WO2020036276A1 - Pcr assay using novel selective nucleotide sequence for abo blood typing of rhesus monkey - Google Patents

Abstract

The present invention relates to a composition for ABO blood typing of rhesus monkeys and a blood typing method using same, the composition comprising primers capable of specifically detecting the gene SNP, which determines ABO blood types of rhesus monkeys, in a polymerase chain reaction. The blood typing composition of the present invention can be used in a polymerase chain reaction, thereby allowing the accurate ABO blood typing of rhesus monkeys.

Description

PCR Assays Using Novel Selective Sequences for ABO Blood Group Analysis in Rhesus Macaques

This application claims priority based on Korean Patent Application No. 10-2018-0096300, filed August 17, 2018, and all the contents disclosed in the specification and drawings of the application are incorporated in this application.

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.

To suppress the fatal immune response after human organ transplantation, organ donors and recipients must be compatible with the ABO blood type. When using 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. Unlike humans, however, 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.

To address this problem, other methods have been introduced to detect A and B antigens in saliva or anti-A and anti-B antibodies in serum. Such methods include erythrocyte aggregation inhibition techniques, saline agglutination, and reverse gel system analysis. However, while this assay is simple and frequently used, non-specific binding with human A + or B + RBC reagents by monkey anti-human biaggregates can lead to erroneous results. Therefore, this assay cannot be used independently for nonhuman primate ABO type analysis.

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. When performed correctly, this method gives a definite result on blood group antigen expression. However, 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. In addition, 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.

One way to overcome the accuracy, technical and logical limitations of serological and IHC tests is the measurement of polymerase chain reaction (PCR) of A, B and O specific single nucleotide polymorphisms (SNPs) in the ABO gene. However, the PCR-based methods reported to date have been limited in detecting type O in rhesus monkeys. Conventional Yamamoto et al . Have reported mutations in exon 6 of ABO that prevent genes from producing functional enzymes, even if they do not specify their exact location. Other studies have revealed that up to 28% of rhesus monkeys are O + by serology, and three other PCR-based studies fail to detect O + rhesus monkeys regardless of whether PCR techniques, IHC, or serological methods are used. It was. It is difficult to detect the O allele of the rhesus monkey only by the conventional PCR method.

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. In addition, 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. We developed a PCR-based algorithm that can determine whether it is a conjugate or a heterozygote. 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.

SEQ ID NO:	primer	set	order
One	PA2-F	SET-1	5′-TGGACGTGGACATGGAGTTCCTT-3 ′
2	PO2-F		5′-AGCCGGGAGGCCTTCACCTGC-3 ′
3	PB-F	SET-2	5′-GGAGATCCTGACTCCGCTCTT-3 ′
4	PO3-F		5′-GCCACCGGGTCCACTACTTT-3 ′
5	CAB		5′-CCCTGGTGAGCCGCTGCACCTCC-3 ′

In one preferred embodiment, 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). Preferably, 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.

In addition, the present invention provides a composition for detecting ABO blood type of rhesus monkey comprising the primer set of the present invention. In the detection composition of the present invention, the detection of the ABO blood type of rhesus monkey can be detected by polymerase chain reaction (PCR).

In addition, 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. In addition, 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.

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.

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, and 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).

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art.

1. Experimental Materials and Methods

(1) the subjects used in the experiment

A total of 66 adult rhesus monkeys (Macaca mulatta) (29 males and 37 females) weighing 3.9-8.5 kg, aged 4-10 years, were used in this experiment. All monkeys were obtained from Gaoyao Kangda Laboratory Animal Sciences & Technology Co., Ltd (Guangdong, China) and maintained at the Seoul National University Hospital (SNUH) Non-human Primate Center, an AAALAC accredited institution. All animals used in this study were managed to strictly follow the guidelines of the National Institutes of Health for the management and use of experimental animals. This study was approved by SNUH's IACUC (no. 14-0002-C2A0).

(2) red blood cell aggregation analysis

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. 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 (-).

(3) flow cytometry

In some cases, the aggregation reactions showed inconsistent results when compared to other ABO type analysis results. Thus, all 66 monkeys underwent 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. Finally the pellet was resuspended in 200 μl PBS and flow cytometry was performed on FACS Calibur (Becton Bioscience, Mountain View, CA, USA). All data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

By inconsistency, all monkey sera were also subjected to FACS analysis after being preabsorbed by human O + RBC. Thus, 5% human O + RBCs were mixed 2: 1 with 100 μl of 10% monkey serum and incubated at 37 ° C. for 30 minutes and centrifuged at 1500 rpm for 30 minutes at 4 ° C. Absorbed serum was obtained and mixed with human A +, B +, and O + RBCs to prepare for FACS.

(4) IHC staining

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. After incubation at room temperature for 60 minutes, washed with PBS, slides were washed with 100 μl of 1: 100-diluted FITC-bound goat anti-mouse IgM (Vector, Burlingame, CA, USA) for 60 minutes at room temperature in dark conditions. Further incubation. Slides were washed with PBS and fixed using Antifade-DAPI (Vector) -1-6-Diamidino-2-phenylindole and stored in dark at 4 ° C. Staining was observed using a fluorescence microscope.

(5) new optional Of primer  Fabrication and Development of PCR-based ABO Type Blood Group Assay

In order to determine the ABO type of the experimental monkey after making a new primer, 66 monkey genomic DNAs were extracted from whole blood using a QIAmp® DNA Blood Mini Kit purchased from QIAGEN (Valencia, CA, USA). To amplify exon 7 of the ABO locus, 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. 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).

To design new selective primers, 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, while SET-2 consists of PB-F, PO3-F, and CAB primers (Table 2).

Primer sequences used for PCR analysis. E7F, exon 7 forward; E7R, exon 7 reverse; CAB, Common AB Reverse Primer.

SEQ ID NO:	primer	set	order
One	PA2-F	SET-1	5′-TGGACGTGGACATGGAGTTCCTT-3 ′
2	PO2-F		5′-AGCCGGGAGGCCTTCACCTGC-3 ′
3	PB-F	SET-2	5′-GGAGATCCTGACTCCGCTCTT-3 ′
4	PO3-F		5′-GCCACCGGGTCCACTACTTT-3 ′
5	CAB		5′-CCCTGGTGAGCCGCTGCACCTCC-3 ′
6	E7F		5′-CCTGCCTTGCAGATACGTG-3 ′
7	E7R		5′-CAGCTGATCACGGGTTCC-3

To analyze the ABO blood group of 66 monkeys, 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.

2. Experimental Results

(1) Rhesus monkey ABO blood type frequency

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.

Results of type ABO analysis in 66 rhesus monkeys using serology, immunohistochemistry, and PCR analysis. Asterisks (*) indicate inconsistencies between assay methods for monkey blood types.

(2) Comparative example  1: Serotype Testing Using Hemagglutination

Aggregation of 66 monkey sera was evaluated by microscopic examination and microscopic examination on plates. Most samples showed obvious positive or negative aggregation both under the microscope and plate. However, the seven monkeys had confusing results such as weak or mixed field aggregation. As a result, the serological phenotypic results of microscopy and plates in these monkeys were inconsistent with one another or partially with IHC and PCR results (Table 3). In particular, in three monkeys (R014, R059, and R065), plate results accurately indicate the B + type, while microscopic results incorrectly indicate the O type. In both monkeys (R058 and R154), when the IHC and PCR results showed type AB, both the microscope and plate results showed either type A or type B. Reversal occurred in the other two monkeys (R076 and R152): both microscopy and plate results show type AB while IHC and PCR results show type A.

(3) FACS analysis of erythrocyte aggregated serum samples

To determine if the mismatched aggregation result was due to the presence of unknown nonspecific antibodies or heterologous reactivity, 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. When 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 (*)). However, FACS analysis after pre-absorption of these mismatched serum samples with human O + RBCs clearly resolved the nonspecific binding (perhaps for unknown aggregates): both exhibit type AB (FIG. 1B). Analysis of FACS data with pre-absorbed serum of 66 monkeys (FIG. 1B) showed that type A and B monkey serum bind to human B + and A + RBCs with at least 1.5% antibody binding, respectively. Five O + type monkey sera bound strongly to both A + and B + RBCs. In contrast, AB + type sera weakly recognized A + and B + RBCs. The exception was that R152 monkeys were incorrectly analyzed as AB + by aggregation analysis and A + by IHC and PCR: its serum bound only weakly to B + RBC (0.97%).

(4) Comparative Example 2: ABO type analysis through IHC staining

IHC staining of oral mucosal cells with anti-A and anti-B antibodies clearly identified the blood types of all monkeys: every two smears in each animal resulted in consistent results (FIG. 2). Although the impurities in the mucosal samples were not completely removed during the staining process, they did not interfere with the determination of the blood type.

(5) Example: Determination of ABO alleles by PCR analysis

In PCR with selective designed new primer sets, 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. Importantly, 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. This is important because the previously reported PCR analysis was unable to detect O homozygotes in macaque monkeys. All these PCR results were in full agreement with the IHC results of oral mucosal cells. In particular, 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.

<110> SEOUL NATIONAL UNIVERSITY R & DB FOUNDATION

<120> PCR assay using novel selective primers for ABO phenotyping of

         rhesus macaques

<130> PCT18-288, SNU-2018-5494

<150> KR 10-2018-0096300

<151> 2018-08-17

<160> 7

<170> KoPatentIn 3.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> PA2-F

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tggacgtgga catggagttc ctt 23

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> PO2-F

<400> 2

agccgggagg ccttcacctg c 21

<210> 3

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<212> DNA

<213> Artificial Sequence

<220>

<223> PB-F

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ggagatcctg actccgctct t 21

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<212> DNA

<213> Artificial Sequence

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<223> PO3-F

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gccaccgggt ccactacttt 20

<210> 5

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<213> Artificial Sequence

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<223> CAB

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ccctggtgag ccgctgcacc tcc 23

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<223> E7F

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Claims (7)

1. A primer set comprising the primers of SEQ ID NOS: 1-4 which amplify exon 7 of the rhesus monkey ABO locus.
2. The method of claim 1, wherein the primer set

   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 comprising PB forward primer (SEQ ID NO: 3) and PO3 forward primer (SEQ ID NO: 4)

   Primer set consisting of.
3. The primer set of claim 1, wherein said Set-1 and Set-2 further comprise a consensus reverse primer (CAB, SEQ ID NO: 5).
4. Composition for detecting ABO blood group of rhesus monkey comprising a primer set of claim 1.
5. The composition for detecting ABO blood type of rhesus monkey according to claim 4, wherein the detection is detected by polymerase chain reaction (PCR).
6. Kit for detecting ABO blood type of rhesus monkey comprising the composition of claim 4.
7. (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) examining the product amplified by PCR in (s2);

   ABO blood group detection method of rhesus monkey comprising a.

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