WO2020036276A1 - Dosage par pcr faisant appel à une nouvelle séquence nucléotidique sélective pour la détermination du groupe sanguin abo du singe rhésus - Google Patents

Dosage par pcr faisant appel à une nouvelle séquence nucléotidique sélective pour la détermination du groupe sanguin abo du singe rhésus Download PDF

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WO2020036276A1
WO2020036276A1 PCT/KR2019/001313 KR2019001313W WO2020036276A1 WO 2020036276 A1 WO2020036276 A1 WO 2020036276A1 KR 2019001313 W KR2019001313 W KR 2019001313W WO 2020036276 A1 WO2020036276 A1 WO 2020036276A1
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abo
pcr
rhesus monkey
primer
seq
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PCT/KR2019/001313
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English (en)
Korean (ko)
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이재일
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서울대학교산학협력단
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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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR

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  • the present invention relates to a composition for detecting Rhesus macaque ABO blood type using a polymerase chain reaction method and a detection method using the same. More specifically, a single gene of a gene for determining the ABO blood type of Rhesus monkey using a polymerase chain reaction method is disclosed.
  • the present invention relates to a rhesus monkey ABO blood type detection composition comprising a primer set capable of specifically detecting single nucleotide polymorphims (SNP) and a detection method using the same.
  • SNP single nucleotide polymorphims
  • organ donors and recipients must be compatible with the ABO blood type.
  • organ transplants of experimental animal models to assess potential immunotherapy strategies it is important to ensure donor and recipient ABO blood group compatibility. This is especially true for non-human primate models that exhibit the same ABH specificity as the human ABO blood group system.
  • most Old World and New World monkeys do not, or weakly, express cohesive A and B antigens on their red blood cell (RBC) surfaces. This means that standard RBC aggregation experiments cannot be used to determine the ABO type of these animals.
  • RBC red blood cell
  • a more objective and accurate way to analyze nonhuman primates is to perform immunohistochemistry (IHC) on vascular tissues (eg kidneys) and / or oral mucosal cells.
  • IHC immunohistochemistry
  • vascular tissues eg kidneys
  • oral mucosal cells e.g kidneys
  • this method gives a definite result on blood group antigen expression.
  • its accuracy depends on the level of blood type antigen expression by the epithelium, which may differ between individuals, and the quality of cells collected from epithelial tissue, such as oral mucosa.
  • unlike serum or DNA it is difficult to keep epithelial cells fresh for a long time.
  • Non-human primates also have the disadvantage of being soothed to obtain oral mucosal cells.
  • PCR polymerase chain reaction
  • SNPs single nucleotide polymorphisms
  • the problem to be solved by the present invention is to provide a primer set that can specifically detect single nucleotide polymorphims (SNP) of genes that determine the ABO blood type of rhesus monkey using polymerase chain reaction method will be.
  • the problem to be solved by the present invention is to provide a rhesus monkey ABO blood type detection composition comprising the primer set and a detection method using them.
  • the present inventors have diligently researched to overcome the problems of the prior arts, and as a result, by utilizing a new primer that can distinguish the A, B, and O alleles of the rhesus monkey, the ABO blood type allele of the rhesus monkey is homologous.
  • the test method of the present invention has completed the present invention by confirming that the Rhesus macaque ABO type can be accurately determined, compared to the conventional serological and immunohistochemical tests (IHC) having a relatively limited point.
  • the present invention provides a primer set comprising primers of SEQ ID NOs: 1 to 4 for amplifying exon 7 of the Rhesus macaque ABO locus of selective nucleotide sequence.
  • the sequences of the primer sets used in the present invention are shown in Table 1 below.
  • the primer set of the present invention comprises a set-1 (SET-1) comprising a PA2 forward primer (SEQ ID NO: 1) and a PO2 forward primer (SEQ ID NO: 2); And set-2 (SET-2) comprising a PB forward primer (SEQ ID NO: 3) and a PO3 forward primer (SEQ ID NO: 4).
  • the set-1 and set-2 may further include a consensus reverse primer (CAB, SEQ ID NO: 5).
  • PCR of exon 7 of the Rhesus macaque ABO blood group locus in combination with the set-1 and set-2 shows a band pattern of various combinations, through which the Rhesus macaque ABO blood type and its homozygotes or heterozygotes
  • the primer set of the present invention can be used to successfully amplify exon7 of the ABO blood group gene of the rhesus monkey without interference between the primers constituting it. Therefore, when real-time PCR is performed using the primer set, the ABO blood type of the rhesus monkey can be detected very reliably and quickly.
  • the present invention provides a composition for detecting ABO blood type of rhesus monkey comprising the primer set of the present invention.
  • the detection of the ABO blood type of rhesus monkey can be detected by polymerase chain reaction (PCR).
  • the present invention provides a kit for detecting ABO blood group of rhesus monkey comprising the composition for detection.
  • the kit of the present invention may further include other probe sets in addition to the primers of SEQ ID NOs.
  • the present invention (s1) collecting a DNA sample from the rhesus monkey; (s2) performing PCR using a primer set comprising primers of SEQ ID NOS: 1 to 4 which amplify exon 7 of a rhesus monkey ABO locus as a template using the DNA collected in step (s1); And (s3) provides a method for detecting the ABO blood type of rhesus macaque comprising the step of (s2) to examine the product amplified by the PCR in (s2).
  • the detection composition of the present invention can specifically detect the ANP blood type of a rhesus monkey by specifically detecting the SNP of a gene that determines the ABO blood type of the rhesus monkey using a polymerase chain reaction method.
  • FIG. 1 shows the results of FACS analysis of 66 Rhesus monkey sera before (A) and after (B) pre-absorption with human O + erythrocytes (RBC).
  • A Two monkeys (asterisk; R058 and R154) showed false-positive antibody binding to human A + and B + RBCs.
  • B Following preabsorption with human O + RBCs, these nonspecific or heteroreactive bindings were removed to correct the blood types of the two monkeys.
  • Figure 2 shows a representative image of the immunohistochemical staining results of oral mucosal cells of the rhesus monkey.
  • Cells from A + and B + monkeys are recognized by FITC-labeled murine anti-A and anti-B antibodies, respectively.
  • Cells from AB + monkeys are recognized by both mouse antibodies.
  • Cells from O + monkeys do not react with both mouse antibodies.
  • DAPI 4 ', 6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
  • FIG. 3 shows PCR band patterns for AA, AO, BB, BO, AB, and OO blood types of Rhesus monkeys.
  • Two sets of ABO allele specific primers were prepared based on monobasic polymorphism at exon 7 of the ABO locus.
  • Whole blood of 66 rhesus monkeys was PCR treated with primers.
  • (A) SET-1 consists of PA2 and PO2.
  • (B) SET-2 is composed of PO3 and PB. PCR assays based on SET-1 and SET-2 successfully determine homozygous and heterozygous for A, B and O.
  • AA homozygotes are represented by PA2 and PO2 bands
  • AO heterozygotes are represented by PA2, PO2, and PO3 bands
  • BB homozygotes are represented by PO2 and PB bands
  • BO heterozygotes are represented by all four bands
  • AB heterozygotes are represented by PA2, PO2, and PB bands
  • OO homozygotes are represented by PA2 and PO3 bands.
  • L ladder; bp, base pairs.
  • 4A-4D show the entire exon 7 sequence obtained from chromosome 15 of Rhesus monkey. Red highlights indicate monobasic polymorphism, blue arrows indicate forward primer sequences (PO3, PA2, PB, and PO2) and black arrows indicate reverse primer sequences (CAB).
  • Erythrocyte aggregation assays were performed with 66 monkey serum and human RBC reagents. That is, blood samples were collected from humans known to have A, B, or O blood types. RBCs were separated by centrifugation at 2500 rpm for 10 minutes. After washing with phosphate-buffered saline (PBS, Sigma, St. Louis, Mo., USA), the RBC suspension was centrifuged again at 2500 rpm for 10 minutes. Supernatant was discarded. This procedure was repeated twice. Finally the RBC was diluted 5% with PBS. Monkey serum was obtained by collecting whole blood from the femoral vein of the experimental animal, and serum was collected by centrifugation at 3500 rpm for 10 minutes.
  • PBS phosphate-buffered saline
  • Serum was diluted to 10% with PBS. Diluted monkey serum was then mixed 2: 1 with human A +, B +, or O + RBC suspension and incubated for 30 minutes at room temperature. Aggregation reactions were observed on plates and under a microscope. Coagulation was graded into strong (+++), medium (++), weak (+), and negative (-).
  • aggregation reactions showed inconsistent results when compared to other ABO type analysis results.
  • FACS analysis which can be identified using flow cytometry.
  • Monkey serum was mixed 2: 1 with the human RBCs and centrifuged at 1500 rpm for 5 minutes after incubation for 30 minutes. The supernatant was removed and the mixture was washed twice. The samples were then stained with fluorescein isothiocyanate (FITC) labeled goat anti-monkey IgG (Santa Cruz Biotechnology, Inc., TX, USA) at 4 ° C. for 30 minutes in cancer conditions. After centrifugation at 1500 rpm for 5 minutes, the supernatant was removed. The mixture was washed twice.
  • FITC fluorescein isothiocyanate
  • ABH phenotypic analysis of oral mucosal smears was performed via IHC. That is, epithelial cells were obtained by rubbing the oral mucosa surface of 66 Rhesus monkeys with a sterile swab tip applicator. Smear from each animal was applied to the microscope slide. Slides were fixed for 10 minutes in cold acetone (Sigma, St Louis, MO, USA), then dried and washed with PBS. Dilutions of 1:50 and 1:20 dilutions of mouse anti-A and anti-B antibodies (Sigma, St. Louis, MO, USA) in PBS, respectively, were prepared and added to the samples.
  • 66 monkey genomic DNAs were extracted from whole blood using a QIAmp® DNA Blood Mini Kit purchased from QIAGEN (Valencia, CA, USA).
  • PCR was performed using 5 ′ CCT GCC TTG CAG ATA CGT G 3 ′ (forward) and 5 ′ CAG CTG ATC ACG GGT TCC 3 ′ (reverse) primers.
  • PCR was denatured in Peltier Thermal Cycler (PTC) -200 for 5 minutes at 95 ° C, followed by 35 cycles of 20 seconds at 95 ° C, 20 seconds at 58 ° C, and 1 minute at 68 ° C.
  • PTC Peltier Thermal Cycler
  • PCR products were given poly (A) tails and then inserted into TA cloning vector pGEM-T Easy (Promega, Madison, Wis., USA).
  • Subcloned plasmids were prepared with AccuPrep Plasmid Mini Extraction Kit (Bioneer, Daejeon, Korea) for sequencing. Nucleotide sequences were confirmed by ABI PRISM® System (model 377). All other chemical reagents were purchased from Duchefa Biochemie (Haarlem, Netherlands).
  • genomic DNA was obtained from 10 out of 66 monkeys that had undergone serometry and ABO blood group analysis by IHC staining. Their total exon 7 sequences were obtained and their SNPs were identified on their ABO loci (FIGS. 4A-4D).
  • Two primer sets were selected for analyzing monkey ABO blood types.
  • SET-1 consists of PA2-F, PO2-F, and CAB primers
  • SET-2 consists of PB-F, PO3-F, and CAB primers (Table 2).
  • PCR was performed with a new selective primer using Takara taq polymerase (Takara, Seoul, Korea). PCR was denatured for 1 minute at 95 ° C. in PTC-200, followed by 30 cycles of 30 seconds at 95 ° C., 30 seconds at 64 ° C., and 30 seconds at 72 ° C. PCR products were then electrophoresed on 2% agarose gel.
  • the ABO blood group of 66 adult rhesus monkeys was analyzed using erythrocyte aggregation assays, IHC, and PCR assays with new primers.
  • the experimental results are shown in Table 3. IHC staining results were in full agreement with PCR results. Type B was the most common (65%), followed by type AB (18%), type A (9%), and type O (8%). Serum tests using hemagglutination assay differed from IHC and PCR results in 7 monkeys (10.6% of all monkeys) (Table 3). Thus, if blood group analysis was determined by serologic testing alone, the results may have affected the frequency of other blood types.
  • Asterisks (*) indicate inconsistencies between assay methods for monkey blood types.
  • the monkey serum: human RBC mixture used in the aggregation assay was FACS analyzed with FITC labeled anti-monkey antibody. FACS results were consistent with IHC and PCR results in 64 of 66 monkeys, including 5 of 7 monkeys that produced inconsistent aggregation assay results.
  • IHC and PCR showed type AB, the remaining two monkeys (R058 and R154), indicated by type A and B, respectively, showed nonspecific antibody binding: one incorrectly indicated type O The other represents type A (FIG. 1A, asterisk (*)).
  • AA homozygotes are represented by PA2 and PO2 bands
  • AO heterozygotes are represented by PA2, PO2, and PO3 bands
  • BB homozygotes are represented by PO2 and PB bands
  • BO The heterozygotes are represented by all four bands and the AB type is represented by the PA2, PO2, and PB bands.
  • the algorithm of the present invention can also detect O homozygotes: they are represented by the PA2 and PO3 bands (FIG. 3). That is, the absence of the PO2 band represented by G-> C SNPs (nucleotide position 5196271) is important for defining O homozygotes.
  • the PCR assay of the present invention has advantages over all other serum assays (up to IHC) tested in the present invention:
  • the PCR assay of the present invention provides that each monkey is heterozygous or homozygous for the A and B alleles. You can decide whether or not.

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Abstract

La présente invention concerne une composition pour la détermination du groupe sanguin ABO de singes rhésus et un procédé de détermination du groupe sanguin faisant appel à celle-ci, la composition comprenant des amorces permettant de détecter spécifiquement le SNP du gène, qui détermine les groupes sanguins ABO de singes rhésus, dans une réaction en chaîne par polymérase. La composition de détermination du groupe sanguin selon la présente invention peut être utilisée dans une réaction en chaîne par polymérase, permettant ainsi la détermination précise du groupe sanguin ABO de singes rhésus.
PCT/KR2019/001313 2018-08-17 2019-01-30 Dosage par pcr faisant appel à une nouvelle séquence nucléotidique sélective pour la détermination du groupe sanguin abo du singe rhésus WO2020036276A1 (fr)

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KR1020180096300A KR102180463B1 (ko) 2018-08-17 2018-08-17 붉은털 원숭이의 abo 혈액형 분석을 위한 새로운 선택적 염기서열을 사용한 pcr 분석법
KR10-2018-0096300 2018-08-17

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561458A (zh) * 2022-04-20 2022-05-31 青岛市中心血站 一种与abo血型系统中b变异型相关的snp位点及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6265557B1 (en) * 1997-05-09 2001-07-24 Loma Linda University Medical Center ABO histo-blood group O alleles of the baboon
WO2017160686A1 (fr) * 2016-03-15 2017-09-21 Georgetown University Séquençage de nouvelle génération pour identifier un groupe sanguin abo

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6265557B1 (en) * 1997-05-09 2001-07-24 Loma Linda University Medical Center ABO histo-blood group O alleles of the baboon
WO2017160686A1 (fr) * 2016-03-15 2017-09-21 Georgetown University Séquençage de nouvelle génération pour identifier un groupe sanguin abo

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHOI, Y.-J. ET AL.: "Comparison of a PCR assay using novel selective primers with current methods in terms of ABO blood phenotyping in rhesus macaques", SCIENTIFIC REPORTS, vol. 8, no. 1957, 2018, pages 1 - 7, XP055688257, DOI: 10.1038/s41598-018-20395-0 *
KIM, T.M. ET AL.: "Comparison of Methods for Determining ABO Blood Type in Cynomolgus Macaques (Macaca fasciculatis", JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE, vol. 54, no. 3, 31 May 2015 (2015-05-31), pages 255 - 260, XP055688254 *
PREMASUTHAN, A. ET AL.: "A simple multiplex polymerase chain reaction to determine ABO blood types of rhesus macaques (Macaca mulatta", TISSUE ANTIGENS, vol. 77, 2011, pages 584 - 588, DOI: 10.1111/j.1399-0039.2010.01602.x *
PREMASUTHAN, A. ET AL.: "Molecular ABO phenotyping in cynomolgus macaques using real-time quantitative PCR", TISSUE ANTIGENS, vol. 80, no. 4, 2012, pages 363 - 367, XP055688251, DOI: 10.1111/j.1399-0039.2012.01935.x *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561458A (zh) * 2022-04-20 2022-05-31 青岛市中心血站 一种与abo血型系统中b变异型相关的snp位点及其应用
CN114561458B (zh) * 2022-04-20 2023-12-29 青岛市中心血站 一种与abo血型系统中b变异型相关的snp位点及其应用

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