 : DRB1*01, B*08, B*14, B*15, B*15(70), B*15(71), B*38, B*39, B*56Detection HLA alleles: DRB1 * 01, B * 08, B * 14, B * 15, B * 15 (70), B * 15 (71), B * 38, B * 39, B * 56
24)24)
- 시발체 쌍 : 서열번호 42, 56, 97, 132-Primer pair: SEQ ID NOs: 42, 56, 97, 132
- 검출 HLA 대립유전자 : DRB1*15, B*46Detection HLA allele: DRB1 * 15, B * 46
25)25)
- 시발체 쌍 : 서열번호 42, 57, 98, 133-Primer pair: SEQ ID NOs: 42, 57, 98, 133
- 검출 HLA 대립유전자 : DRB1*16, B*07, B*14, B*38, B*42, B*55, B*56, B*67, B*78, B*81, B*82, B*83Detection HLA alleles: DRB1 * 16, B * 07, B * 14, B * 38, B * 42, B * 55, B * 56, B * 67, B * 78, B * 81, B * 82, B * 83
26)26)
- 시발체 쌍 : 서열번호 43, 58, 99, 134-Primer pair: SEQ ID NOs: 43, 58, 99, 134
- 검출 HLA 대립유전자 : DRB1*03, B*13, B*15, B*15(75), B*15(77), B*18, B*35, B*51, B*55Detection HLA alleles: DRB1 * 03, B * 13, B * 15, B * 15 (75), B * 15 (77), B * 18, B * 35, B * 51, B * 55
27)27)
- 시발체 쌍 : 서열번호 44, 59, 99, 135-Primer pair: SEQ ID NO: 44, 59, 99, 135
- 검출 HLA 대립유전자 : DRB1*04, B*13, B*15, B*15(62), B*15(76), B*27, B*40, B*44, B*52Detection HLA alleles: DRB1 * 04, B * 13, B * 15, B * 15 (62), B * 15 (76), B * 27, B * 40, B * 44, B * 52
28)28)
- 시발체 쌍 : 서열번호 45, 60, 100, 136-Primer pair: SEQ ID NO: 45, 60, 100, 136
- 검출 HLA 대립유전자 : DRB1*07, B*07, B*13, B*38, B*44, B*48, B*51, B*53, B*57Detection HLA alleles: DRB1 * 07, B * 07, B * 13, B * 38, B * 44, B * 48, B * 51, B * 53, B * 57
29)29)
- 시발체 쌍 : 서열번호 46, 61, 101, 137-Primer pair: SEQ ID NOs: 46, 61, 101, 137
- 검출 HLA 대립유전자 : DRB1*08, B*15, B*18, B*35, B*37, B*40, B*44, B*49, B*51, B*52, B*53, B*78Detection HLA alleles: DRB1 * 08, B * 15, B * 18, B * 35, B * 37, B * 40, B * 44, B * 49, B * 51, B * 52, B * 53, B * 78
30)30)
- 시발체 쌍 : 서열번호 47, 62, 97, 138-Primer pair: SEQ ID NOs: 47, 62, 97, 138
- 검출 HLA 대립유전자 : DRB1*09, B*15, B*15(75), B*55Detection HLA alleles: DRB1 * 09, B * 15, B * 15 (75), B * 55
31)31)
- 시발체 쌍 : 서열번호 48, 63, 102, 133-Primer pair: SEQ ID NOs: 48, 63, 102, 133
- 검출 HLA 대립유전자 : DRB1*10, B*54Detection HLA allele: DRB1 * 10, B * 54
32)32)
- 시발체 쌍 : 서열번호 43, 64, 103, 139-Primer pair: SEQ ID NOs: 43, 64, 103, 139
- 검출 HLA 대립유전자 : DRB1*03, DRB1*11, B*08, B*15(B70), B*15(B72), B*27, B*37, B*38, B*39, B*40(B60). B*48, B*52, B*55Detection HLA alleles: DRB1 * 03, DRB1 * 11, B * 08, B * 15 (B70), B * 15 (B72), B * 27, B * 37, B * 38, B * 39, B * 40 (B60). B * 48, B * 52, B * 55
33)33)
- 시발체 쌍 : 서열번호 46, 65, 104, 140-Primer pair: SEQ ID NOs: 46, 65, 104, 140
- 검출 HLA 대립유전자 : DRB1*08, DRB1*12, B*07, B*08, B*14, B*15, B*15(B70), B*15(B71), B*15(B72), B*18, B*35, B*39, B*40, B*40(B60), B*48, B*58Detection HLA alleles: DRB1 * 08, DRB1 * 12, B * 07, B * 08, B * 14, B * 15, B * 15 (B70), B * 15 (B71), B * 15 (B72) , B * 18, B * 35, B * 39, B * 40, B * 40 (B60), B * 48, B * 58
34)34)
- 시발체 쌍 : 서열번호 43, 66, 97, 141-Primer pair: SEQ ID NOs: 43, 66, 97, 141
- 검출 HLA 대립유전자 : DRB1*11, DRB1*13, DRB1*14, B*13, B*15(B77), B*44Detection HLA alleles: DRB1 * 11, DRB1 * 13, DRB1 * 14, B * 13, B * 15 (B77), B * 44
35)35)
- 시발체 쌍 : 서열번호 49, 67, 105, 142-Primer pair: SEQ ID NOs: 49, 67, 105, 142
- 검출 HLA 대립유전자 : DRB1*08, DRB1*11, DRB1*13, DRB1*14, B*07, B*08, B*15, B*27, B*35, B*40, B*40(B60), B*40(B61), B*41, B*44, B*45, B*50Detection HLA alleles: DRB1 * 08, DRB1 * 11, DRB1 * 13, DRB1 * 14, B * 07, B * 08, B * 15, B * 27, B * 35, B * 40, B * 40 ( B60), B * 40 (B61), B * 41, B * 44, B * 45, B * 50
36)36)
- 시발체 쌍 : 서열번호 43, 68, 106, 123-Primer pair: SEQ ID NO: 43, 68, 106, 123
- 검출 HLA 대립유전자 : DRB1*03, DRB1*08, DRB1*13, DRB1*14, B*15(B76)Detection HLA alleles: DRB1 * 03, DRB1 * 08, DRB1 * 13, DRB1 * 14, B * 15 (B76)
37)37)
- 시발체 쌍 : 서열번호 50, 69, 83, 143-Primer pair: SEQ ID NOs: 50, 69, 83, 143
- 검출 HLA 대립유전자 : DRB1*11, DRB1*14, B*41, B*45Detection HLA alleles: DRB1 * 11, DRB1 * 14, B * 41, B * 45
38)38)
- 시발체 쌍 : 서열번호 50, 70, 107, 144-Primer pair: SEQ ID NOs: 50, 70, 107, 144
- 검출 HLA 대립유전자 : DRB1*14, B*18Detection HLA allele: DRB1 * 14, B * 18
39)39)
- 시발체 쌍 : 서열번호 43, 71, 108, 125-Primer pair: SEQ ID NOs: 43, 71, 108, 125
- 검출 HLA 대립유전자 : DRB1*03, DRB1*13, DRB1*14, B*13, B*15(B62), B*40(B61), B*44, B*45, B*46, B*49, B*50, B*51, B*52, B*54, B*56, B*59, B*82Detection HLA alleles: DRB1 * 03, DRB1 * 13, DRB1 * 14, B * 13, B * 15 (B62), B * 40 (B61), B * 44, B * 45, B * 46, B * 49, B * 50, B * 51, B * 52, B * 54, B * 56, B * 59, B * 82
40)40)
- 시발체 쌍 : 서열번호 50, 71, 109, 125-Primer pair: SEQ ID NOs: 50, 71, 109, 125
- 검출 HLA 대립유전자 : DRB1*03, DRB1*13, DRB1*14, B*15, B*15(B62), B*35, B*40, B*44, B*49, B*50, B*51, B*52, B*56, B*58, B*78Detection HLA alleles: DRB1 * 03, DRB1 * 13, DRB1 * 14, B * 15, B * 15 (B62), B * 35, B * 40, B * 44, B * 49, B * 50, B * 51, B * 52, B * 56, B * 58, B * 78
41)41)
- 시발체 쌍 : 서열번호 44, 72, 110, 143-Primer pair: SEQ ID NO: 44, 72, 110, 143
- 검출 HLA 대립유전자 : DRB1*04, DRB1*14, B*08, B*13, 15, B*35, B*39, B*41, B*42, B*45, B*46, B*54, B*55, B*59, B*78Detection HLA alleles: DRB1 * 04, DRB1 * 14, B * 08, B * 13, 15, B * 35, B * 39, B * 41, B * 42, B * 45, B * 46, B * 54, B * 55, B * 59, B * 78
42)42)
- 시발체 쌍 : 서열번호 43, 73, 111, 145-Primer pair: SEQ ID NOs: 43, 73, 111, 145
- 검출 HLA 대립유전자 : DRB1*08, DRB1*11, DRB1*14, B*38, B*39, B*40, B*58, B*67Detection HLA alleles: DRB1 * 08, DRB1 * 11, DRB1 * 14, B * 38, B * 39, B * 40, B * 58, B * 67
43)43)
- 시발체 쌍 : 서열번호 51, 74, 112, 125-Primer pair: SEQ ID NOs: 51, 74, 112, 125
- 검출 HLA 대립유전자 : DRB1*13, DRB1*14, B*57, B*58Detection HLA allele: DRB1 * 13, DRB1 * 14, B * 57, B * 58
44)44)
- 시발체 쌍 : 서열번호 52, 75, 86, 146-Primer pair: SEQ ID NOs: 52, 75, 86, 146
- 검출 HLA 대립유전자 : DRB3, B*13, B*15(B62), B*18, B*35, B*40, B*40(B60), B*40(B61), B*44, B*48, B*49, B*53, B*54, B*55, B*56, B*57, B*58Detection HLA alleles: DRB3, B * 13, B * 15 (B62), B * 18, B * 35, B * 40, B * 40 (B60), B * 40 (B61), B * 44, B * 48, B * 49, B * 53, B * 54, B * 55, B * 56, B * 57, B * 58
45)45)
- 시발체 쌍 : 서열번호 53, 76, 106, 125-Primer pair: SEQ ID NO: 53, 76, 106, 125
- 검출 HLA 대립유전자 : DRB4, B*13, B*15, B*15(B62), B*15(B63), B*15(B70), B*15(B71), B*15(B72), B*15(B75), B*15(B76), B*15(B77), B*18, B*35, B*44, B*45, B*46, B*48, B*49, B*51, B*53, B*54, B*56, B*57, B*58Detection HLA alleles: DRB4, B * 13, B * 15, B * 15 (B62), B * 15 (B63), B * 15 (B70), B * 15 (B71), B * 15 (B72) , B * 15 (B75), B * 15 (B76), B * 15 (B77), B * 18, B * 35, B * 44, B * 45, B * 46, B * 48, B * 49, B * 51, B * 53, B * 54, B * 56, B * 57, B * 58
46)46)
- 시발체 쌍 : 서열번호 54, 77, 91, 133-Primer pair: SEQ ID NOs: 54, 77, 91, 133
- 검출 HLA 대립유전자 : DRB5, B*7, B*15, B*35, B*40, B*44, B*45, B*54, B*55, B*56, B*83Detection HLA alleles: DRB5, B * 7, B * 15, B * 35, B * 40, B * 44, B * 45, B * 54, B * 55, B * 56, B * 83
47)47)
- 시발체 쌍 : 서열번호 113, 114, 115, 116, 147-Primer pair: SEQ ID NOs: 113, 114, 115, 116, 147
- 검출 HLA 대립유전자 : Bw4Detection HLA allele: Bw4
48)48)
- 시발체 쌍 : 서열번호 113, 114, 115, 116, 148-Primer pair: SEQ ID NO: 113, 114, 115, 116, 148
- 검출 HLA 대립유전자 : Bw6Detection HLA allele: Bw6
또한, 형광탐침자는 상기 시발체를 통해 증폭된 산물을 모두 검출할 수 있도록 HLA 유전자 중 다형성이 없는 부위를 선택하여 27내지 33 nt의 길이로 작재할 수 있으며 구체적으로, 서열번호 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 또는 162에 기재된 서열을 가질 수 있다.In addition, the fluorescence probe may select a region without polymorphism in the HLA gene so as to detect all of the products amplified by the primers, and may operate in a length of 27 to 33 nt. Specifically, SEQ ID NOs: 151, 152, 153, It may have the sequence described in 154, 155, 156, 157, 158, 159, 160, 161 or 162.
보다 구체적으로, HLA-A 대립유전자에 대해서는 서열번호 151, 152, 153, 154 또는 155에 기재된 서열을 갖는 형광탐침자를 통해 증폭 유무를 검출할 수 있다. HLA-B 대립유전자에 대해서는 서열번호 156, 157, 158, 159 또는 160에 기재된 서열을 갖는 형광탐침자를 통해 증폭 유무를 검출할 수 있다. HLA-DRB1 대립유전자에 대해서는 서열번호 161 또는 162에 기재된 서열을 갖는 형광탐침자를 통해 증폭 유무를 검출할 수 있다. 이때 형광탐침자의 Tm 값은 60 내지 65℃ 일 수 있다.More specifically, for the HLA-A allele, the presence or absence of amplification can be detected through a fluorescent probe having a sequence set forth in SEQ ID NO: 151, 152, 153, 154 or 155. For the HLA-B allele, the presence or absence of amplification can be detected through a fluorescent probe having a sequence set forth in SEQ ID NO: 156, 157, 158, 159 or 160. For the HLA-DRB1 allele, the presence or absence of amplification can be detected through a fluorescent probe having a sequence set forth in SEQ ID NO: 161 or 162. At this time, the Tm value of the fluorescent probe may be 60 to 65 ℃.
상기 형광탐침자는 탐침자의 5' 및 3' 말단에 형광 표지 인자로 표지되어 있는 것으로, HLA 대립유전자의 검출세트의 종류에 따라 각각의 탐침자는 서로 상이한 형광 표지 인자로 표지된다. 형광 표지 인자는 종류에 따라 여기 및 방사 파장이 다르며, 사용방법 또한 상이하므로, 이를 고려하여 하나의 PCR 반응물에 함께 사용하는 형광 표지 인자는 별개로 검출가능한지 여부를 판단하여 선택 사용하여야 하며, 서로 다른 색상을 사용할 수 있다. 상기 형광 표지 인자에 대한 구체적인 사항 및 선택은 본원 발명에 속하는 기술분야의 당업자들에게 자명한 것이다.The fluorescent probe is labeled with fluorescent labeling factors at the 5 'and 3' ends of the probe, and each probe is labeled with a different fluorescent labeling factor according to the type of detection set of the HLA allele. Fluorescent labeling factors differ in excitation and emission wavelengths depending on the type, and the method of use is also different. Therefore, the fluorescent labeling factors used together in one PCR reaction should be selected and used separately in consideration of this. Colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
일 구체예에 따르면, 형광 표지 인자는 통상의 방법으로 본 발명에 따른 HLA 대립유전자 검사세트에 포함된 탐침자에 표지되며, 표지 방법은 인터컬레이팅 (interchelating) 방법, TaqManTM 프로브법 및 분자 비콘(Molecualr beacon) 방법들이 있다. 본 발명에서 사용하고 있는 TaqManTM 프로브법은 5'말단을 형광 표지인자(FAM 등)로 3'말단을 quencher 물질(TAMRA 등)로 수식한 올리고뉴클레오티드(TaqMan probe)를 PCR 반응액에 첨가하는 방법으로, TaqManTM 프로브가 어닐링 단계에서 주형 DNA에 특이적으로 혼성화하지만, 프로브상의 형광 억제 물질(Quencher)에 의해 형광 발생이 억제되고, 연장 단계에서 Taq DNA 중합효소가 갖는 5'→3' 엑소뉴클레아제 활성으로 주형에 혼성화한 TaqManTM 프로브만 분해되어 형광색소가 프로브에서 유리됨으로써 quencher에 의한 억제가 해제되어 형광을 발하게 된다.According to one embodiment, the fluorescent labeling factor is labeled on a probe included in the HLA allele test set according to the present invention in a conventional manner, and the labeling method is an interchelating method, a TaqMan ™ probe method and a molecular beacon (Molecualr beacon) methods are available. Method for adding TaqMan TM probe method was 5 used in this invention in a raised the reaction solution of PCR oligonucleotide (TaqMan probe) modified with "a terminal fluorescent marker (FAM, etc.) to the 3 'end a quencher substance (TAMRA etc.) As a TaqMan ™ probe specifically hybridizes to the template DNA in the annealing step, the fluorescence is suppressed by the fluorescence inhibitor (Quencher) on the probe, and the 5 '→ 3' exonew of the Taq DNA polymerase in the extension step Only the TaqMan TM probe hybridized to the template by clease activity is decomposed to release the fluorescent dye from the probe, thereby releasing the inhibition by the quencher and causing fluorescence.
이때 상기 프로브는 5'말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3' 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ(molecular grove binding non-fluorescence quencher)로 이루어진 군으로부터 선택된 1종의 형광 억제 물질(Quencher)로 표지될 수 있다.In this case, the probe 5 'end is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3' The terminus may be labeled with one fluorescence inhibitor (Quencher) selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and molecular grove binding non-fluorescence quencher (MGBNFQ).
또한, 본 발명의 검출세트는 검사의 신뢰도를 높이고, 실험 과정에서 사용되는 전체 PCR 튜브에 포함되어 주형 및 PCR 조건의 상태 확인을 위해 내부 양성대조군 유전자를 증폭하기 위한 시발체를 더 포함할 수 있다. 바람직하게는, -글로빈, 인간 -액틴, GAPDH(glyceraldehydes-3-phosphate dehydrogenase), 또는 Homo sapiens adenomatous polyposis coli (APC) 유전자 등을 사용할 수 있다. 보다 바람직하게는, Homo sapiens adenomatous polyposis coli (APC) 유전자를 증폭할 수 있는 서열번호 149 및 150에 기재된 서열로 이루어진 시발체 쌍을 사용하는 것이 좋다.In addition, the detection set of the present invention may further include a primer for amplifying the internal positive control gene to increase the reliability of the test and to be included in the entire PCR tube used in the experiment process to check the status of the template and the PCR conditions. Preferably, -globin, human -actin, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), or Homo sapiens adenomatous polyposis coli (APC) gene can be used. More preferably, a primer pair consisting of the sequences set forth in SEQ ID NOs: 149 and 150 capable of amplifying the Homo sapiens adenomatous polyposis coli (APC) gene may be used.
따라서, 본 발명의 검출세트는 양성대조군 유전자의 증폭 유무를 탐지하기 위한 형광탐침자를 추가로 포함할 수 있으며, 바람직하게는 서열번호 163에 기재된 서열을 갖는 것을 사용할 수 있다.Therefore, the detection set of the present invention may further include a fluorescent probe for detecting the presence or absence of the amplification of the positive control gene, preferably those having the sequence set forth in SEQ ID NO: 163.
일 구체예에 따르면, HLA-A, B, DRB1 대립유전자를 위한 시발체 및 형광탐침자를 포함하는 검출세트의 상세한 사항은 다음과 같다. 또한, 모든 검출세트에서 양성대조군 시발체 쌍(서열번호 149 및 150)과 형광탐침자(서열번호 163)의 종류는 동일하다.According to one embodiment, details of a detection set comprising primers and fluorescent probes for the HLA-A, B, DRB1 alleles are as follows. In addition, the types of positive control primer pairs (SEQ ID NOs: 149 and 150) and fluorescence probes (SEQ ID NO: 163) are the same in all detection sets.
1)One)
- 시발체 쌍 : 서열번호 1, 21, 78, 117-Primer pair: SEQ ID NO: 1, 21, 78, 117
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 151, 156HLA fluorescent probes: SEQ ID NOs: 151, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 103bp, 257bp-Amplification product size: 103bp, 257bp
2)2)
- 시발체 쌍 : 서열번호 2, 22, 79, 118-Primer pair: SEQ ID NO: 2, 22, 79, 118
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 156HLA fluorescent probes: SEQ ID NOs: 152, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 147bp, 226bp-Amplification product size: 147bp, 226bp
3)3)
- 시발체 쌍 : 서열번호 3, 23, 80, 119-Primer pair: SEQ ID NO: 3, 23, 80, 119
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 157HLA fluorescent probes: SEQ ID NOs: 155, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 206bp, 216bp-Amplification product size: 206bp, 216bp
4)4)
- 시발체 쌍 : 서열번호 4, 24, 81, 119-Primer pair: SEQ ID NO: 4, 24, 81, 119
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 157HLA fluorescent probes: SEQ ID NOs: 155, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 216bp, 101bp-Amplification product size: 216bp, 101bp
5)5)
- 시발체 쌍 : 서열번호 5, 25, 82, 120-Primer pair: SEQ ID NOs: 5, 25, 82, 120
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 157HLA fluorescent probes: SEQ ID NOs: 155, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 190bp, 147bp-Amplification product size: 190bp, 147bp
6)6)
- 시발체 쌍 : 서열번호 6, 26-Primer pair: SEQ ID NO: 6, 26
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152HLA fluorescent probe: SEQ ID NO: 152
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 234bp-Size of amplified product: 234bp
7)7)
- 시발체 쌍 : 서열번호 7, 27, 81, 121-Primer pair: SEQ ID NO: 7, 27, 81, 121
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 154, 157HLA fluorescent probes: SEQ ID NOs: 154, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 116bp, 98bp-Amplification product size: 116bp, 98bp
8)8)
- 시발체 쌍 : 서열번호 8, 28, 83, 122-Primer pair: SEQ ID NOs: 8, 28, 83, 122
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 156HLA fluorescent probes: SEQ ID NOs: 152, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 201bp, 213bp-Size of amplified product: 201bp, 213bp
9)9)
- 시발체 쌍 : 서열번호 9, 27, 84, 122-Primer pair: SEQ ID NOs: 9, 27, 84, 122
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 153, 156HLA fluorescence probes: SEQ ID NOs: 153, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 274bp, 208bp-Amplification product size: 274bp, 208bp
10)10)
- 시발체 쌍 : 서열번호 10, 29, 85, 123-Primer pair: SEQ ID NOs: 10, 29, 85, 123
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 156HLA fluorescent probes: SEQ ID NOs: 152, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 189bp, 189bp-Amplification product size: 189bp, 189bp
11)11)
- 시발체 쌍 : 서열번호 11, 26, 86, 124-Primer pair: SEQ ID NOs: 11, 26, 86, 124
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 156HLA fluorescent probes: SEQ ID NOs: 152, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 240bp, 186bp-Amplification product size: 240bp, 186bp
12)12)
- 시발체 쌍 : 서열번호 8, 30, 87, 125-Primer pair: SEQ ID NOs: 8, 30, 87, 125
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 156HLA fluorescent probes: SEQ ID NOs: 152, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 201bp, 176bp-Size of amplified product: 201bp, 176bp
13)13)
- 시발체 쌍 : 서열번호 12, 31, 88, 126-Primer pair: SEQ ID NOs: 12, 31, 88, 126
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 157HLA fluorescent probes: SEQ ID NOs: 155, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 131bp, 254bp-Amplification product size: 131bp, 254bp
14)14)
- 시발체 쌍 : 서열번호 13, 32, 89, 122-Primer pair: SEQ ID NO: 13, 32, 89, 122
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 156HLA fluorescent probes: SEQ ID NOs: 152, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 182bp, 212bp-Size of amplified product: 182bp, 212bp
15)15)
- 시발체 쌍 : 서열번호 14, 33, 90, 126-Primer pair: SEQ ID NOs: 14, 33, 90, 126
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 157HLA fluorescent probes: SEQ ID NOs: 155, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 180bp, 206bp-Amplification product size: 180bp, 206bp
16)16)
- 시발체 쌍 : 서열번호 15, 34, 91, 127-Primer pairs: SEQ ID NOs: 15, 34, 91, 127
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 160-HLA fluorescence probe: SEQ ID NOs: 155, 160
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 191bp, 100bp-Size of amplified product: 191bp, 100bp
17)17)
- 시발체 쌍 : 서열번호 16, 35-Primer pair: SEQ ID NOs: 16, 35
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152HLA fluorescent probe: SEQ ID NO: 152
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 199bp-Size of amplified product: 199bp
18)18)
- 시발체 쌍 : 서열번호 17, 36, 92, 128-Primer pair: SEQ ID NOs: 17, 36, 92, 128
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 157HLA fluorescent probes: SEQ ID NOs: 155, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 185bp, 232bp-Size of amplified product: 185bp, 232bp
19)19)
- 시발체 쌍 : 서열번호 18, 37, 93, 129-Primer pair: SEQ ID NOs: 18, 37, 93, 129
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 154, 156HLA fluorescent probes: SEQ ID NOs: 154, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 156bp, 173bp-Amplification product size: 156bp, 173bp
20)20)
- 시발체 쌍 : 서열번호 19, 38, 94, 118-Primer pair: SEQ ID NOs: 19, 38, 94, 118
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152, 158HLA fluorescent probes: SEQ ID NOs: 152, 158
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 186bp, 127bp-Amplification product size: 186bp, 127bp
21)21)
- 시발체 쌍 : 서열번호 20, 39, 95, 130-Primer pair: SEQ ID NO: 20, 39, 95, 130
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 155, 160-HLA fluorescence probe: SEQ ID NOs: 155, 160
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 177bp, 127bp-Size of amplified product: 177bp, 127bp
22)22)
- 시발체 쌍 : 서열번호 6, 40-Primer pair: SEQ ID NO: 6, 40
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 152HLA fluorescent probe: SEQ ID NO: 152
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 207bp-Size of amplified product: 207bp
23)23)
- 시발체 쌍 : 서열번호 41, 55, 96, 131-Primer pair: SEQ ID NO: 41, 55, 96, 131
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 196bp, 142bp-Size of amplified product: 196bp, 142bp
24)24)
- 시발체 쌍 : 서열번호 42, 56, 97, 132-Primer pair: SEQ ID NOs: 42, 56, 97, 132
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 207bp, 97bp-Amplification product size: 207bp, 97bp
25)25)
- 시발체 쌍 : 서열번호 42, 57, 98, 133-Primer pair: SEQ ID NOs: 42, 57, 98, 133
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 213bp, 98bp-Amplification product size: 213bp, 98bp
26)26)
- 시발체 쌍 : 서열번호 43, 58, 99, 134-Primer pair: SEQ ID NOs: 43, 58, 99, 134
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 222bp, 91bp-Size of amplification products: 222bp, 91bp
27)27)
- 시발체 쌍 : 서열번호 44, 59, 99, 135-Primer pair: SEQ ID NO: 44, 59, 99, 135
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 162, 157HLA fluorescent probes: SEQ ID NOs: 162, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 176bp, 89bp-Amplification product size: 176bp, 89bp
28)28)
- 시발체 쌍 : 서열번호 45, 60, 100, 136-Primer pair: SEQ ID NO: 45, 60, 100, 136
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 230bp, 248bp-Amplification product size: 230bp, 248bp
29)29)
- 시발체 쌍 : 서열번호 46, 61, 101, 137-Primer pair: SEQ ID NOs: 46, 61, 101, 137
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 213bp, 102bp-Amplification product size: 213bp, 102bp
30)30)
- 시발체 쌍 : 서열번호 47, 62, 97, 138-Primer pair: SEQ ID NOs: 47, 62, 97, 138
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 195bp, 100bp-Size of amplified product: 195bp, 100bp
31)31)
- 시발체 쌍 : 서열번호 48, 63, 102, 133-Primer pair: SEQ ID NOs: 48, 63, 102, 133
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 206bp, 100bp-Amplification product size: 206bp, 100bp
32)32)
- 시발체 쌍 : 서열번호 43, 64, 103, 139-Primer pair: SEQ ID NOs: 43, 64, 103, 139
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 206bp, 91bp-Amplification product size: 206bp, 91bp
33)33)
- 시발체 쌍 : 서열번호 46, 65, 104, 140-Primer pair: SEQ ID NOs: 46, 65, 104, 140
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 162bp, 189bp-Amplification product size: 162bp, 189bp
34)34)
- 시발체 쌍 : 서열번호 43, 66, 97, 141-Primer pair: SEQ ID NOs: 43, 66, 97, 141
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 188bp, 139bp-Size of amplified product: 188bp, 139bp
35)35)
- 시발체 쌍 : 서열번호 49, 67, 105, 142-Primer pair: SEQ ID NOs: 49, 67, 105, 142
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 157HLA fluorescent probes: SEQ ID NOs: 161, 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 194bp, 181bp-Size of amplified product: 194bp, 181bp
36)36)
- 시발체 쌍 : 서열번호 43, 68, 106, 123-Primer pair: SEQ ID NO: 43, 68, 106, 123
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 173bp, 278bp-Size of amplified product: 173bp, 278bp
37)37)
- 시발체 쌍 : 서열번호 50, 69, 83, 143-Primer pair: SEQ ID NOs: 50, 69, 83, 143
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 148bp, 234bp-Amplification product size: 148bp, 234bp
38)38)
- 시발체 쌍 : 서열번호 50, 70, 107, 144-Primer pair: SEQ ID NOs: 50, 70, 107, 144
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 159HLA fluorescent probes: SEQ ID NOs: 161, 159
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 148bp, 113bp-Amplification product size: 148bp, 113bp
39)39)
- 시발체 쌍 : 서열번호 43, 71, 108, 125-Primer pair: SEQ ID NOs: 43, 71, 108, 125
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 160bp. 177bp-Amplification product size: 160bp. 177 bp
40)40)
- 시발체 쌍 : 서열번호 50, 71, 109, 125-Primer pair: SEQ ID NOs: 50, 71, 109, 125
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 111bp, 161bp-Amplification product size: 111bp, 161bp
41)41)
- 시발체 쌍 : 서열번호 44, 72, 110, 143-Primer pair: SEQ ID NO: 44, 72, 110, 143
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 270bp, 240bp-Amplification product size: 270bp, 240bp
42)42)
- 시발체 쌍 : 서열번호 43, 73, 111, 145-Primer pair: SEQ ID NOs: 43, 73, 111, 145
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 224bp, 162bp-Amplification product size: 224bp, 162bp
43)43)
- 시발체 쌍 : 서열번호 51, 74, 112, 125-Primer pair: SEQ ID NOs: 51, 74, 112, 125
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 248bp, 235bp-Amplification product size: 248bp, 235bp
44)44)
- 시발체 쌍 : 서열번호 52, 75, 86, 146-Primer pair: SEQ ID NOs: 52, 75, 86, 146
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 225bp, 207bp-Size of amplified product: 225bp, 207bp
45)45)
- 시발체 쌍 : 서열번호 53, 76, 106, 125-Primer pair: SEQ ID NO: 53, 76, 106, 125
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 156HLA fluorescence probes: SEQ ID NOs: 161, 156
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 213bp, 176bp-Amplification product size: 213bp, 176bp
46)46)
- 시발체 쌍 : 서열번호 54, 77, 91, 133-Primer pair: SEQ ID NOs: 54, 77, 91, 133
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 161, 160-HLA fluorescence probe: SEQ ID NOs: 161, 160
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 263bp, 201bp-Amplification product size: 263bp, 201bp
47)47)
- 시발체 쌍 : 서열번호 113, 114, 115, 116, 147-Primer pair: SEQ ID NOs: 113, 114, 115, 116, 147
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 157HLA fluorescence probe: SEQ ID NO: 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 181bp-Size of amplified product: 181bp
48)48)
- 시발체 쌍 : 서열번호 113, 114, 115, 116, 148-Primer pair: SEQ ID NO: 113, 114, 115, 116, 148
- 양성대조군 시발체 쌍 : 서열번호 149과 150Positive control primer pairs: SEQ ID NOs: 149 and 150
- HLA 형광탐침자 : 서열번호 157HLA fluorescence probe: SEQ ID NO: 157
- 양성대조군 형광탐침자 : 서열번호 163Positive control fluorescent probe: SEQ ID NO: 163
- 증폭산물의 크기 : 181bp-Size of amplified product: 181bp
본 발명의 검출세트는 HLA 대립유전자의 종류에 특이적인 적어도 둘 이상의 시발체와 형광탐침자를 한 세트로 하여, 이들의 1종 이상의 조합을 동시에 사용하여 멀티플렉스 실시간 PCR을 수행할 수 있다.The detection set of the present invention can perform multiplex real-time PCR using at least two or more primers and fluorescent probes specific to the type of HLA allele, using one or more combinations thereof simultaneously.
본 발명은 또한 본 발명의 검출세트를 포함하는 HLA 대립유전자 형별 선별 키트에 관한 것이다.The invention also relates to an HLA allele type selection kit comprising the detection set of the invention.
본 발명의 HLA 대립유전자 형별 선별 키트를 통해 형별할 수 있는 HLA 대립유전자는 HLA-A, HLA-B, HLA-DRB1 대립유전자이다.HLA alleles that can be typed through the HLA allele type selection kit of the present invention are HLA-A, HLA-B, HLA-DRB1 alleles.
상기 시발체 쌍 및 형광탐침자는 스트립 또는 마이크로플레이트에 패키징 될 수 있으며, 당업계에 공지된 방법으로 패키징 된다. 또한, 본 발명의 키트는 텍 중합효소(Taq polymerase), MgCl2를 포함한 반응 완충액, dNTP 및 안정화제(stabilizer)로 이루어진 군으로부터 선택된 1종 이상을 더 포함할 수 있으며, 그 외에도 당업계에 공지된 다른 시약, 예컨대 실시간 PCR용 마스터 믹스를 추가적으로 포함할 수 있다.The primer pair and the fluorescence probe may be packaged in strips or microplates and packaged by methods known in the art. In addition, the kit of the present invention may further include one or more selected from the group consisting of Taq polymerase, a reaction buffer including MgCl 2 , dNTP, and a stabilizer, in addition to those known in the art. Other reagents, such as a master mix for real-time PCR.
본 발명의 HLA 대립유전자 형별 선별 키트를 사용할 경우, 주조직적합성항원 복합체를 유전자 수준에서 구별할 수 있으므로, 보다 정확한 조직적합성 판단에 널리 활용될 수 있다.When using the HLA allele type selection kit of the present invention, since the major histocompatibility antigen complex can be distinguished at the gene level, it can be widely used for more accurate histocompatibility determination.
본 발명은 또한,The present invention also provides
시료에서 추출한 DNA를 대상으로 본 발명의 HLA 대립유전자용 검출세트를 이용하여 실시간 중합효소연쇄반응(real time PCR)을 실시하는 단계; 및 상기 실시간 PCR 결과를 확인하여 HLA 대립유전자를 형별하는 단계를 포함하는 HLA 대립유전자의 형별 방법에 관한 것이다.Performing real time PCR on DNA extracted from a sample using a detection set for the HLA allele of the present invention; And identifying the HLA allele by identifying the real-time PCR result.
본 발명의 HLA 대립유전자의 형별 방법을 단계별로 보다 구체적으로 설명하면 다음과 같다.Referring to the method of typing the HLA allele of the present invention in more detail as follows.
제1단계는 HLA 대립유전자 증폭을 위한 시발체를 제작하는 단계로, HLA 대립유전자의 다양성을 나타내는 위치를 기점으로 하여 17 내지 24 nt 길이의 시발체를 작재한다.The first step is to prepare a primer for amplifying the HLA allele, and construct a primer having a length of 17 to 24 nt based on a position indicating the diversity of the HLA allele.
바람직하게는, 상기 시발체는 HLA-A 대립유전자를 선택적으로 증폭할 수 있는 서열번호 1 내지 40에 기재된 서열로부터 선택된 2종을 사용, HLA-DRB1 대립유전자를 선택적으로 증폭할 수 있는 서열번호 41 내지 77에 기재된 서열로부터 선택된 2종을 사용, HLA-B 대립유전자를 선택적으로 증폭할 수 있는 서열번호 78 내지 148에 기재된 서열로부터 선택된 2종 이상을 사용한다.Preferably, the primer is SEQ ID NO: 41 to which can selectively amplify the HLA-DRB1 allele using two species selected from the sequence set forth in SEQ ID NO: 1 to 40 to selectively amplify the HLA-A allele Two kinds selected from the sequences set forth in 77 are used, and two or more kinds selected from the sequences set forth in SEQ ID NOS: 78 to 148 capable of selectively amplifying the HLA-B allele are used.
상기 시발체는 검사의 신뢰도를 높이고, 실험 과정에서 사용되는 전체 PCR 튜브에 포함되어 주형 및 PCR 조건의 상태 확인을 위해 내부 양성대조군 유전자를 증폭하기 위한 서열번호 149 및 150에 기재된 서열로 이루어진 시발체 쌍을 더 포함할 수 있다.The primers include a primer pair consisting of the sequences set forth in SEQ ID NOs: 149 and 150 for amplification of the internal positive control genes to increase the reliability of the test and to be included in the entire PCR tube used during the experiment to check the status of the template and the PCR conditions. It may further include.
제2단계는 형광탐침자의 제작단계로 상기에서 작재된 시발체를 증폭하고, 이 증폭 산물을 검출할 수 있는 형광탐침자를 HLA 각 유전자별로 HLA-A는 5종류, HLA-B는 5종류, HLA-DRB1은 2종류 작재한다. 작재된 형광탐침자는 HLA 유전자 중 다형성이 없는 부위를 선택하여 27 내지 33 nt 길이로 작재한다.In the second step, a fluorescent probe capable of amplifying the primers described above, and detecting the amplified product, is classified into five types of HLA-A, five types of HLA-B, and HLA- for each HLA gene. Two types of DRB1 exist. The constructed fluorescence probe selects a region without polymorphism in the HLA gene and constructs it with a length of 27 to 33 nt.
구체적으로, HLA-A 대립유전자의 증폭 유무를 검출할 수 있는 서열번호 151, 152, 153, 154, 155에 기재된 서열일 수 있다. 또한, HLA-B 대립유전자의 증폭 유무를 검출할 수 있는 서열번호 156, 157, 158, 159, 160에 기재된 서열일 수 있다. 또한, HLA-DRB1 대립유전자의 증폭 유무를 검출할 수 있는 서열번호 161 또는 162에 기재된 서열일 수 있다.Specifically, it may be a sequence described in SEQ ID NOs: 151, 152, 153, 154, and 155 capable of detecting the amplification of the HLA-A allele. It may also be the sequence set forth in SEQ ID NO: 156, 157, 158, 159, 160 capable of detecting the amplification of the HLA-B allele. It may also be the sequence set forth in SEQ ID NO: 161 or 162 capable of detecting the amplification of the HLA-DRB1 allele.
또한, 양성대조군 유전자의 증폭 유무를 탐지하기 위한 서열번호 163에 기재된 서열을 갖는 형광탐침자를 더 포함할 수 있다.In addition, the method may further include a fluorescent probe having a sequence set forth in SEQ ID NO: 163 for detecting amplification of a positive control gene.
제3단계는 실시간 PCR을 통해 상기 시발체를 증폭하는 단계와 형광탐침자로 증폭 시 증폭 유무를 실시간 측정하는 단계가 동시에 이루어지는 단계이다.The third step is the step of amplifying the primer through real-time PCR and the step of real-time measuring the presence or absence of amplification when amplified by a fluorescent probe.
본 발명의 HLA 대립유전자를 형별하는 방법에 있어, 단일 또는 멀티플렉스 실시간 PCR 반응 조건은 통상적인 조건을 취할 수 있다. 또한, 단일 실시간 PCR과 멀티플렉스 실시간 PCR 반응은 동일한 조건으로 수행될 수 있으며, 일 예로 초기 변성(initial denaturation)을 95℃에서 10분간 수행한 후, 1단계로 변성(denaturation, 95℃에서 25초), 결합(annealing, 70℃에서 45초) 및 연장(extension, 72℃에서 30초) 을 5회 실시하고, 2단계로 변성(denaturation, 95℃에서 25초), 결합(annealing, 70℃에서 45초) 및 연장(extension, 72℃에서 30초) 을 30회 실시하여 총 35회 실시하는 조건을 취할 수 있다. 실시간 PCR 중 2단계의 연장단계에서 형광탐침자에 의해 생성된 형광을 측정한다.In the method for screening the HLA alleles of the present invention, single or multiplex real-time PCR reaction conditions can take conventional conditions. In addition, single real-time PCR and multiplex real-time PCR reaction may be performed under the same conditions. For example, initial denaturation is performed at 95 ° C. for 10 minutes and then denatured in one step (denaturation, 25 seconds at 95 ° C.). ), Annealing (45 seconds at 70 ° C.) and extension (30 seconds at 72 ° C.), five times, denaturation (25 seconds at 95 ° C.), annealing, at 70 ° C. 45 seconds) and extension (30 seconds at 72 ° C.) may be carried out 30 times to achieve a total of 35 times. Fluorescence generated by the fluorescence probe is measured in the extension step of the real time PCR.
또한, 본 발명의 HLA 대립유전자 형별 방법은 통상적인 실시간 PCR 방법 및 장치를 사용하여 실시할 수 있다. 상기 실시간 PCR 방법은 DNA 중합효소와 형광공명 에너지이동 (Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 PCR의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 이러한 방법은 특이적인 증폭 산물을 비 특이적인 증폭산물로부터 구별하여 확인할 수 있으며, 자동화된 양상으로 분석 결과를 쉽게 입수할 수 있다.In addition, the HLA allele typing method of the present invention can be carried out using conventional real-time PCR methods and apparatus. The real-time PCR method is a method of detecting and quantifying fluorescence performed in real time every cycle of PCR based on the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). This method distinguishes specific amplification products from non-specific amplification products and makes it easy to obtain analysis results in an automated manner.
본 발명에 사용 가능한 실시간 PCR 기기로는 AB 사의 Real-time PCR 기기 7900, 7500, 7300, Roche 사의 LightCycler 480, Stratagene사의 Mx3000p, 및 BioRad 사의 Chromo 4 기기 등이 있으나, 이로 한정되는 것은 아니다. PCR 종료 시 이러한 실시간 PCR 기기의 레이저는 증폭된 PCR 산물의 형광탐침자에 표지된 형광 표지 인자를 감지하여 도 2 와 같은 피크를 구현한다. 이에, 전기영동 과정 없이 기기에 내장된 프로그램을 실행시켜 자동으로 결과를 분석할 수 있다.The real-time PCR devices that can be used in the present invention include, but are not limited to, AB Real-time PCR devices 7900, 7500, 7300, LightCycler 480 from Roche, Mx3000p from Stratagene, and Chromo 4 from BioRad. At the end of PCR, the laser of the real-time PCR device senses the fluorescent labeling factor labeled on the fluorescent probe of the amplified PCR product to implement the peak as shown in FIG. 2. Thus, the results can be automatically analyzed by executing a program embedded in the device without the electrophoresis process.
본 발명에서는 일 예로, 자동화 프로그램으로 KoRASTM (코젠바이오텍)을 사용할 수 있다. 상기 자동화 프로그램을 사용하는 경우 분석된 결과를 O/X 타입 또는 Ct 타입으로 나타낼 수 있어 비숙련자라도 분석된 결과를 쉽게 알 수 있다.In the present invention, for example, KoRASTM (Kozen Biotech) can be used as an automation program. In the case of using the automated program, the analyzed result can be represented by the O / X type or the Ct type, so that even the unskilled person can easily know the analyzed result.
본 발명의 HLA 대립유전자용 검출세트 및 이를 이용한 HLA 대립유전자 형별 방법에 의해, 기존의 HLA 대립유전자를 식별하기 위한 PCR 과정을 현저하게 단축시킬 수 있을 뿐만 아니라, 그 결과를 실시간으로 신속하게 확인할 수 있다.The detection set for the HLA allele of the present invention and the HLA allele typing method using the same not only significantly shorten the PCR process for identifying the existing HLA allele, but also can quickly check the result in real time. have.
이러한 방법은 전기영동법으로는 분별하기 어려웠던 적은 농도의 DNA 절편의 유무나 100 bp 미만의 PCR 산물의 확인을 레이저 감광으로 구분할 수 있고, 증폭된 두 유전자간의 PCR산물의 크기가 유사한 경우, PCR 산물에 의한 오염에 대한 우려가 없으며, 결과가 분석되어 검사가 종료되기까지 모든 시험과정이 자동 시스템으로 운영되기 때문에 시험자의 실수, 검사의 오류 등이 발생할 확률이 낮으며, 데이터의 수집, 분석이 용이하고 소요되는 검사시간과 노동력을 최소화시킬 수 있다는 점에서 매우 유용하다.This method can be distinguished by laser sensitization of the presence of small concentrations of DNA fragments or less than 100 bp PCR products that were difficult to discern by electrophoresis. There is no concern about contamination due to contamination, and all the test processes are operated as an automatic system until the results are analyzed and the test is completed. Therefore, there is a low probability of error or error of the tester. This is very useful in minimizing the inspection time and labor required.
이하, 본 발명에 따르는 실시예 및 본 발명에 따르지 않는 비교 예를 통하여 본 발명을 보다 상세히 설명하나, 본 발명의 범위가 하기 제시된 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.
<실시예 1> 특이 HLA-A, B, DRB1 대립 유전자 증폭을 위한 시발체 제조Example 1 Preparation of Primer for Amplifying Specific HLA-A, B, DRB1 Alleles
HLA-A 대립유전자의 다양성을 나타내는 위치 부위를 3' 말단으로 하여 17 mer에서 24 mer의 시발체를 작재하였다. 각 시발체에 사용한 염기서열은 다음과 같다:From 17 mer to 24 mer primers were constructed with the 3 'end as the site region representing the diversity of the HLA-A allele. The base sequences used for each primer were as follows:
HLA-AHLA-A
시발체 1 : GACGCCGCGAGCCAGAA (서열번호 1)Primer 1: GACGCCGCGAGCCAGAA (SEQ ID NO: 1)
시발체 2 : AGCCCCGCTTCATCGCA(서열번호 2)Primer 2: AGCCCCGCTTCATCGCA (SEQ ID NO: 2)
시발체 3 : TGGCTGCGACGTGGGGT (서열번호 3)Primer 3: TGGCTGCGACGTGGGGT (SEQ ID NO: 3)
시발체 4 : GTTCTCACACCATCCAGATA (서열번호 4)Primer 4: GTTCTCACACCATCCAGATA (SEQ ID NO: 4)
시발체 5 : CACACCCTCCAGATGATGTT (서열번호 5)Primer 5: CACACCCTCCAGATGATGTT (SEQ ID NO: 5)
시발체 6 : CCCATGAGGTATTTCTACACC (서열번호 6)Precursor 6: CCCATGAGGTATTTCTACACC (SEQ ID NO: 6)
시발체 7 : GGAGTATTGGGACCGGAAC (서열번호 7)Precursor 7: GGAGTATTGGGACCGGAAC (SEQ ID NO: 7)
시발체 8 : CCACTCCATGAGGTATTTCAC (서열번호 8)Precursor 8: CCACTCCATGAGGTATTTCAC (SEQ ID NO: 8)
시발체 9 : CACAGACTGACCGAGTGG (서열번호 9)Primer 9: CACAGACTGACCGAGTGG (SEQ ID NO: 9)
시발체 10 : CCGTGTCCCGGCCCGGCAGT (서열번호 10)Primer 10: CCGTGTCCCGGCCCGGCAGT (SEQ ID NO: 10)
시발체 11 : CCACTCCATGAGGTATTTCTT (서열번호 11)Primer 11: CCACTCCATGAGGTATTTCTT (SEQ ID NO: 11)
시발체 12 : CGGGTACCAGCAGGACGCT (서열번호 12)Primer 12: CGGGTACCAGCAGGACGCT (SEQ ID NO: 12)
시발체 13 : CCCACTCCATGAGGTATTTCA (서열번호 13)Precursor 13: CCCACTCCATGAGGTATTTCA (SEQ ID NO: 13)
시발체 14 : CCTCCGCGGGTACCGG (서열번호 14)Precursor 14: CCTCCGCGGGTACCGG (SEQ ID NO: 14)
시발체 15 : TCCTCCGCGGGTACCGGC (서열번호 15)Precursor 15: TCCTCCGCGGGTACCGGC (SEQ ID NO: 15)
시발체 16 : CTCCATGAGGTATTTCTACTCC (서열번호 16)Precursor 16: CTCCATGAGGTATTTCTACTCC (SEQ ID NO: 16)
시발체 17 : GCGACGTGGGGTCGGACT (서열번호 17)Primer 17: GCGACGTGGGGTCGGACT (SEQ ID NO: 17)
시발체 18 : ACGCAGTTCGTGCGGTTT (서열번호 18)Primer 18: ACGCAGTTCGTGCGGTTT (SEQ ID NO: 18)
시발체 19 : GAGCCCCGCTTCATCGCA (서열번호 19)Precursor 19: GAGCCCCGCTTCATCGCA (SEQ ID NO: 19)
시발체 20 : CACACCCTCCAGATGATGTT (서열번호 20)Primer 20: CACACCCTCCAGATGATGTT (SEQ ID NO: 20)
시발체 21 : TCTGTGAGTGGGCCTTCAT (서열번호 21)Precursor 21: TCTGTGAGTGGGCCTTCAT (SEQ ID NO: 21)
시발체 22 : CACTTTCCGTGTCTCCCC (서열번호 22)Primer 22: CACTTTCCGTGTCTCCCC (SEQ ID NO: 22)
시발체 23 : CCACTCCACGCACGTGCCA (서열번호 23)Primer 23: CCACTCCACGCACGTGCCA (SEQ ID NO: 23)
시발체 24 : GCCCTCCAGGTAGGCTCTCT (서열번호 24)Promoter 24: GCCCTCCAGGTAGGCTCTCT (SEQ ID NO: 24)
시발체 25 : CCTCCAGGTAGGCTCTCTG (서열번호 25)Precursor 25: CCTCCAGGTAGGCTCTCTG (SEQ ID NO: 25)
시발체 26 : GCGCGATCCGCAGGCTCT (서열번호 26)Precursor 26: GCGCGATCCGCAGGCTCT (SEQ ID NO: 26)
시발체 27 : CCGGGGTCACTCACCGT (서열번호 27)Precursor 27: CCGGGGTCACTCACCGT (SEQ ID NO: 27)
시발체 28 : CTTCACATTCCGTGTCTGCA (서열번호 28)Precursor 28: CTTCACATTCCGTGTCTGCA (SEQ ID NO: 28)
시발체 29 : GTCTGTGAGTGGGCCTTCAC (서열번호 29)Precursor 29: GTCTGTGAGTGGGCCTTCAC (SEQ ID NO: 29)
시발체 30 : CTTCACATTCCGTGTGTTCCG (서열번호 30)Precursor 30: CTTCACATTCCGTGTGTTCCG (SEQ ID NO: 30)
시발체 31 : CTCCGCCTCATGGGCCGT (서열번호 31)Precursor 31: CTCCGCCTCATGGGCCGT (SEQ ID NO: 31)
시발체 32 : GGTCCCAATACTCAGGCCT (서열번호 32)Primer 32: GGTCCCAATACTCAGGCCT (SEQ ID NO: 32)
시발체 33 : GAGCCACTCCACGCACGT (서열번호 33)Primer 33: GAGCCACTCCACGCACGT (SEQ ID NO: 33)
시발체 34 : AGGTATCTGCGGAGCCCG (서열번호 34)Precursor 34: AGGTATCTGCGGAGCCCG (SEQ ID NO: 34)
시발체 35 : CCTTCATATTCCGTGTCTGCA (서열번호 35)Precursor 35: CCTTCATATTCCGTGTCTGCA (SEQ ID NO: 35)
시발체 36 : CCTCCAGGTAGGCTCTCAA (서열번호 36)Precursor 36: CCTCCAGGTAGGCTCTCAA (SEQ ID NO: 36)
시발체 37 : GCAGGGTCCCCAGGTCCA (서열번호 37)Precursor 37: GCAGGGTCCCCAGGTCCA (SEQ ID NO: 37)
시발체 38 : TCCCCAGGTTCGCTCGGTT (서열번호 38)Precursor 38: TCCCCAGGTTCGCTCGGTT (SEQ ID NO: 38)
시발체 39 : TCTCAACTGCTCCGCCACAC (서열번호 39)Precursor 39: TCTCAACTGCTCCGCCACAC (SEQ ID NO: 39)
시발체 40 : CACTCGGTCAGTCTGTGAC (서열번호 40)Precursor 40: CACTCGGTCAGTCTGTGAC (SEQ ID NO: 40)
HLA-DRBHLA-DRB
시발체 41 : CTTGTGGCAGCTTAAGTTTGAA (서열번호 41)Precursor 41: CTTGTGGCAGCTTAAGTTTGAA (SEQ ID NO: 41)
시발체 42 : CCTGTGGCAGCCTAAGAGG (서열번호 42)Precursor 42: CCTGTGGCAGCCTAAGAGG (SEQ ID NO: 42)
시발체 43 : GTTTCTTGGAGTACTCTACGTC (서열번호 43)Primer 43: GTTTCTTGGAGTACTCTACGTC (SEQ ID NO: 43)
시발체 44 : GTTTCTTGGAGCAGGTTAAACA (서열번호 44)Primer 44: GTTTCTTGGAGCAGGTTAAACA (SEQ ID NO: 44)
시발체 45 : CCTGTGGCAGGGTAAGTATA (서열번호 45)Precursor 45: CCTGTGGCAGGGTAAGTATA (SEQ ID NO: 45)
시발체 46 : AGTACTCTACGGGTGAGTGTT (서열번호 46)Precursor 46: AGTACTCTACGGGTGAGTGTT (SEQ ID NO: 46)
시발체 47 : GACGGAGCGGGTGCGGTA (서열번호 47)Precursor 47: GACGGAGCGGGTGCGGTA (SEQ ID NO: 47)
시발체 48 : GCGGTTGCTGGAAAGACGCG (서열번호 48)Primer 48: GCGGTTGCTGGAAAGACGCG (SEQ ID NO: 48)
시발체 49 : GTTTCTTGGAGTACTCTACGTC (서열번호 49)Precursor 49: GTTTCTTGGAGTACTCTACGTC (SEQ ID NO: 49)
시발체 50 : GGTTCCTGGACAGATACTTCC (서열번호 50)Precursor 50: GGTTCCTGGACAGATACTTCC (SEQ ID NO: 50)
시발체 51 : TACTCTACGTCTGAGTGTCAA (서열번호 51)Primer 51: TACTCTACGTCTGAGTGTCAA (SEQ ID NO: 51)
시발체 52 : ACAGCACGTTTCTTGGAGCT (서열번호 52)Precursor 52: ACAGCACGTTTCTTGGAGCT (SEQ ID NO: 52)
시발체 53 : GAGCGAGTGTGGAACCTGAT (서열번호 53)Primer 53: GAGCGAGTGTGGAACCTGAT (SEQ ID NO: 53)
시발체 54 : GTTTCTTGCAGCAGGATAAGTA (서열번호 54)Precursor 54: GTTTCTTGCAGCAGGATAAGTA (SEQ ID NO: 54)
시발체 55 : CCGCCTCTGCTCCAGGAG (서열번호 55)Precursor 55: CCGCCTCTGCTCCAGGAG (SEQ ID NO: 55)
시발체 56 : TCCACCGCGGCCCGCGC (서열번호 56)Precursor 56: TCCACCGCGGCCCGCGC (SEQ ID NO: 56)
시발체 57 : TAGGTGTCCACCGCGGCG (서열번호 57)Promoter 57: TAGGTGTCCACCGCGGCG (SEQ ID NO: 57)
시발체 58 : GCAGTAGTTGTCCACCCGGC (서열번호 58)Primer 58: GCAGTAGTTGTCCACCCGGC (SEQ ID NO: 58)
시발체 59 : CGCACGTACTCCTCTTGGTG (서열번호 59)Precursor 59: CGCACGTACTCCTCTTGGTG (SEQ ID NO: 59)
시발체 60 : CCCGTAGTTGTGTCTGCACAC (서열번호 60)Precursor 60: CCCGTAGTTGTGTCTGCACAC (SEQ ID NO: 60)
시발체 61 : TGCAGTAGGTGTCCACCAG (서열번호 61)Precursor 61: TGCAGTAGGTGTCCACCAG (SEQ ID NO: 61)
시발체 62 : ACCCCGTAGTTGTGTCTGCACAC (서열번호 62)Precursor 62: ACCCCGTAGTTGTGTCTGCACAC (SEQ ID NO: 62)
시발체 63 : GCTGCACTGTGAAGCTCTCAC (서열번호 63)Primer 63: GCTGCACTGTGAAGCTCTCAC (SEQ ID NO: 63)
시발체 64 : CTGGCTGTTCCAGTACTCCT (서열번호 64)Primer 64: CTGGCTGTTCCAGTACTCCT (SEQ ID NO: 64)
시발체 65 : CTGTTCCAGGACTCGGCGA (서열번호 65)Primer 65: CTGTTCCAGGACTCGGCGA (SEQ ID NO: 65)
시발체 66 : CCCGCTCGTCTTCCAGGAT (서열번호 66)Primer 66: CCCGCTCGTCTTCCAGGAT (SEQ ID NO: 66)
시발체 67 : CCCGCCTGTCTTCCAGGAA (서열번호 67)Precursor 67: CCCGCCTGTCTTCCAGGAA (SEQ ID NO: 67)
시발체 68 : CTGGCTGTTCCAGTACTCGG (서열번호 68)Primer 68: CTGGCTGTTCCAGTACTCGG (SEQ ID NO: 68)
시발체 69 : CACCTCGGCCCGCCTCC (서열번호 69)Precursor 69: CACCTCGGCCCGCCTCC (SEQ ID NO: 69)
시발체 70 : CACCGCGGCCCGCCTCT (서열번호 70)Primer 70: CACCGCGGCCCGCCTCT (SEQ ID NO: 70)
시발체 71 : CTGTTCCAGTGCTCCGCAG (서열번호 71)Precursor 71: CTGTTCCAGTGCTCCGCAG (SEQ ID NO: 71)
시발체 72 : GCTYACCTCGCCKCTGCAC (서열번호 72)Primer 72: GCTYACCTCGCCKCTGCAC (SEQ ID NO: 72)
시발체 73 : TCTGCAGTAGGTGTCCACCAG (서열번호 73)Precursor 73: TCTGCAGTAGGTGTCCACCAG (SEQ ID NO: 73)
시발체 74 : GCTGCACTGTGAAGCTCTCCA (서열번호 74)Precursor 74: GCTGCACTGTGAAGCTCTCCA (SEQ ID NO: 74)
시발체 75 : GCAGTAATTGTCCACCCGGC (서열번호 75)Primer 75: GCAGTAATTGTCCACCCGGC (SEQ ID NO: 75)
시발체 76 : CTGCACTGTGAAGCTCTCCA (서열번호 76)Precursor 76: CTGCACTGTGAAGCTCTCCA (SEQ ID NO: 76)
시발체 77 : CCGCTGCACTGTGAAGCTCT (서열번호 77)Primer 77: CCGCTGCACTGTGAAGCTCT (SEQ ID NO: 77)
HLA-BHLA-B
시발체 78: GGTCTCACACCCTCCAGT (서열번호 78)Primer 78: GGTCTCACACCCTCCAGT (SEQ ID NO: 78)
시발체 79: GCCTCCTCCGCGGGCATA (서열번호 79)Primer 79: GCCTCCTCCGCGGGCATA (SEQ ID NO: 79)
시발체 80: CCCACTCCATGAGGTATTTCGA (서열번호 80)Primer 80: CCCACTCCATGAGGTATTTCGA (SEQ ID NO: 80)
시발체 81: CGCGAGTCCGAGGACGGA (서열번호 81)Primer 81: CGCGAGTCCGAGGACGGA (SEQ ID NO: 81)
시발체 82: GCTACGTGGACGACACGCT (서열번호 82)Primer 82: GCTACGTGGACGACACGCT (SEQ ID NO: 82)
시발체 83: GGTCTCACACTTGGCAGAG (서열번호 83)Primer 83: GGTCTCACACTTGGCAGAG (SEQ ID NO: 83)
시발체 84: CACACCATCCAGAGGATGTC (서열번호 84)Primer 84: CACACCATCCAGAGGATGTC (SEQ ID NO: 84)
시발체 85: TCCGCGGGTATGACCAGGA (서열번호 85)Primer 85: TCCGCGGGTATGACCAGGA (SEQ ID NO: 85)
시발체 86: GCTGCGACCTGGGGCCC (서열번호 86)Primer 86: GCTGCGACCTGGGGCCC (SEQ ID NO: 86)
시발체 87: CCGCGGGTACCACCAGGA (서열번호 87)Primer 87: CCGCGGGTACCACCAGGA (SEQ ID NO: 87)
시발체 88: CCCACTCCATGAGGTATTTCC (서열번호 88)Primer 88: CCCACTCCATGAGGTATTTCC (SEQ ID NO: 88)
시발체 89: GTCTCACACCCTCCAGAGC (서열번호 89)Primer 89: GTCTCACACCCTCCAGAGC (SEQ ID NO: 89)
시발체 90: GGAGCCCCGCTTCATTG (서열번호 90)Precursor 90: GGAGCCCCGCTTCATTG (SEQ ID NO: 90)
시발체 91: GAGGTATTTCYACACCGCCA (서열번호 91)Primer 91: GAGGTATTTCYACACCGCCA (SEQ ID NO: 91)
시발체 92: GAGGTATTTCTACACCGCCA (서열번호 92)Primer 92: GAGGTATTTCTACACCGCCA (SEQ ID NO: 92)
시발체 93: GTCTCACACCCTCCAGAGC (서열번호 93)Primer 93: GTCTCACACCCTCCAGAGC (SEQ ID NO: 93)
시발체 94: AGATCACCCAGCGCAAGTG (서열번호 94)Primer 94: AGATCACCCAGCGCAAGTG (SEQ ID NO: 94)
시발체 95: CATGAGGTATTTCCACACCT (서열번호 95)Primer 95: CATGAGGTATTTCCACACCT (SEQ ID NO: 95)
시발체 96: CTACGTGGACGACACGCA (서열번호 96)Primer 96: CTACGTGGACGACACGCA (SEQ ID NO: 96)
시발체 97: CGCGAGTCCGAGGATGGC (서열번호 97)Primer 97: CGCGAGTCCGAGGATGGC (SEQ ID NO: 97)
시발체 98: GCCGCGAGTCCGAGAGA (서열번호 98)Precursor 98: GCCGCGAGTCCGAGAGA (SEQ ID NO: 98)
시발체 99: CGCCGCGAGTCCGAGGAT (서열번호 99)Primer 99: CGCCGCGAGTCCGAGGAT (SEQ ID NO: 99)
시발체 100: ATGAGGTATTTCTACACCTCCG (서열번호 100)Precursor 100: ATGAGGTATTTCTACACCTCCG (SEQ ID NO: 100)
시발체 101: GCCGCGAGTCCGAGGAC (서열번호 101)Precursor 101: GCCGCGAGTCCGAGGAC (SEQ ID NO: 101)
시발체 102: ACGCCGCGAGTCCGACAGG (서열번호 102)Primer 102: ACGCCGCGAGTCCGACAGG (SEQ ID NO: 102)
시발체 103: GCCGCGAGTCCGAGAGA (서열번호 103)Precursor 103: GCCGCGAGTCCGAGAGA (SEQ ID NO: 103)
시발체 104: GGGGAGCCCCGCTTCATCT (서열번호 104)Precursor 104: GGGGAGCCCCGCTTCATCT (SEQ ID NO: 104)
시발체 105: GCTACGTGGACGACACGCT (서열번호 105)Primer 105: GCTACGTGGACGACACGCT (SEQ ID NO: 105)
시발체 106: CCGCGGGCATGACCAGTC (서열번호 106)Precursor 106: CCGCGGGCATGACCAGTC (SEQ ID NO: 106)
시발체 107: CTCAGTGGGCTACGTGGACGG (서열번호 107)Primer 107: CTCAGTGGGCTACGTGGACGG (SEQ ID NO: 107)
시발체 108: TCCGCGGGCATAACCAGTTA (서열번호 108)Primer 108: TCCGCGGGCATAACCAGTTA (SEQ ID NO: 108)
시발체 109: AGTACGCCTACGACGGCAAA (서열번호 109)Primer 109: AGTACGCCTACGACGGCAAA (SEQ ID NO: 109)
시발체 110: GGCCAGGGTCTCACACTTGG (서열번호 110) Primer 110: GGCCAG GGTCTCACACTTGG (SEQ ID NO: 110)
시발체 111: CCGCGGGCATAACCAGTTC (서열번호 111)Precursor 111: CCGCGGGCATAACCAGTTC (SEQ ID NO: 111)
시발체 112: GGGTCTCACATCATCCAGGT (서열번호 112)Primer 112: G GGTCTCACATCATCCAGGT (SEQ ID NO: 112)
시발체 113: GACGACACGCAGTTCGTGA (서열번호 113)Primer 113: GACGACACGCAGTTCGTGA (SEQ ID NO: 113)
시발체 114: GACGACACGCTGTTCGTGA (서열번호 114)Primer 114: GACGACACGCTGTTCGTGA (SEQ ID NO: 114)
시발체 115: GACGACACCCAGTTCGTGA (서열번호 115)Precursor 115: GACGACACCCAGTTCGTGA (SEQ ID NO: 115)
시발체 116: GACGGCACCCAGTTCGTGA (서열번호 116)Primer 116: GACGGCACCCAGTTCGTGA (SEQ ID NO: 116)
시발체 117: CTTCCCGTTCTCCAGGTG (서열번호 117)Primer 117: CTTCCCGTTCTCCAGGTG (SEQ ID NO: 117)
시발체 118: CTAGCGCGCTCCAGCTTGT (서열번호 118)Primer 118: CTAGCGCGCTCCAGCTTGT (SEQ ID NO: 118)
시발체 119: CAGTCTGTGTGTTGGTCTTGA (서열번호 119)Primer 119: CAGTCTGTGTGTTGGTCTTGA (SEQ ID NO: 119)
시발체 120: GGTCAGTCTGTGCCTTGGC (서열번호 120)Primer 120: GGTCAGTCTGTGCCTTGGC (SEQ ID NO: 120)
시발체 121: TCTGCGCGGAGGCCTTCAT (서열번호 121)Primer 121: TCTGCGCGGAGGCCTTCAT (SEQ ID NO: 121)
시발체 122: CCTCCAGGTAGGCTCTGTCC (서열번호 122)Primer 122: CCTCCAGGTAGGCTCTGTCC (SEQ ID NO: 122)
시발체 123: CCAGGTATCTGCGGAGCGA (서열번호 123)Primer 123: CCAGGTATCTGCGGAGCGA (SEQ ID NO: 123)
시발체 124: CCTCCAGGTAGGCTCTGTCA (서열번호 124)Primer 124: CCTCCAGGTAGGCTCTGTCA (SEQ ID NO: 124)
시발체 125: GGAGCCACTCCACGCACAG (서열번호 125)Primer 125: GGAGCCACTCCACGCACAG (SEQ ID NO: 125)
시발체 126: CTTGTAGTAGCGGAGCGCGA (서열번호 126)Primer 126: CTTGTAGTAGCGGAGCGCGA (SEQ ID NO: 126)
시발체 127: CGCTGTCGAACCTCACGAACA (서열번호 127)Primer 127: CGCTGTCGAACCTCACGAACA (SEQ ID NO: 127)
시발체 128: GAGCAGGGTCCGCAGGTC (서열번호 128)Primer 128: GAGCAGGGTCCGCAGGTC (SEQ ID NO: 128)
시발체 129: CCTCCAACTTGCGCTGGGA (서열번호 129)Primer 129: CCTCCAACTTGCGCTGGGA (SEQ ID NO: 129)
시발체 130: CTCCTTCCTCGGACTCGT (서열번호 130)Primer 130: CTCCTTCCTCGGACTCGT (SEQ ID NO: 130)
시발체 131: AGTCTGTGTGTTGGTCTTGC (서열번호 131)Primer 131: AGTCTGTGTGTTGGTCTTGC (SEQ ID NO: 131)
시발체 132: CTGTGCCTGGCGCTTGTACT (서열번호 132)Primer 132: CTGTGCCTGGCGCTTGTACT (SEQ ID NO: 132)
시발체 133: TGTGCCTGGGCCTTGTA (서열번호 133)Primer 133: TGTGCCTGGGCCTTGTA (SEQ ID NO: 133)
시발체 134: GGTCTTGDAGATCTGTGTGTTC (서열번호 134)Primer 134: GGTCTTGDAGATCTGTGTGTTC (SEQ ID NO: 134)
시발체 135: TGGTCTTGGAGATCTGTGTCTC (서열번호 135)Primer 135: TGGTCTTGGAGATCTGTGTCTC (SEQ ID NO: 135)
시발체 136: TGGTTGTAGTAGCGGAGCGC (서열번호 136)Primer 136: TGGTTGTAGTAGCGGAGCGC (SEQ ID NO: 136)
시발체 137: AGTCTGTGTGTTGGTCTTGG (서열번호 137)Primer 137: AGTCTGTGTGTTGGTCTTGG (SEQ ID NO: 137)
시발체 138: GTCTGTGTGTTGGTCTTGTAG (서열번호 138)Precursor 138: GTCTGTGTGTTGGTCTTGTAG (SEQ ID NO: 138)
시발체 139: TGGTCTTGGAGATCTGTGTCTC (서열번호 139)Primer 139: TGGTCTTGGAGATCTGTGTCTC (SEQ ID NO: 139)
시발체 140: GTTCCGCAGGCTCTCTCGGTA (서열번호 140)Primer 140: GTTCCGCAGGCTCTCTCGGTA (SEQ ID NO: 140)
시발체 141: GTTGTAGTAGCGGAGCGCGG (서열번호 141)Primer 141: GTTGTAGTAGCGGAGCGCGG (SEQ ID NO: 141)
시발체 142: TTGTAGTAGCCGCGCAGGT (서열번호 142)Primer 142: TTGTAGTAGCCGCGCAGGT (SEQ ID NO: 142)
시발체 143: GGAGCCACTCCACGCACGT (서열번호 143)Primer 143: GGAGCCACTCCACGCACGT (SEQ ID NO: 143)
시발체 144: CCCAATACTCCGGCCCCTCT (서열번호 144)Primer 144: CCCAATACTCCGGCCCCTCT (SEQ ID NO: 144)
시발체 145: CACGTGCCCTCCAGGTAGGT (서열번호 145)Primer 145: CACGTGCCCTCCAGGTAGGT (SEQ ID NO: 145)
시발체 146: GGAGCCACTCCACGCACAG (서열번호 146)Primer 146: GGAGCCACTCCACGCACAG (SEQ ID NO: 146)
시발체 147: CGCTCTGGTTGTAGTAGCG (서열번호 147)Primer 147: CGCTCTGGTTGTAGTAGCG (SEQ ID NO: 147)
시발체 148: CGCTCTGGTTGTAGTAGCC (서열번호 148)Primer 148: CGCTCTGGTTGTAGTAGCC (SEQ ID NO: 148)
<실시예 2> 증폭 유무를 측정하기 위한 형광 탐침자의 제조Example 2 Preparation of Fluorescence Probe for Measuring Amplification
HLA-A, B, DRB1 유전자의 증폭유무를 측정하기 위하여 보편적인 형광탐침자를 12종류 작재하고 양성대조군 표준유전자 증폭 유무를 측정할 수 있는 보편적인 형광탐침자 1종류를 작재하였다. 각 형광탐침자에 사용한 염기 서열과 형광물질은 다음과 같다:To measure the amplification of the HLA-A, B, and DRB1 genes, 12 types of universal fluorescent probes were constructed and one type of universal fluorescent probes capable of measuring the presence or absence of positive control standard gene amplification were constructed. The base sequence and fluorescent material used for each fluorescent probe are as follows:
HLA-A 형광탐침자1: FAM 5'-CGT GGA TAG AGC AGG AGG GGC CGG AGT 3' BHQ1(서열번호 151)HLA-A fluorescence probe 1: FAM 5'-CGT GGA TAG AGC AGG AGG GGC CGG AGT 3 'BHQ1 (SEQ ID NO: 151)
HLA-A 형광탐침자2: FAM 5'-CGA CAC GCA GTT CGT GCG GTT CGA CAG C 3' BHQ1(서열번호 152)HLA-A fluorescence probe 2: FAM 5'-CGA CAC GCA GTT CGT GCG GTT CGA CAG C 3 'BHQ1 (SEQ ID NO: 152)
HLA-A 형광탐침자3: FAM 5'-CCC TGC GCG GCT ACT ACA ACC AGA GC 3' BHQ1(서열번호 153)HLA-A Fluorescent Probe 3: FAM 5'-CCC TGC GCG GCT ACT ACA ACC AGA GC 3 'BHQ1 (SEQ ID NO: 153)
HLA-A 형광탐침자4: FAM 5'-TGA AGG CCC ACT CAC AGA CTG ACC GA 3' BHQ1(서열번호 154)HLA-A fluorescence probe 4: FAM 5'-TGA AGG CCC ACT CAC AGA CTG ACC GA 3 'BHQ1 (SEQ ID NO: 154)
HLA-A 형광탐침자5: FAM 5'-AGG ACC TGC GCT CTT GGA CCG CGG CGG AC 3' BHQ1(서열번호 155)HLA-A Fluorescent Probe 5: FAM 5'-AGG ACC TGC GCT CTT GGA CCG CGG CGG AC 3 'BHQ1 (SEQ ID NO: 155)
HLA-B 형광탐침자1: Cy5 5'-CAA RGA TTA CAT CGC CCT GAA CGA GGA CC 3' BHQ1(서열번호 156)HLA-B Fluorescent Probe 1: Cy5 5'-CAA RGA TTA CAT CGC CCT GAA CGA GGA CC 3 'BHQ1 (SEQ ID NO: 156)
HLA-B 형광탐침자2: Cy5 5'-AGA GCA RGA GGG GCC GGA RTA TTG GGA C 3' BHQ1(서열번호 157)HLA-B fluorescence probe 2: Cy5 5'-AGA GCA RGA GGG GCC GGA RTA TTG GGA C 3 'BHQ1 (SEQ ID NO: 157)
HLA-B 형광탐침자3: Cy5 5'-TGG AGT GGC TCC GCA GAT ACC TGG AGA ACG 3' BHQ1(서열번호 158)HLA-B Fluorescence Probe 3: Cy5 5'-TGG AGT GGC TCC GCA GAT ACC TGG AGA ACG 3 'BHQ1 (SEQ ID NO: 158)
HLA-B 형광탐침자4: Cy5 5'-CGT GAG GTT CGA CAG CGA CGC CGC GAG TCC 3' BHQ1(서열번호 159)HLA-B fluorescent probe 4: Cy5 5'-CGT GAG GTT CGA CAG CGA CGC CGC GAG TCC 3 'BHQ1 (SEQ ID NO: 159)
HLA-B 형광탐침자5: Cy5 5'-CCC GGC CGC GGG GAG CCC CGC TTC ATC 3' BHQ1(서열번호 160)HLA-B fluorescent probe 5: Cy5 5'-CCC GGC CGC GGG GAG CCC CGC TTC ATC 3 'BHQ1 (SEQ ID NO: 160)
HLA-DRB1 형광탐침자1: FAM 5'-CGC TTC GAC AGC GAC GTG GGG GAG TWC CG 3' BHQ1(서열번호 161)HLA-DRB1 Fluorescent Probe 1: FAM 5'-CGC TTC GAC AGC GAC GTG GGG GAG TWC CG 3 'BHQ1 (SEQ ID NO: 161)
HLA-DRB1 형광탐침자2: FAM 5'-TCA TTT CTT CAA CGG GAC GGA GCG GGT GC 3' BHQ1(서열번호 162)HLA-DRB1 fluorescent probe 2: FAM 5'-TCA TTT CTT CAA CGG GAC GGA GCG GGT GC 3 'BHQ1 (SEQ ID NO: 162)
양성대조군 표준유전자 형광탐침자 2: Cyanine 3 5'-CCA CAC AGA ACT AAC CTC CAA CCA ACA ATC AGC 3'BHQ2(서열번호 163)를 작재하였다.Positive control standard gene fluorescent probe 2: Cyanine 3 5'-CCA CAC AGA ACT AAC CTC CAA CCA ACA ATC AGC 3'BHQ2 (SEQ ID NO: 163).
<실시예 3> 실시간 중합효소연쇄반응을 이용한 HLA-A, B, DRB1 대립 유전자 형별Example 3 HLA-A, B, DRB1 Allele Types Using Real-Time Polymerase Chain Reaction
혈액에서 분리한 DNA를 2mM dATP, dGTP, dCTP, dTTP와 HLA-A, B, DRB1 대립유전자를 증폭할 수 있는 특이 시발체 쌍과 양성대조군 표준유전자를 증폭하기 위한 시발체 쌍, 증폭 유무를 측정하기 위한 HLA-A, B, DRB1 형광탐침자, 양성대조군 표준유전자 형광탐침자, 중합반응 완충용액, 중합반응효소, DNA를 넣고 실시간 중합효소연쇄반응 기기를 이용하여 96에서 1분간 1회, 1단계로 변성(denaturation, 95℃에서 25초), 결합(annealing, 70℃에서 45초) 및 연장(extension, 72℃에서 30초) 을 5회 실시하고, 2단계로 변성(denaturation, 95℃에서 25초), 결합(annealing, 70℃에서 45초) 및 연장(extension, 72℃에서 30초) 을 30회 실시하여 총 35회 실시하고 실시간 증폭 유무 측정은 2단계 72℃에서 30초 되는 단계에서 측정하였다. 실시간 중합효소연쇄반응 기기에서의 증폭 유무 측정은 FAM은 490 nm에서, Cyanine 3은 532 nm, cyanine 5은 670 nm 로 측정하였다.DNA isolated from blood was analyzed for specific amplification pairs capable of amplifying 2 mM dATP, dGTP, dCTP, dTTP and HLA-A, B, and DRB1 alleles, primer pairs for amplifying positive control standard genes, and for measuring amplification. HLA-A, B, DRB1 fluorescence probe, positive control standard gene fluorescence probe, polymerization buffer solution, polymerase, DNA, and using a real-time polymerase chain reaction device once in 96 minutes for 1 minute, in one step Denaturation (25 seconds at 95 ° C.), annealing (45 seconds at 70 ° C.) and extension (30 seconds at 72 ° C.) are performed five times, denaturation (25 seconds at 95 ° C.) in two steps. ), Annealing (45 seconds at 70 ° C.) and extension (30 seconds at 72 ° C.) were performed 30 times in total, and real-time amplification was measured at 30 seconds at 72 ° C. in 2 steps. . In the real-time polymerase chain reaction device, the amplification was measured at 490 nm for FAM, 532 nm for Cyanine 3, and 670 nm for cyanine 5.
이때 사용된 특이 시발체의 농도는 각각 1.0 M로 실시하였고 양성대조군은 0.5 M로 사용하였다. 또한 사용한 중합효소 연쇄반응 완충용액은 트리스염기(Tris base) 67 mM, 암모늄 설페이트(ammonium sulphate) 16.6 mM Tween 0.1%, MgCl2 0.2 mM이다. 사용된 dNTP농도는 0.2 mM로 사용하였고 Taq 폴리머라아제(GLS)를 사용하였다. 사용된 real time PCR 기기는 ABI 7500의 제품을 사용하였다.At this time, the concentration of the specific primer used was carried out at 1.0 M and the positive control group was used at 0.5 M. In addition, the polymerase chain reaction buffer used was Tris base 67 mM, ammonium sulphate 16.6 mM Tween 0.1%, and MgCl 2 0.2 mM. The dNTP concentration used was 0.2 mM and Taq polymerase (GLS) was used. The real time PCR instrument used was a product of ABI 7500.
도 3에 나타난 바와 같이, 시작점이 측정된 튜브의 번호는 A1(HLA-A CT값 : 23.383, IC CT값 : 25.039), A3(HLA-A CT값 : 22.186, IC CT값 : 27.259), A10(HLA-B CT값: 22.092, IC CT값: 22.092), B3(HLA-A CT값 : 20.735, IC CT값 : 24.040), B4(HLA-A CT값 : 22.013, HLA-B CT값 : 28.033, IC CT값 : 26.093), C4(HLA-DR CT값 : 23.597, IC CT값 : 25.301), C10 (HLA-DR CT값 : 24.170, IC CT값 : 25.495), D7 (HLA-B CT값 : 23.106, IC CT값 : 26.308), D8 (HLA-DR CT값 : 21.199, IC CT값 : 26.180), D9 (HLA-B CT값 : 22.568, HLA-DR CT값 : 22.433, IC CT값 : 25.708), D11 (HLA-B CT값 : 22.735, IC CT값 : 24.678)이 측정되었고 그 외 튜브에서는 IC 유전자에서만 CT값이 20~28사이로 측정되었다. 이를 분석표 도1을 보고 분석하면 HLA-A 유전자에서 A1, A3, B3, B4 튜브가 증폭되어 HLA-A*01, HLA-A*03으로 판명되었고, HLA-B 유전자에서 보면 A10, B4, D7, D9, D11 튜브가 증폭되어 HLA-B*44, HLA-B*57, HLA-Bw4 양성으로 판명되고, C4, C10, C12, D8, D9 튜브가 증폭되어 HLA-DRB1*07, HLA-DRB1*13, HLA-DRB3 양성, HLA-DRB4 양성으로 판명되었다.As shown in Figure 3, the number of the tube whose starting point was measured is A1 (HLA-A CT value: 23.383, IC CT value: 25.039), A3 (HLA-A CT value: 22.186, IC CT value: 27.259), A10 (HLA-B CT value: 22.092, IC CT value: 22.092), B3 (HLA-A CT value: 20.735, IC CT value: 24.040), B4 (HLA-A CT value: 22.013, HLA-B CT value: 28.033 , IC CT value: 26.093), C4 (HLA-DR CT value: 23.597, IC CT value: 25.301), C10 (HLA-DR CT value: 24.170, IC CT value: 25.495), D7 (HLA-B CT value: 23.106, IC CT value: 26.308), D8 (HLA-DR CT value: 21.199, IC CT value: 26.180), D9 (HLA-B CT value: 22.568, HLA-DR CT value: 22.433, IC CT value: 25.708) , D11 (HLA-B CT value: 22.735, IC CT value: 24.678) was measured. In other tubes, CT value was measured between 20 and 28 only in IC gene. Analysis Table 1 shows that the A1, A3, B3, and B4 tubes were amplified in the HLA-A gene to be identified as HLA-A * 01, HLA-A * 03, and in the HLA-B gene, A10, B4, and D7. , D9, D11 tubes are amplified to HLA-B * 44, HLA-B * 57, HLA-Bw4 positive, C4, C10, C12, D8, D9 tubes are amplified to HLA-DRB1 * 07, HLA-DRB1 * 13, HLA-DRB3 positive and HLA-DRB4 positive were found.

Claims (8)

  1. HLA-A, HLA-B 및 HLA-DRB로 이루어진 군에서 선택된 2 이상의 유전자에 대한 HLA 대립유전자를 실시간 PCR에 의해 특이적으로 증폭할 수 있는 시발체 세트; 및A set of primers capable of specifically amplifying HLA alleles for at least two genes selected from the group consisting of HLA-A, HLA-B and HLA-DRB by real-time PCR; And
    상기 실시간 PCR에 의해 증폭된 산물을 탐침 할 수 있는 탐침자 세트를 포함하는 HLA 대립유전자 형별 키트.HLA allele type kit comprising a set of probes that can probe the product amplified by the real-time PCR.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 탐침자 세트는 유전자 종류의 구별이 가능하도록 다른 색을 발광하는 2 이상의 형광탐침자인 것을 특징으로 하는 키트.The probe set is a kit, characterized in that two or more fluorescent probes that emit different colors to distinguish the type of gene.
  3. 제 1 항에 있어서,The method of claim 1,
    상기 HLA 유전자의 대립유전자는The allele of the HLA gene is
    HLA-A*01, A*02, A*03, A*11, A*23, A*24, A*25, A*26, A*29, A*30, A*31, A*32, A*33, A*34, A*36, A*43, A*66, A*68, A*69, A*74, A*80;HLA-A * 01, A * 02, A * 03, A * 11, A * 23, A * 24, A * 25, A * 26, A * 29, A * 30, A * 31, A * 32, A * 33, A * 34, A * 36, A * 43, A * 66, A * 68, A * 69, A * 74, A * 80;
    B*07, B*08, B*13, B*14, B*15(B62), B*15(63), B*15(70), B*15(71), B*15(72), B*15(75), B*15(76), B*15(77), B*15, B*18, B*27, B*35, B*37, B*38, B*39, B*40, B*40(B60), B*40(B61), B*41, B*42, B*44, B*45, B*46, B*47, B*48, B*49, B*50, B*51, B*52, B*53, B*54, B*55, B*56, B*57, B*58, B*59, B*67, B*78, B*81, B*82, B*83, Bw4, Bw6;B * 07, B * 08, B * 13, B * 14, B * 15 (B62), B * 15 (63), B * 15 (70), B * 15 (71), B * 15 (72) , B * 15 (75), B * 15 (76), B * 15 (77), B * 15, B * 18, B * 27, B * 35, B * 37, B * 38, B * 39, B * 40, B * 40 (B60), B * 40 (B61), B * 41, B * 42, B * 44, B * 45, B * 46, B * 47, B * 48, B * 49, B * 50, B * 51, B * 52, B * 53, B * 54, B * 55, B * 56, B * 57, B * 58, B * 59, B * 67, B * 78, B * 81, B * 82, B * 83, Bw4, Bw6;
    DRB1*01, DRB1*15, DRB1*16, DRB1*03, DRB1*04, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB3, DRB4, 및 DRB5로 이루어진 그룹으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 HLA 대립유전자 형별 키트.DRB1 * 01, DRB1 * 15, DRB1 * 16, DRB1 * 03, DRB1 * 04, DRB1 * 11, DRB1 * 12, DRB1 * 13, DRB1 * 14, DRB1 * 07, DRB1 * 08, DRB1 * 09, DRB1 * HLA allele type kit, characterized in that any one or more selected from the group consisting of 10, DRB3, DRB4, and DRB5.
  4. 제 1 항에 있어서,The method of claim 1,
    상기 시발체는 HLA 대립유전자의 다양성을 나타내는 위치를 기점으로 17 내지 24 nt의 길이로 작재한 것을 특징으로 하는 키트.The primer is characterized in that the kit was constructed in a length of 17 to 24 nt from the position showing the diversity of the HLA allele.
  5. 제 2 항에 있어서,The method of claim 2,
    상기 형광탐침자는 HLA-A 대립유전자의 증폭 유무를 검출할 수 있는 서열번호 151, 152, 153, 154 또는 155에 기재된 서열을 갖는 형광탐침자;The fluorescent probe may include a fluorescent probe having a sequence set forth in SEQ ID NO: 151, 152, 153, 154 or 155 capable of detecting the amplification of the HLA-A allele;
    HLA-B 대립유전자의 증폭 유무를 검출할 수 있는 서열번호 156, 157, 158, 159 또는 160에 기재된 서열을 갖는 형광탐침자;A fluorescent probe having a sequence set forth in SEQ ID NO: 156, 157, 158, 159 or 160 capable of detecting the amplification of the HLA-B allele;
    HLA-DRB1 대립유전자의 증폭 유무를 검출할 수 있는 서열번호 161 또는 162에 기재된 서열인 것을 특징으로 하는 키트.A kit comprising the sequence set forth in SEQ ID NO: 161 or 162 capable of detecting the amplification of the HLA-DRB1 allele.
  6. 제 1 항에 있어서,The method of claim 1,
    양성대조군 유전자를 증폭하기 위한 시발체 및 양성대조군 유전자의 증폭 유무를 탐지하기 위한 형광탐침자를 추가로 포함하는 것을 특징으로 하는 키트.And a fluorescent probe for detecting the presence or absence of the amplification of the positive control gene and the primer for amplifying the positive control gene.
  7. 제 6 항에 있어서,The method of claim 6,
    대조군 유전자를 증폭하기 위한 시발체는 서열번호 149 및 150에 기재된 서열을 갖고, 대조군 유전자의 증폭 유무를 탐지하기 위한 형광탐침자는 서열번호 163에 기재된 서열을 갖는 것을 특징으로 하는 키트.A primer for amplifying a control gene has a sequence set forth in SEQ ID NOs: 149 and 150, and a fluorescence probe for detecting the amplification of a control gene has a sequence set forth in SEQ ID NO: 163.
  8. 시료로부터 DNA를 추출 정제하는 단계;Extracting and purifying DNA from the sample;
    상기 정제된 DNA를 대상으로 제 1 항에 따른 키트를 사용하여 실시간 중합효소연쇄반응(PCR)을 실시하는 단계; 및Performing real-time polymerase chain reaction (PCR) on the purified DNA using the kit according to claim 1; And
    실시간 PCR 결과를 판독하여 HLA 대립유전자를 형별하는 단계를 포함하는, HLA 대립유전자의 형별 방법.A method of typing HLA alleles, comprising the steps of classifying HLA alleles by reading real-time PCR results.
PCT/KR2014/001097 2013-03-28 2014-02-11 Method and kit for testing hla alleles using speed multiplex pcr WO2014157825A1 (en)

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* Cited by examiner, † Cited by third party
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