KR102180463B1 - PCR assay using novel selective primers for ABO phenotyping of rhesus macaques - Google Patents

Abstract

The present invention relates to a composition for detecting ABO blood type in rhesus monkeys comprising a primer capable of specifically detecting the SNP of a gene that determines the ABO blood type of rhesus monkeys using a polymerase chain reaction method, and a detection method using the same will be. The detection composition of the present invention can accurately detect the ABO blood type of rhesus monkeys using a polymerase chain reaction method.

Description

PCR assay using novel selective primers for ABO phenotyping of rhesus macaques}

The present invention relates to a composition for detecting a rhesus monkey ABO blood type using a polymerase chain reaction method and a detection method using the same, and more specifically, a single gene that determines the ABO blood type of rhesus monkeys using a polymerase chain reaction method The present invention relates to a composition for detecting a rhesus monkey ABO blood type including a primer set capable of specifically detecting single nucleotide polymorphims (SNP) and a detection method using the same.

In order to suppress a lethal immune response after human organ transplantation, organ donors and recipients must have compatible ABO blood types. When using organ transplantation in laboratory animal models to evaluate potential immunotherapy strategies, it is important to ensure donor and recipient ABO blood type compatibility. This is especially the case for non-human primate models that exhibit the same ABH specificity as the human ABO blood group system. However, unlike humans, most Old and New World monkeys do not or weakly express aggregated A and B antigens on the surface of their red blood cells (RBCs). This means that standard RBC aggregation experiments cannot be used to determine the ABO form of these animals.

To solve this problem, other methods of detecting A and B antigens in saliva or anti-A and anti-B antibodies in serum have been introduced. These methods include hemagglutination inhibition techniques, saline aggregation methods, and reverse gel system analysis. However, although this assay is simple and often used, erroneous results may occur due to nonspecific binding with human A+ or B+ RBC reagents by monkey anti-human diagglobin. Therefore, this assay cannot be used independently for the analysis of non-human primate ABO type.

A more objective and accurate way to analyze non-human primates is to perform immunohistochemistry (IHC) of vascular tissue (eg, kidney) and/or oral mucosa cells. When performed correctly, this method provides reliable results for blood type antigen expression. However, its accuracy depends on the level of expression of blood type antigens by the epithelium, which may differ between individuals, and the quality of cells collected from epithelial tissues such as the oral mucosa. In addition, unlike serum or DNA, it is difficult to keep epithelial cells fresh for a long time. In addition, non-human primates have the disadvantage that they must be sedated to obtain oral mucosal cells.

One way to overcome the accuracy, technical and logical limitations of serological and IHC assays is the polymerase chain reaction (PCR) measurement of A, B and O specific mononucleotide polymorphisms (SNPs) in the ABO gene. However, the PCR-based method reported to date has limitations in detecting type O in rhesus monkeys. Conventional Yamamoto et al . reported that there is a mutation in exon 6 of ABO that prevents the gene from producing a functional enzyme (even if they do not specify its exact location). Other studies have shown that up to 28% of rhesus monkeys are O+ through serological testing, and three other PCR-based studies fail to detect O+ rhesus monkeys regardless of the use of PCR techniques, IHC or serological methods. I did. It is difficult to detect the O allele of rhesus monkeys only with conventional PCR methods.

The problem to be solved by the present invention is to provide a primer set capable of specifically detecting single nucleotide polymorphims (SNPs) of genes that determine the ABO blood type of rhesus monkeys using a polymerase chain reaction method. will be. In addition, the problem to be solved by the present invention is to provide a composition for detecting rhesus monkey ABO blood type including the primer set and a detection method using the same.

As a result of intensive research efforts to overcome the problems of the prior art, the present inventors utilized a new primer capable of distinguishing the A, B, and O alleles of rhesus monkeys, and the ABO blood type alleles of rhesus monkeys are homogeneous. We have developed a PCR-based algorithm that can determine whether it is a conjugate or a heterozygous. The present invention was completed by confirming that the test method of the present invention can accurately determine the presence of ABO type in rhesus monkeys compared to the conventional serological test and tissue immunochemical test (IHC) having relatively limitations.

The present invention provides a primer set comprising the primers of SEQ ID NOs: 1 to 4 for amplifying exon 7 of the rhesus monkey ABO locus of a selective nucleotide sequence. The sequence of the primer set used in the present invention is shown in Table 1 below.

Sequence number 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 a preferred embodiment, the primer set of the present invention is set-1 (SET-1) comprising a PA2 forward primer (SEQ ID NO: 1) and a PO2 forward primer (SEQ ID NO: 2); And 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 additionally include a common reverse primer (CAB, SEQ ID NO: 5). When PCR is performed for exon 7 of the rhesus monkey ABO blood type locus by combining the above set-1 and set-2, various combinations of band patterns appear, through which the ABO blood type of the rhesus monkey and whether or not it is homozygous or heterozygous The primer set of the present invention can be used to successfully amplify exon 7 of the ABO blood type gene of the rhesus monkey without interference between the primers constituting it. Therefore, when real-time PCR is performed using the above primer set, ABO blood type of rhesus monkeys can be detected very reliably and quickly.

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

In addition, the present invention provides a kit for detecting ABO blood type of rhesus monkeys comprising the detection composition. The kit of the present invention may further include other probe sets in addition to the primers of SEQ ID NOs: 1 to 4. In addition, the present invention (s1) collecting a DNA sample from the rhesus monkey; (s2) performing PCR using a primer set including primers of SEQ ID NOs: 1 to 4 for amplifying exon 7 of the rhesus monkey ABO locus as a template using the DNA collected in step (s1); And (s3) it provides a method for detecting ABO blood type in rhesus monkeys comprising the step of examining the product amplified by 　 PCR in (s2).

The detection composition of the present invention specifically detects the SNP of a gene that determines the ABO blood type of rhesus monkeys by using a polymerase chain reaction method, thereby accurately detecting the ABO blood type of rhesus monkeys.

1 is a FACS analysis result of 66 rhesus monkey sera before (A) and after (B) pre-absorption with human O+ red blood cells (RBC). (A) Two monkeys (asterisks; R058 and R154) showed false-positive antibody binding to human A+ and B+ RBCs. (B) After pre-absorption with human O+ RBC, these non-specific or heteroreactive bonds were removed to correct the blood types of the two monkeys.
2 shows a representative image of the results of immunohistochemical staining of oral mucosa cells of rhesus monkeys. 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 monopolymorphism in exon 7 of the ABO locus. Whole blood of 66 rhesus monkeys was PCR-treated with primers. (A) SET-1 is composed of PA2 and PO2. (B) SET-2 is composed of PO3 and PB. The SET-1 and SET-2 based PCR assays 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, and BO heterozygotes are represented by all four bands. , AB heteroconjugates are represented by PA2, PO2, and PB bands, and OO homozygotes are represented by PA2 and PO3 bands. L, ladder; bp, base pair.
4 shows the total exon 7 sequence obtained from chromosome 15 of rhesus monkey. Red highlighting indicates mononucleotide polymorphism, blue arrows indicate forward primer sequences (PO3, PA2, PB, and PO2), and black arrows indicate reverse primer sequences (CAB).

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

1. Experimental materials and methods

(1) Objects used in the experiment

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

(2) erythrocyte aggregation analysis

Hemagglutination assay was performed with 66 monkey serum and human RBC reagent. 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. The supernatant was discarded. This process was repeated twice. Finally RBC was diluted to 5% with PBS. Monkey serum was obtained by collecting whole blood from the femoral vein of the experimental animal, and the serum was collected by centrifugation at 3500 rpm for 10 minutes. Serum was diluted 10% with PBS. The diluted monkey serum was then mixed 2:1 with human A+, B+, or O+ RBC suspension, and incubated at room temperature for 30 minutes. The aggregation reaction was observed on a plate and under a microscope. Aggregation was graded into strong (+++), medium (++), weak (+), and negative (-).

(3) Flow cytometry analysis

In some cases, the aggregation reaction showed inconsistent results when compared to other ABO type assay results. Thus, all 66 monkeys were subjected to FACS analysis, which can be confirmed using flow cytometry. Monkey serum was mixed with the human RBC 2:1, incubated for 30 minutes, and then centrifuged at 1500 rpm for 5 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 dark 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 of PBS and flow cytometric analysis was performed on FACS Calibur (Becton Bioscience, Mountain View, CA, USA). All data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

By case of discrepancy, all monkey serum was also preabsorbed by human O+ RBCs followed by FACS analysis. Thus, 5% human O+ RBC was mixed with 100 μl of 10% monkey serum 2:1, 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 and prepared 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 cotton swab tip applicator. Smear from each animal was applied to a microscope slide. Slides were fixed in cold acetone (Sigma, St Louis, MO, USA) for 10 minutes, then dried and washed with PBS. Each of 1:50 and 1:20 dilutions of mouse anti-A and anti-B antibodies (Sigma, St. Louis, MO, USA) in PBS were prepared and added to the samples. After incubation at room temperature for 60 minutes, washed with PBS, and slides were washed with 100 μl of 1:100-diluted FITC-conjugated goat anti-mouse IgM (Vector, Burlingame, CA, USA) in dark conditions for 60 minutes at room temperature. It was further cultured in. The slides were washed with PBS, fixed with Antifade-DAPI (Vector)-1-6-Diamidino-2-phenylindole, and stored in dark conditions at 4°C. Staining was observed using a fluorescence microscope.

(5) new optional Of the primer Fabrication and development of PCR-based ABO blood type measurement method

After making a new primer, to determine the ABO type of the experimental monkey, the genomic DNA of 66 monkeys was extracted from whole blood using the QIAmp® DNA Blood Mini Kit purchased from QIAGEN (Valencia, CA, USA). To amplify exon 7 of the ABO locus, PCR was performed using primers 5′ CCT GCC TTG CAG ATA CGT G 3′ (forward) and 5′ CAG CTG ATC ACG GGT TCC 3′ (reverse). PCR was performed in Peltier Thermal Cycler (PTC)-200 for 5 minutes at 95°C, followed by 35 cycles at 95°C for 20 seconds, 58°C for 20 seconds, and 68°C for 1 minute. The PCR product was given a poly(A) tail and then inserted into the TA cloning vector pGEM-T Easy (Promega, Madison, WI, USA). The sub-cloned plasmid was prepared with AccuPrep Plasmid Mini Extraction Kit (Bioneer, Daejeon, Korea) for sequencing. The nucleotide sequence was confirmed by ABI PRISM® System (model 377). All other chemical reagents were purchased from Duchefa Biochemie (Haarlem, Netherlands).

In order to design a new selective primer, genomic DNA was obtained from 10 monkeys out of 66 animals that were subjected to ABO blood typing by serology and IHC staining. All of these exon 7 sequences were obtained, and SNPs were identified on their ABO locus (FIG. 4). Two sets of primers were selected to analyze monkey ABO blood types. SET-1 consists of PA2-F, PO2-F, and CAB primers, whereas SET-2 consists of PB-F, PO3-F, and CAB primers (Table 2).

Primer sequence used for PCR analysis. E7F, exon 7 forward; E7R, exon 7 reverse; CAB, common AB reverse primer. Sequence number 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 type of 66 monkeys, PCR was performed with a new selective primer using Takara taq polymerase (Takara, Seoul, Korea). PCR was denatured in PTC-200 at 95° C. for 1 minute, followed by 30 cycles at 95° C. for 30 seconds, 64° C. for 30 seconds, and 72° C. for 30 seconds. Subsequently, the PCR product was electrophoresed on a 2% agarose gel.

2. Experiment result

(One) Rhesus monkey ABO blood group frequency

The ABO blood types of 66 adult rhesus monkeys were analyzed using hemagglutination assay, IHC, and PCR assay with new primers. The experimental results are shown in Table 3. The IHC staining results were completely consistent with the PCR results. In the above two experimental results, type B was the most common (65%), followed by type AB (18%), type A (9%), and type O (8%). Serological tests using hemagglutination assay differed from the IHC and PCR results in 7 monkeys (10.6% of all monkeys) (Table 3). Thus, if blood type analysis was determined by serology alone, the results may have influenced the frequency of other blood types.

Results of ABO type analysis in 66 rhesus monkeys using serology, immunohistochemistry, and PCR analysis. An asterisk (*) indicates a discrepancy between the methods of analysis for monkey blood types.

(2) Comparative example 1: Serotype test using erythrocyte aggregation reaction

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

(3) FACS analysis of erythrocyte aggregated serum samples

To determine if the mismatched aggregation result was due to the presence of an unknown nonspecific antibody 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 resulted in inconsistent aggregation assay results. When IHC and PCR indicated AB type, the remaining two monkeys (R058 and R154), where the results of each microscope and plate were indicated as type A and type B, respectively, showed non-specific antibody binding: one incorrectly indicated type O. The other showed type A (Fig. 1A, asterisk (*)). However, when these mismatched serum samples were preabsorbed with human O+ RBCs and then FACS analysis, non-specific binding (probably for unknown agglutinates) was clearly resolved: both showed AB type (FIG. 1B ). Analysis of FACS data with preabsorbed sera from 66 monkeys (FIG. 1B) showed that type A and type B monkey sera bind to human B+ and A+ RBCs, each with at least 1.5% antibody binding. Monkey sera of the 5 O+ types strongly bound to both A+ and B+ RBCs. In contrast, AB+ type serum weakly recognized A+ and B+ RBCs. Exceptionally R152 monkeys were incorrectly analyzed as AB+ by aggregation analysis and A+ by IHC and PCR: their serum bound relatively weakly to only B+ RBCs (0.97%).

(4) Comparative example 2: Analysis of ABO type through IHC staining

IHC staining of oral mucosal cells with anti-A and anti-B antibodies clearly confirmed the blood types of all monkeys: all two smears in each animal produced consistent results (FIG. 2 ). Although impurities from the mucous membrane sample were not completely removed during the staining process, it did not interfere with blood type determination.

(5) Example : Determination of ABO allele through PCR analysis

In PCR using a new selectively designed primer set, AA homozygotes are indicated by PA2 and PO2 bands, AO heterozygotes are indicated by PA2, PO2, and PO3 bands, BB homozygotes are indicated by PO2 and PB bands, and BO Heteroconjugates are represented by all four bands, and type AB is represented by the PA2, PO2, and PB bands. Significantly, the algorithm of the present invention can also detect O homozygotes: they are represented by the PA2 and PO3 bands (Figure 3). In other words, the absence of the PO2 band represented by G -> C SNP (nucleotide position 5196271) is important in defining the O homozygous. This is important because previously reported PCR analysis was unable to detect O homozygotes in macaque monkeys. All of these PCR results were in perfect agreement with the IHC results of oral mucosal cells. In particular, the PCR assay of the present invention also has advantages over all other serological assays (up to IHC) tested in the present invention: the PCR assay of the present invention indicates whether each monkey is a 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> P16-168, SNU-2018-5494 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PA2-F <400> 1 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 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB-F <400> 3 ggagatcctg actccgctct t 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PO3-F <400> 4 gccaccgggt ccactacttt 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CAB <400> 5 ccctggtgag ccgctgcacc tcc 23 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> E7F <400> 6 cctgccttgc agatacgtg 19 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> E7R <400> 7 cagctgatca cgggttcc 18

Claims (7)

Set-1 (SET-1) comprising a PA2 forward primer (SEQ ID NO: 1) and a PO2 forward primer (SEQ ID NO: 2); And
Consisting of set-2 (SET-2) containing PB forward primer (SEQ ID NO: 3) and PO3 forward primer (SEQ ID NO: 4),
Primer set to amplify exon 7 of rhesus monkey ABO locus. delete The primer set of claim 1, wherein the set-1 and set-2 additionally include a common reverse primer (CAB, SEQ ID NO: 5). A composition for detecting ABO blood type of rhesus monkeys comprising the primer set of claim 1. The composition for detecting ABO blood type of rhesus monkeys according to claim 4, wherein the detection is detected by polymerase chain reaction (PCR). A kit for detecting ABO blood type of rhesus monkeys comprising the composition of claim 4. (s1) taking a DNA sample from the rhesus monkey;
(s2) performing PCR using a primer set including primers of SEQ ID NOs: 1 to 4 for amplifying exon 7 of the rhesus monkey ABO locus as a template using the DNA collected in step (s1); And
(s3) examining the product amplified by PCR in (s2);
Including,
The primer set is set-1 (SET-1) including a PA2 forward primer (SEQ ID NO: 1) and a PO2 forward primer (SEQ ID NO: 2); And
A method for detecting ABO blood type in rhesus monkeys consisting of SET-2 (SET-2) comprising PB forward primer (SEQ ID NO: 3) and PO3 forward primer (SEQ ID NO: 4).

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