CN106967808B

CN106967808B - Primer group for detecting RhD negative blood type and application thereof

Info

Publication number: CN106967808B
Application number: CN201710230590.0A
Authority: CN
Inventors: 胡彬; 迟晓云; 朱于莉; 韩斌
Original assignee: Qingdao Blood Center (qingdao Blood Transfusion Institute Of Medicine Qingdao Citizens Blood Donation Office)
Current assignee: Qingdao Blood Center (qingdao Blood Transfusion Institute Of Medicine Qingdao Citizens Blood Donation Office)
Priority date: 2017-04-11
Filing date: 2017-04-11
Publication date: 2020-11-27
Anticipated expiration: 2037-04-11
Also published as: CN106967808A

Abstract

The invention provides a primer group for detecting RhD negative blood type, which can detect more than ten kinds of RHD genotypes of individuals which are detected as RhD negative or D variation in 226 cases of serological confirmation experiments, and find 1 case of new SNP sites, wherein the SNP sites are that the 208 th base of an RHD gene coding region from an initiation codon is mutated, and C is mutated into T; the insertion of G into the 211 th base results in a subsequent frameshift mutation. The primer pair comprises an upstream primer and a downstream primer for PCR amplification of 1-10 exons and 10 sequencing primers. The invention not only verifies a plurality of discovered genotypes, but also discovers a new SNP locus, thereby providing an effective RhD negative blood type identification way, and the application effect shows that the detection primer provided by the invention can be effectively used for clinical patients and blood donors in blood stations to carry out rapid detection on the RHD gene, thereby accurately determining the RhD blood type.

Description

Primer group for detecting RhD negative blood type and application thereof

Technical Field

The invention belongs to the technical field of gene diagnosis products, and particularly relates to a primer group for detecting RhD negative blood type and application thereof.

Background

The Rh blood group system (Rhesus monkeys) was first discovered from the red blood cells of Rhesus monkeys by a famous scientist, Nobel prize-winning Karl Landsteiner, and was thus named. The Rh blood group system is the most complex of the current 36 blood group systems of human, has importance second to the ABO system, and plays an important role in clinical blood transfusion and diagnosis and treatment of neonatal hemolytic disease. The D antigen in Rh blood group system is the most immunogenic and the most complex polymorphic. For Han population in continental China, the RhD negative population accounts for about three thousandths, and is rare, so the panda blood is also named. RhD-negative individuals cannot receive RhD-positive blood due to their strong antigenicity, because the RhD antigen will stimulate production of anti-RhD antibodies in RhD-negative individuals. If RhD positive blood is reinfused, hemolytic transfusion reactions can result. Similarly, if RhD-negative women develop anti-RhD antibodies in vivo as a result of pregnancy or transfusion stimuli, re-pregnancy can lead to the development of hemolytic disease in the newborn.

The RhD blood group system has many variants, including weak d (week d), partial d (partial d), and DEL (diffuse), in addition to the normal RhD positivity and negativity. These variants have two main characteristics, because incomplete or variant RhD antigens are present on the surface of erythrocytes and can stimulate the production of antibodies when infused into negative individuals: firstly, they are treated differently as recipients and donors, and as recipients, they can only input RhD negative blood; as donors, their blood can only be used as RhD positive blood. And secondly, the reagent is easy to miss detection, and the conventional antibody can not react with the RhD antigen during conventional blood type serology detection due to the variation of the RhD antigen, so that the reagent is easy to be mistaken to be RhD negative, and the reagent has great harm to patients needing multiple blood transfusions and pregnant women due to no effective measure for clearing the irregular antibody at present. Accurate typing is therefore a prerequisite to ensure correct infusion. The conventional method for detecting blood type is serological experiment, but the experiment is limited by various factors, such as sample quality, interference of autoantibody or irregular antibody, disease influence and the like, and the DEL type which can be detected only by absorption and diffusion test cannot be judged, so that the DEL type needs to be diagnosed at gene level by using molecular biological method.

The RH gene is located in the short arm 34.3-36.1 region of the first human chromosome and consists of RHD gene (encoding RhD antigen) and RHCE gene (encoding RHC, c, E and E antigens) which are closely connected in series. The RHD and RHCE genes are highly structurally homologous, consisting of 10 exons and 9 introns, of which exon 8 is identical, with the major differences being exons 3, 4, 5, 7, 9 and intron 4. Therefore, the RHD gene detection system is established by adopting a molecular biological means, is applied to clinical and blood collection work, is beneficial to accurately detecting RhD negative individuals, reducing the generation of irregular antibodies of blood recipients, preventing and monitoring prenatal irregular antibodies and reducing the occurrence of hemolytic disease of newborn.

Disclosure of Invention

The invention aims to provide a primer group for detecting RhD negative blood type, which can more effectively confirm the RhD negative blood type, thereby making up the defects of the prior art.

The applicant carried out RHD gene sequencing on 226 individuals tested as RhD negative or D variant by serological confirmation experiments, 166 samples (73.45%) with all negative exons of the RHD gene 1-10, 60 samples (26.55%) with partial or all existing exons, and obtained 11 allelic types in total, respectively, RHD1227G > A28 case (12.39%), RHD-CE- (2-9) -D19 case (8.41%), RHD845G > A4 case (1.77%), RHD711delC 2 case (0.88%), RHD697G > A1 case (0.44%), RHD-CE- (3-7) -D1 case (0.44%), RHD3G > A1 case (0.44%), RHD1013T > C1 case (0.44%), RHD 1227A/G1 case (0.44%), RHD 1-10 exon no mutation found 1 case (0.44%), and new SNP mutations resulting in RHD negative 1 case (0.44%). The new SNP locus is that the 208 th base of the RHD gene coding region from the initiation codon is mutated, and the C is mutated into T; the insertion of G into the 211 th base results in a subsequent frame shift mutation and premature termination. The RHD blood type of the blood sample to be tested can be accurately determined by detecting the RHD gene, thereby promoting the invention.

The invention firstly provides a primer pair for detecting RhD negative blood type, the upstream and downstream primer sequences of the primer pair are in SEQ ID NO. 1-20,

the specific primer information is as follows:

RHD-1F:5’-AACTCCATAGAGAGGCCAGCAC-3’SEQ ID NO:1

RHD-1R:5’-AGATGGGGGAATCTTTTTCCTT-3’SEQ ID NO:2

RHD-2F:5’-TCCCCCTCGTCCTTCTCG-3’SEQ ID NO:3

RHD-2R:5’-CAGGATGCCCAGTTAATTTGAAT-3’SEQ ID NO:4

RHD-3F:5’-CCACAGAAAGTAGGTGCCCAA-3’SEQ ID NO:5

RHD-3R:5’-TCTTTATTTTTCAAAACCCTGGAAA-3’SEQ ID NO:6

RHD-4F:5’-TCCAAGGACTATCAGGGCTTG-3’SEQ ID NO:7

RHD-4R:5’-CTTCAGACACCCAGGGGAAC-3’SEQ ID NO:8

RHD-5F:5’-CAGCCCTAGGATTCTCATCCAA-3’SEQ ID NO:9

RHD-5R:5’-GCAGAAATGGGGTTCAAACTG-3’SEQ ID NO:10

RHD-6F:5’-CCCATCATGTCCACTGATGAA-3’SEQ ID NO:11

RHD-6R:5’-AGCCAAAGCAGAGGAGGTTAGTT-3’SEQ ID NO:12

RHD-7F:5’-AACTCCCCGATGATGTGAGTG-3’SEQ ID NO:13

RHD-7R:5’-CAAGGTAGGGGCTGGACAG-3’SEQ ID NO:14

RHD-8F:5’-TGGAGGCTCTGAGAGGTTGAG-3’SEQ ID NO:15

RHD-8R:5’-TCCTGGCAATGGTGGAAGAA-3’SEQ ID NO:16

RHD-9F:5’-TACTGTCGTTTTGACACACAATATTTC-3’SEQ ID NO:17

RHD-9R:5’-GGGAAGTGTGGCCAGTGAAA-3’SEQ ID NO:18

RHD-10F：5’-TCAGTATGTGGGTTCATCTGCAA-3’SEQ ID NO:19

RHD-10R:5’-TGGATGACCACCATCATATATGC-3’SEQ ID NO:20；

the invention secondly provides a sequencing primer for detecting RhD negative blood type, wherein the primer sequence is one of the amplification sequences, and the primer information is as follows:

RHD-1:5’-AACTCCATAGAGAGGCCAGCAC-3’SEQ ID NO:1

RHD-2:5’-CAGGATGCCCAGTTAATTTGAAT-3’SEQ ID NO:4

RHD-3:5’-CCACAGAAAGTAGGTGCCCAA-3’SEQ ID NO:5

RHD-4:5’-TCCAAGGACTATCAGGGCTTG-3’SEQ ID NO:7

RHD-5:5’-CAGCCCTAGGATTCTCATCCAA-3’SEQ ID NO:9

RHD-6:5’-CCCATCATGTCCACTGATGAA-3’SEQ ID NO:11

RHD-7:5’-AACTCCCCGATGATGTGAGTG-3’SEQ ID NO:13

RHD-8:5’-TGGAGGCTCTGAGAGGTTGAG-3’SEQ ID NO:15

RHD-9:5’-TACTGTCGTTTTGACACACAATATTTC-3’SEQ ID NO:17

RHD-10:5’-TGGATGACCACCATCATATATGC-3’SEQ ID NO:20；

the primer pair is used for preparing a product for detecting RhD negative blood type;

the product is preferably a molecular detection kit, and comprises any one or more of the primer pairs.

The invention provides a primer group for detecting RHD gene, thereby providing an identification way for effectively carrying out RhD negative blood type, and the application effect shows that the detection primer provided by the invention can be effectively used for clinical patients and blood donors in blood stations to carry out rapid detection on the RHD gene, thereby accurately determining the RhD blood type.

Drawings

FIG. 1: the sequencing diagrams of the partial RhD-negative or mutant RHD genes of example 1 are RHD711delC, RHD3G > A, RHD1013T > C, RHD845G > A, RHD1227G > A, RHD697G > A and RHD208C > T, 211insG, respectively.

FIG. 2: SNP mutation proband its parental RHD gene second exon sequencing map of example 2, where a: the father of the first person carries the RHD complete deletion gene with the normal RhD positive phenotype; b: firstly, the mother of the patient carries the mutant gene with the normal RhD positive phenotype; c: proband, RhD negative phenotype. In the family, the 208 th base of the two RHD gene coding regions from the initiation codon is mutated, C is mutated into T, the 211 th base is subjected to insertion mutation and G insertion, and the subsequent frame shift mutation is caused.

Detailed Description

The applicant carried out RHD gene sequencing on 226 individuals tested as RhD negative or D variant by serological confirmation experiments, found 166 samples with all negative RHD gene 1-10 exons and 60 samples (26.55%) with partial or all existing samples, and obtained 11 allelic genotypes, 10 of which are reported and 1 of which are not reported, and the testing of new genotypes can be used for identifying the individuals with RhD negative or D variant, thereby leading to the invention.

The RH gene is located in the chromosome 1p34.3-1p36.1 region, can be transcribed into 2837bp mRNA (NCBI accession No. NM-016124.4), and finally translated into 417 amino acid protein.

The present invention will be described in detail with reference to examples.

Example 1: screening of mutant sites of RHD gene from serological RhD negative or mutant blood donors

1. Extracting peripheral blood genome DNA:

on the basis of meeting the relevant national policy regulations and agreeing with a sampling object, extracting 2-5mL of peripheral venous blood of a RhD negative or variant blood donor, and putting the peripheral venous blood into an EDTA anticoagulant tube to be frozen at-80 ℃ for later use; after the frozen EDTA anticoagulation blood is melted at room temperature, 500 mu L of the EDTA anticoagulation blood is put into a centrifuge tube, equal volume of TE (pH8.0) is added into the centrifuge tube, the mixture is mixed evenly, the mixture is centrifuged for 10 minutes at 10000rpm at 4 ℃, and the supernatant is discarded.

Add 180. mu.L TE, 20. mu.L LSDS (10%), 8. mu.L proteinase K (L0mg/ml), mix well and place in a 37 ℃ water bath overnight. The sample was removed from the water bath and the sample was pelleted by instantaneous centrifugation. An equal volume of Tris-saturated phenol (about 300. mu.L) was added to the reaction tube, mixed well, centrifuged at 10000rpm for 10 minutes at room temperature, and the supernatant (about 300. mu.L) was pipetted into a new centrifuge tube. Phenol extraction was repeated once and the supernatant was aspirated into a new centrifuge tube.

Adding equal volume of Tris saturated phenol and chloroform mixed solution (150 μ L of phenol and chloroform respectively), mixing, centrifuging at room temperature of 10000rpm for 10 minutes, and transferring the supernatant to a new centrifuge tube.

Adding equal volume of Tris saturated phenol, chloroform and isoamyl alcohol mixed solution (100 μ L of each of phenol, chloroform and isoamyl alcohol), mixing, centrifuging at room temperature of 10000rpm for 10 minutes, and transferring the supernatant to a new centrifuge tube.

Add L/10 volume of 3mol/L, pH5.2 sodium acetate (about 30. mu.L), 2 volumes of pre-cooled 100% ethanol, mix gently to see white flocculent precipitate. The DNA was precipitated at the bottom of the tube by centrifugation at 10000rpm for 10 minutes at room temperature, and the supernatant was discarded.

To the DNA precipitation adding 70% ethanol, rinsing, room temperature 7000rpm centrifugation for 5 minutes, abandoning the supernatant, placed in room temperature to volatilize the ethanol, finally adding 50 u L TE (pH8.0), 4 degrees overnight dissolved DNA.

And (3) performing agarose gel electrophoresis on the extracted DNA, and performing color comparison at 260nm and 280nm by using an ultraviolet spectrophotometer to detect the purity and the concentration of the DNA.

2. Direct sequencing method for searching mutation of RHD gene of blood donor

PCR amplification of the fragment of interest: reaction conditions and reaction system:

(1) and (3) PCR reaction conditions: 5m at 95 ℃; 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 60s, 35 cycles; 5m at 72 ℃.

(2) Reaction system: (Onelambda Fast startTaqpolymerase)

The reaction system is used for respectively carrying out amplification reaction of the genome DNA template of each RhD negative blood donor and the RhD primer.

Sequencing a PCR product: the PCR products were sequenced by conventional Sanger sequencing to obtain 11 kinds of alleles, including RHD1227G > A, RHD-CE- (2-9) -D, RHD845G > A, RHD711delC, RHD697G > A, RHD-CE- (3-7) -D, RHD3G > A, RHD1013T > C, RHD1227A/G, RHD 1-10 exon, and 1 new SNP (FIG. 1). The SNP locus is detected by amplifying the second exon of the RHD gene through RHD-2F 5'-TCCCCCTCGTCCTTCTCG-3' SEQ ID NO 3 and RHD-2R 5'-CAGGATGCCCAGTTAATTTGAAT-3' SEQ ID NO 4, sequencing by using the RHD-2R 5'-CAGGATGCCCAGTTAATTTGAAT-3' SEQ ID NO 4, and finding that the base C at the 208 th position is mutated into T, the base G at the 211 th position is subjected to insertion mutation, so that the subsequent frame shift mutation is caused, the base coding at the 475 sand 477 position is TAA stop code, the second exon is only translated into 159 amino acids, so that the subsequent 8 exons cannot be translated into protein, the translation of the RhD protein is terminated early, and the RhD negative blood group is caused. Multiple sequencing results indicated that the mutation site was not introduced due to amplification or sequencing errors. The mutation is a nascent mutation. This mutation is not present in the following four databases: single nucleotide polymorphism databases (ftp:// ftp. ncbi. nih. gov/snp/database /), thousand human genome project (ftp:// ftp-trace. ncbi. nih. gov/1000 genes/ftp /), Hapmap8 database (http:// Hapmap. ncbi. nlm. nih. gov /), and Yanhuang database (http:// yh. genomics. org. cn /), indicate that the mutation is very rare. In contrast, in the peripheral blood genomic DNA samples of 200 RhD-positive blood donors, the site was screened for the mutation, and the mutation was not found.

Through the analysis, the RHD gene amplification and sequencing primer can be used for accurately determining the RHD gene, so that the RhD blood type of a person to be detected can be more accurately determined, and the method has important significance for a patient to formulate a blood transfusion policy.

Example 2: genetic verification of parents of SNP mutation proband

1. Extracting peripheral blood genome DNA:

2. Direct sequencing method for searching mutation of second exon of proband parent RHD gene

(2) Reaction system: (Onelambda Fast startTaqpolymerase)

The reaction system is applied to carry out amplification reaction of the genome DNA template of the proband parents and the primers RHD-2F: 5'-TCCCCCTCGTCCTTCTCG-3' SEQ ID NO:3 and RHD-2R: 5'-CAGGATGCCCAGTTAATTTGAAT-3' SEQ ID NO:4 for amplifying the second exon of the RHD gene respectively.

Sequencing a PCR product: sequencing the PCR product by a conventional Sanger sequencing method and using RHD-2R: 5'-CAGGATGCCCAGTTAATTTGAAT-3' SEQ ID NO:4 (figure 2), wherein the proband father (A) is a normal RhD positive phenotype and carries an RHD complete deletion gene; the proband mother (B) is of a normal RhD positive phenotype, carries a mutant gene and shows a heterozygous peak of an RHD normal gene and an SNP mutant gene; proband (C) inherits the RHD complete deletion gene of the father and the SNP mutant gene of the mother, and shows RhD negative phenotype. This mutation is not present in the following four databases: single nucleotide polymorphism databases (ftp:// ftp. ncbi. nih. gov/snp/database /), thousand human genome project (ftp:// ftp-trace. ncbi. nih. gov/1000 genes/ftp /), Hapmap8 database (http:// Hapmap. ncbi. nlm. nih. gov /), and Yanhuang database (http:// yh. genomics. org. cn /), indicate that the mutation is very rare. In contrast, in the peripheral blood genomic DNA samples of 200 RhD-positive blood donors, the site was screened for the mutation, and the mutation was not found.

The experiment result shows that the SNP mutation site can accurately determine the RhD blood type of a person to be detected, and has important significance for a patient to formulate a blood transfusion policy.

SEQUENCE LISTING

<110> Qingdao City central blood station (Qingdao city national obligation blood donation office Qingdao city transfusion medical research institute)

<120> primer group for detecting RhD negative blood type and application thereof

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Claims

1. The application of a primer pair for detecting SNP loci in preparing a product for detecting RhD negative blood type; the SNP locus is a 208 th base in the RHD gene coding region from an initiation codon, and is mutated from C to T; g is inserted into the 211 th base;

the sequences of the primer pair are SEQ ID NO. 3 and SEQ ID NO. 4.

2. The use of claim 1, wherein the article of manufacture is a molecular assay kit.

3. A molecular detection kit is characterized in that the kit comprises a primer pair for detecting SNP loci; the SNP locus is a 208 th base in the RHD gene coding region from an initiation codon, and is mutated from C to T; g is inserted into the 211 th base;

the sequences of the primer pair are SEQ ID NO. 3 and SEQ ID NO. 4.