WO2014157825A1 - Procédé et trousse d'analyse d'allèles hla utilisant une pcr multiplex rapide - Google Patents

Procédé et trousse d'analyse d'allèles hla utilisant une pcr multiplex rapide Download PDF

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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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seq
hla
primer
nos
drb1
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PCT/KR2014/001097
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English (en)
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/ko
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Publication of WO2014157825A1 publication Critical patent/WO2014157825A1/fr

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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.

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Abstract

La présente invention concerne un procédé et une trousse permettant d' analyser simultanément des allèles HLA-A, -B et -DR en utilisant une réaction en chaîne par polymérase multiplex en temps réel et, plus précisément, un procédé et une trousse de typage d'allèle HLA par le biais d'une valeur de fluorescence qui s'affiche au moyen d'amorces permettant d'établir une liaison précise avec les allèles HLA pour l'ADN extrait d'un échantillon et une sonde fluorescente permettant de détecter des produits d'amplification amplifiés par les amorces.
PCT/KR2014/001097 2013-03-28 2014-02-11 Procédé et trousse d'analyse d'allèles hla utilisant une pcr multiplex rapide WO2014157825A1 (fr)

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KR1020130033330A KR101320622B1 (ko) 2013-03-08 2013-03-28 스피드 멀티플렉스 pcr법을 이용한 hla 대립유전자 검사방법 및 검사키트
KR10-2013-0033330 2013-03-28

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624676A (zh) * 2018-05-23 2018-10-09 健路生物科技（苏州）有限公司 用于检测hla-b*5801等位基因的试剂盒及其检测方法和应用
CN115404278A (zh) * 2021-05-26 2022-11-29 四川大学华西医院 八位点多重pcr基因分型试剂盒

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KR20100131350A (ko) * 2009-06-05 2010-12-15 가톨릭대학교 산학협력단 실시간 중합효소연쇄반응을 이용한 ｈｌａ 대립유전자 자동검사 키트

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624676A (zh) * 2018-05-23 2018-10-09 健路生物科技（苏州）有限公司 用于检测hla-b*5801等位基因的试剂盒及其检测方法和应用
CN108624676B (zh) * 2018-05-23 2022-04-01 健路生物科技（苏州）有限公司 用于检测hla-b*5801等位基因的试剂盒及其检测方法和应用
CN115404278A (zh) * 2021-05-26 2022-11-29 四川大学华西医院 八位点多重pcr基因分型试剂盒

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