CN108410972B - Genotyping detection kit for 23 genetic loci of human Rh blood group - Google Patents
Genotyping detection kit for 23 genetic loci of human Rh blood group Download PDFInfo
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Abstract
The invention belongs to the technical field of human nucleic acid in-vitro detection, and particularly relates to a genotyping detection kit for 23 gene loci of human chromosomes 5, 19, 21 and X/Y. The invention firstly designs a composite PCR amplification system for simultaneously genotyping 23 gene loci distributed on human chromosomes 5, 19, 21 and X/Y, wherein the PCR amplification primer group comprises: SEQ ID No.1 to SEQ ID No. 53; the genotyping detection kit comprises a primer composition container and a PCR reaction mother liquor container; the primer composition container comprises primers SEQ ID No. 1-SEQ ID No.53 composition stock solution. The invention realizes the single-tube amplification of 23 gene loci, the amplification of the gene loci in each fluorescence channel and in different fluorescence channels is balanced, and the typing map is good; but also can obviously save cost, manpower and time and improve the working efficiency.
Description
Technical Field
The invention belongs to the technical field of human nucleic acid in-vitro detection, and particularly relates to detection of genotypes of polymorphic genetic loci in human genome DNA, in particular to a kit for carrying out Rh blood group genotyping detection on 23 genetic loci on human chromosomes 5, 19, 21 and X/Y by adopting multiple polymerase chain reaction.
Background
Two blood types, Rh negative and Rh positive, can be distinguished according to the existence of Rh factor, and the blood type system is called Rh blood type system. Rh is the first two letters of the Rhesus monkey (Rhesuus Macacus) foreign language name. Scientists such as landstein et al, in 1940, found that there were antigenic substances of Rh blood group on erythrocytes in rhesus monkeys and most humans, and so named. Rh-positive was found in all cases where Rh was present on erythrocytes in human blood. Otherwise, the result is negative.
The Rh blood group system is the most polymorphic blood group system in the human erythrocyte blood group system, has high immunogenicity, is an important blood group system next to the ABO blood group, and has important clinical significance. More than 40 Rh systemic antigens can be identified at present, but in the clinically common antigen 5, the antigenicity is sequentially D > C > E > C > E from strong to weak, wherein the existence of the D antigen is often used to indicate that Rh is positive or negative, written as Rh (D) + or Rh (D) -, and the letter D plus a small bracket indicates the antigen (without the small bracket, the corresponding gene is indicated by italics). Has important significance in clinical transfusion therapy of Rh (D) -negative population and neonatal hemolysis caused by maternal-fetal immunity. The proportion of Rh (D) negative people in Chinese Han population is about 0.3-0.5%, but the proportion of Rh (D) negative people in part of minority population can be as high as 10%. Foreign literature reports that the proportion of Rh (D) -negative individuals in the Caucasian population can be as high as 10-18%.
In recent years, along with cloning and sequencing of RhD and RhCE genes encoding Rh protein, great progress is made in research of molecular genetic mechanism of Rh blood group system and typing detection of Rh blood group. By Southern hybridization analysis, it has been determined that the Rh "locus" consists of two homologous structural genes: one encoding the rh (d) polypeptide and the other encoding the (CcEe) polypeptide. The research shows that after 1 single cDNA of RhCE is transferred to K562 cells, the full-length peptide chain can express E/E and C/C antigens. The RhD and RhCE genes are highly homologous, differing in nucleotide sequence by only about 7%, and the scholars believe that this may be a duplication of origin in a common ancestral gene.
The genetic polymorphism of RhD is complex and rich, and comprises complete deletion of RhD gene, partial deletion of RhD gene, point mutation of gene, RhD Ψ pseudogene and the like; the Rh (D) antigen has various genetic mechanisms, and the Rh (D) antigen has various variants, including weak D type, partial D type and other D weak phenotypes, at least 42 weak D types and 6 classes of partial D are clarified at present. Many scholars at home and abroad find that the case of mistakenly transfusing blood of a weak D-type donor to Rh (D) -negative recipients by a hematology serology method to generate primary anti-D is also reported, and the anti-D antibody is generated by partial D immunization 15-20 times. Research studies on caucasian populations have found that rh (D) negative populations are predominant due to RhD gene deletion, with only 0.2-1% of caucasian populations exhibiting weak D-type, with about 95% weak DI-IV. However, due to the lack of a mature detection method in China, the Rh blood group molecular background and population distribution of the domestic population are lack of systematic research, but researches indicate that the genetic mechanism of the Rh blood group in China is various and complex, and researches in different areas in China find that only about 50% of Rh (D) negative populations in China are caused by RhD gene deletion, about 20-30% of Rh (D) negative populations in China are in a diffuse Type, wherein more than 95% of Rh (D) negative populations are in 1227G > A mutation, and the rest 10-20% of Rh (D) negative populations are in a weak D phenotype, wherein more than 60% of Rh blood group molecular background and more than 30% of Rh blood group molecular background and population distribution are in lack of systematic research, and are in a part of DVI.
In view of the importance of Rh blood groups, fraction D, weak D in clinical Transfusion and Rh blood groups in fetal and neonatal hemolytic diseases caused by maternal-fetal blood group incompatibility, and the uncertainty of these Rh (D) antigen variants in routine serological testing, the 2015 American society of Pathologists (CAP), American society of Transfusion and Medical Resources Committee (TMRC) have been commonly referred to as enhancing the clinical application of Rh blood groups, particularly weak D, genotyping technology.
Disclosure of Invention
The invention aims to provide a kit for simultaneously carrying out Rh blood group genotyping detection on 23 gene loci on human chromosomes 5, 19, 21 and X/Y, aiming at the requirements of Rh blood group genotyping on gene genotyping of part D, weak D and the like and the requirements of clinical Rh blood group common genotyping detection.
The invention provides a composite PCR amplification system for simultaneously genotyping 23 gene loci of human No.5, No.19, No.21 and X/Y chromosomes, wherein the 23 gene loci are as follows: AMEL, SID1, SID2, SID3, SID4, Ref19, Box, pEx01, IN01, IN02, Ex03, IN03, IN07, IN08, wD1_ gT, wD2_ Gc, wD3_ Cg, wD4_ Cg, wD15_ aG, 1227Ga, Ex10, Dc, Ee. According to the length distribution range of the amplicon of each gene locus, the 23 gene loci are divided into two groups, the first group is 12 gene loci: box, IN07, pEx01, IN02, IN08, Ref19, SID1, SID2, Ex03, IN01, IN03, SID3, and the second group is 11 loci: AMEL, 1227Ga, wD2_ Gc, wD15_ aG, wD1_ gT, Dc, wD3_ Cg, wD4_ Cg, Ex10, Ee, SID 4. The composite PCR amplification system comprises a first group and a second group of PCR amplification primers, wherein the first group comprises the PCR amplification primers corresponding to the following groups of sites: the corresponding primer sequences of the site Box are shown as SEQ ID No.1 and SEQ ID No. 2; the sequences of corresponding primers of the IN07 site are shown as SEQ ID No.3 and SEQ ID No. 4; at the position pEx01, the sequences of the corresponding primers are shown as SEQ ID No.5 and SEQ ID No. 6; the sequences of corresponding primers of the IN02 site are shown as SEQ ID No.7 and SEQ ID No. 8; the sequences of corresponding primers of the IN08 site are shown as SEQ ID No.9 and SEQ ID No. 10; the primer sequence is shown as SEQ ID No.11 and SEQ ID No.12 at the position Ref 19; the site SID1, the corresponding primer sequence is shown as SEQ ID No.13, SEQ ID No. 14; the site SID2, the corresponding primer sequence is shown as SEQ ID No.15 and SEQ ID No. 16; the corresponding primer sequences of the Ex03 site are shown as SEQ ID No.17 and SEQ ID No. 18; the sequences of corresponding primers of the IN01 site are shown as SEQ ID No.19 and SEQ ID No. 20; the sequences of corresponding primers of the IN03 site are shown as SEQ ID No.21 and SEQ ID No. 22; the site SID3, the corresponding primer sequence is shown as SEQ ID No.23 and SEQ ID No. 24;
wherein one primer in the PCR amplification primers corresponding to each locus in the first group of gene loci is labeled by FAM fluorescein; wherein, the upstream primers corresponding to the gene loci Box, pEx01, IN02, IN08, Ref19, SID1, SID2 and SID3 are all marked by FAM fluorescein; downstream primers corresponding to gene sites IN07, Ex03, IN01 and IN03 were labeled with FAM fluorescein.
The second group includes PCR amplification primers corresponding to the following group of sites: site AMEL, corresponding primer sequences are shown as SEQ ID No.25 and SEQ ID No. 26; a site 1227Ga, the corresponding primer sequences are shown as SEQ ID No.27, SEQ ID No.28 and SEQ ID No. 29; the corresponding primer sequences of the position wD2_ Gc are shown as SEQ ID No.30, SEQ ID No.31 and SEQ ID No. 32; position wD15_ aG, the corresponding primer sequence is shown as SEQ ID No.33, SEQ ID No.34 and ID No. 35; position wD1_ gT, the corresponding primer sequence is shown as SEQ ID No.36, SEQ ID No.37, SEQ ID No. 38; the sequences of the corresponding primers of the site Dc are shown as SEQ ID No.39 and SEQ ID No. 40; position wD3_ Cg, the corresponding primer sequence is shown as SEQ ID No.41, SEQ ID No.42, SEQ ID No. 43; position wD4_ Cg, the corresponding primer sequence is shown as SEQ ID No.44, SEQ ID No.45, SEQ ID No. 46; the corresponding primer sequences of the Ex10 site are shown as SEQ ID No.47 and SEQ ID No. 48; the corresponding primer sequences of the site Ee are shown as SEQ ID No.49, SEQ ID No.50 and SEQ ID No. 51; the site SID4, the corresponding primer sequence is shown as SEQ ID No.52 and SEQ ID No. 53;
wherein at least one primer in the PCR amplification primers corresponding to each locus in the second group of gene loci is marked by TAMRA fluorescein; wherein downstream primers corresponding to gene loci AMEL, 1227Ga, wD2_ Gc, wD1_ gT, wD3_ Cg, Ex10 and SID4 are marked by TAMRA; the upstream primers corresponding to gene positions wD15_ aG, Dc, wD4_ Cg, Ee were labeled with TAMRA.
Preferably, the composite PCR amplification primers and the fluorescein modification characteristics corresponding to the 23 gene loci are shown in table 1 and table 2:
TABLE 1
TABLE 2
The lengths of PCR amplification products of each group of gene loci do not overlap with each other. The working concentration of PCR primers of each gene locus is adjusted to ensure that the difference of the peak heights of fragments between homozygotes or heterozygotes in the same group is within 40 percent. The working concentration of the PCR amplification primers corresponding to the first group of gene loci and the second group of gene loci is 80-900 nmol/L.
In the PCR amplification system of the present invention, the primer consists of dry powder or solution of the PCR amplification primer of the oligonucleotide sequence shown by SEQ ID No.1 to SEQ ID No. 53.
The 23 chromosome gene locus composite amplification system provided by the invention comprises 18 genetic marker quality control loci, 4 genetic marker quality control loci, 1 individual quality control locus and 1 quality control locus on other chromosomes, wherein the 4 genetic marker quality control loci are used for respectively detecting RHD gene exons and common mutation loci on RHD genes.
(1)18 detection sites respectively detect common mutation types of Rh blood types, including total deletion of RHD genes, partial deletion of common RhD genes, 1227G > A, weak D1-4 and weak D15 types.
(2) Ref19 site: the site adopts a pair of PCR primers to amplify chromosome 19 segments, and chromosome 19 is used as PCR system quality control.
(3) The 4 Short Tandem Repeat (STR) polymorphism genetic markers are respectively positioned on chromosomes 5, 20 and 21, STR loci are 3 nucleotides or 4 nucleotides core repeat units, show high polymorphism in Han population, can be used for sample confirmation and linkage genetic tracing, and are also experimental quality control sites.
(4)1 the individual locus is used for determining the sex of the detected sample and the quality control of the experiment.
The 23 gene loci detected by the multiplex PCR amplification system provided by the invention are respectively distributed on a plurality of human specific chromosomes to form 6 functional modules: respectively comprises a sample molecular label, PCR internal quality control, a RhD gene full-deletion detection functional region, a partial deletion detection functional region, a weak D and diffusion type detection functional region, and a C/C and E/E detection functional region. Wherein, the full deletion detection function and partial deletion detection function regions of the RhD gene are positioned in an FAM fluorescence channel, the genetic markers of the RhD gene have a semi-quantitative detection function, each genetic marker has a reference peak, and the copy number of the RhD specific amplification fragment is calculated according to the peak height of the reference peak. The tests of weak D type, diffusion type, C/C and E/E are positioned in a TAMRA fluorescence channel and only used for qualitative detection, and the wild type allele is used for supplementary verification of the test result of total deletion or partial deletion while the corresponding site mutant type qualitative detection is carried out.
The second aspect of the invention provides the use of the PCR amplification system of the first aspect of the invention in the preparation of a kit for Rh blood group genotyping detection of 23 loci on human chromosomes 5, 19, 21 and X/Y.
The third aspect of the present invention provides a kit for Rh blood group genotyping detection of 23 loci on human chromosomes 5, 19, 21 and X/Y, comprising a primer composition container containing the PCR amplification system of the first aspect of the present invention and a PCR reaction mother liquor container,
the primer composition container contains a storage solution of primers with sequences from SEQ ID No.1 to SEQ ID No.53, and the concentration of each primer in the storage solution is 4 times of that of the corresponding primer working solution;
the PCR reaction mother liquor container contains 2 times of reaction concentration of PCR reaction mother liquor, and the PCR reaction mother liquor contains DNA polymerase with enzyme activity of 0.5-5U, magnesium ions with concentration of 1-5 mmol/L, dNTP with concentration of 40-400 mu mol/L and Tris-HCl buffer solution with concentration of 20-100 mmol/L.
The fourth aspect of the present invention provides a method for using the detection kit of the third aspect of the present invention, which comprises the following steps:
(1) extracting human genome DNA, and performing ultraviolet light splitting to quantify the concentration of the genome DNA to be 2-10 ng/mu L;
(2) multiplex PCR amplification: the total reaction system is 20 mu L, and the reaction system comprises the following components:
(3) the PCR reaction is carried out on a common PCR instrument (such as ABI9700, ABI9600, Bio-Rad C1000 and the like) under the following conditions: after the starting at 95 ℃ for 15 minutes, 28 cycles of 95 ℃ for 30 seconds, 60 ℃ for 40 seconds and 72 ℃ for 60 seconds, then keeping the temperature at 72 ℃ for 20 minutes, and then keeping the temperature at 4 ℃;
(4) mu.L of PCR product was subjected to capillary electrophoresis on a genetic analyzer according to the conventional procedure.
(5) The data after electrophoresis is analyzed by using a dedicated data analysis software (e.g., GeneMapper, etc.), and a typing map and data of each gene site can be obtained.
Wherein the DNA template refers to human genomic DNA. Human genomic DNA can be extracted from the following tissues or samples by various conventional methods (e.g., Chelex-100 method, magnetic bead method, phenol chloroform method, and various commercially available human genomic DNA extraction kits, etc.): human blood, blood stain, semen stain, saliva stain, amniotic fluid, hair, muscle tissue, bone, etc. Preferably, in 20. mu.L PCR amplification system, the amount of the DNA template is in the range of 2ng to 5ng, which can obtain better amplification and typing results.
According to a fifth aspect of the invention there is provided the use of a test kit according to the third aspect of the invention for the non-diagnostic genotyping of Rh blood groups.
The PCR amplification product of the 23-gene locus composite PCR amplification system provided by the invention can be subjected to capillary electrophoresis analysis by using a Genetic Analyzer (such as AB3130XL and AB3500Genetic Analyzer), and the data after electrophoresis can be analyzed on data analysis software such as GeneMapper and the like to obtain the typing map and data of each gene locus.
The invention has the beneficial effects that:
as the number of the detected gene sites is increased in the process of establishing the multiplex PCR amplification system, the mutual interference between each pair of primers is also increased. According to the invention, through optimized design, single-tube amplification of 23 gene loci is realized, the amplification of the gene loci in each fluorescence channel and in different fluorescence channels is balanced, and the typing map is good.
By adopting the gene typing detection kit for the 23 gene loci of the human Rh blood type, the confirmation work of the common Rh blood type typing and sample tracing can be effectively solved through one-time reaction, and the cost, the labor and the time can be remarkably saved no matter in a PCR amplification link or a genetic analyzer detection link, so that the working efficiency is improved.
Drawings
FIG. 1 is a 23-gene locus typing map obtained by using the kit of the present invention on an example of a single-copy total deletion carrier DNA sample of RhD gene.
FIG. 2 is a 23-gene locus typing map obtained by using the kit of the present invention on an example of a RhD-RhCE fusion gene carrier DNA sample.
FIG. 3 is an example of a 23-gene locus typing map obtained by using the kit of the present invention on a DNA sample of a RhD gene total-deletion Rh blood group negative person.
FIG. 4 is a 23-locus typing map of DNA samples of wD15 mutant carriers obtained by using the kit of the present invention, wherein the RhD gene is not deleted.
Detailed Description
In order to better understand the content of the present invention, the following examples are further described with respect to the detection of 23 loci in a human blood sample. It should be understood that the following specific examples are illustrative of the invention only and are not limiting.
The primer composition in the PCR reaction system reagent comprises primers with nucleotide sequences of SEQ ID No.1 to SEQ ID No.53, is synthesized by a DNA synthesis company according to a general synthesis method commonly used in the field through primer design, and then is prepared into a working concentration of 80-900 nmol/L.
In this example, the amplification reaction was performed on an ABI9700 thermal cycler, electrophoresis and detection were performed on an ABI 3500genetic analyzer, and data analysis was performed using GeneMapper ID v3.2 software. Other reagents and materials used such as internal standard, POP7, capillary electrophoresis buffer, Hi-Di, allele ladder (ladder) are all conventional materials commonly used by those skilled in the art.
Examples
1: human blood sample genomic DNA preparation
The test specimens were donated by volunteers under informed consent. Collecting 1mL of peripheral venous blood according to medical routine, and performing EDTA anticoagulation. Genomic DNA was extracted using a human peripheral blood genome extraction kit from QIAGEN, the elution volume was 100. mu.L, and the concentration of genomic DNA was diluted to 2.5 ng/. mu.L by UV light spectroscopy.
2: preparation of PCR System
A PCR reaction system (20 μ L of total reaction system) was prepared as follows:
and taking out the primer composition container and the PCR reaction mother liquor container from the kit. Calculating the amount of the PCR reaction mother liquor, the amount of the primer composition, the amount of the PCR reaction auxiliary liquor and ddH required by each reaction in the reaction system according to the multiplication of the total reaction number and the required amount of each reaction in the reaction system2O amount, the above reagents were mixed uniformly in a 1.5mL EP tube, 19. mu.L of each PCR reaction tube was dispensed and numbered, and 1. mu.L of the sample DNA template was added and mixed uniformly again in accordance with the sample number.
3: PCR reaction
PCR was performed using an ABI9700 PCR instrument.
The PCR conditions were as follows: after 15 minutes at 95 ℃ for 28 cycles of 30 seconds at 95 ℃, 40 seconds at 60 ℃ and 60 seconds at 72 ℃, the mixture is then incubated for 20 minutes at 72 ℃ and then for 4 ℃.
4: capillary electrophoresis typing detection
(1) Taking (1 mu L internal standard +10 mu L Hi-Di) × (sample number +1) to prepare a mixed solution, mixing, and then respectively filling 10 mu L of mixed solution in the holes of a 96-hole plate according to each tube. One well was loaded with 1. mu.L of the allele Ladder (Ladder).
(2) 1 μ L of PCR product was added to the wells of the corresponding 96-well plate according to the sample number.
(3) Samples were denatured at 95 ℃ for 5 minutes and then rapidly cooled on ice for 4 minutes.
(4) The samples were placed in the sample trays of a gene analyzer and subjected to capillary electrophoresis according to the conventional parameters (see the instructions of the manufacturer of the genetic analyzer).
(5) The capillary electrophoresis was terminated and the experimental data were analyzed by GeneMapper software to obtain a typing map, as shown in fig. 1, 2, 3 and 4.
As shown IN FIG. 1, the typing map of RhD single copy deletion carrier (typing ccDdEe: RhD-) genomic DNA, FAM-tagged 12 gene loci (Box, IN07, pEx01, IN02, Ref19, IN08, SID1, SID2, Ex03, IN01, IN03, SID3 from left to right), TAMRA-tagged 11 gene loci (AMEL, 1227Ga, wD15_ aG, wD2_ Gc, wD3_ Cg, wD4_ Cg, wD1_ gT, Dc, Ex10, Ee, SID4 from left to right), and the average patterns are clear.
And (3) analysis:
(1) quality control of the PCR process: the corresponding amplification product peaks appear in SID1, SID2, SID3, SID4, Ref19 and C1401L, and the AMEL locus presents XY. The PCR process was normal.
(2) Box is approximately 1:2 peak type, which indicates that the RhD gene is completely deleted IN single copy, pEx01, IN01, Ex03, IN03, IN07 and IN08 are consistent with Box sites, which indicates that the RhD gene is deleted IN single copy. IN02 was an approximate 2:1 peak (cc form IN RhCE), supporting RhD single copy deletion.
(3) TAMRA channel: 1227Ga, wD15_ aG, wD2_ Gc, wD3_ Cg, wD4_ Cg and wD1_ gT are all wild homozygous, Ex09 and Ex10 amplification peaks exist, and Dc RhD specific peaks and c specific peaks exist. The Ee site is heterozygous. The C site has no peak of amplification product.
The typing profile of the carrier of the RhD-RhCE fusion gene (typed as CcDdEe: RhD-CE (2-9) D2) is shown in FIG. 2. 12 gene loci marked by FAM and 11 gene loci marked by TAMRA are all clearly typed.
And (3) analysis:
(1) quality control of the PCR process: the corresponding amplification product peaks appear in SID1, SID2, SID3, SID4, Ref19 and C1401L, and the AMEL locus presents XY. The PCR process was normal.
(2) Box is approximate to 1:1 peak type, which indicates that RhD gene is completely deleted; pEx01 is approximately 1:1 peak type; IN01 was approximately 1:1 peak; IN02 was an approximate 3:1 peak (Cc-type IN RhCE), indicating a single copy deletion of RhD intron 2; ex03, IN03, IN07 and IN08 all have approximate 2:1 peak patterns, indicating that the corresponding region has single copy deletion.
(3) TAMRA channel: 1227Ga, wD15_ aG, wD2_ Gc, wD3_ Cg, wD4_ Cg and wD1_ gT are all wild homozygous, Ex09 and Ex10 amplification peaks exist, and Dc RhD specific peaks and c specific peaks exist. The Ee site is heterozygous. The peak of the C site amplification product exists.
The typing map of the RhD gene total deletion (typing ccddEe: RhD. about. D-/RhD. about. D-) Rh negative blood type is shown in FIG. 3. 12 gene loci marked by FAM and 11 gene loci marked by TAMRA are all clearly typed.
And (3) analysis:
(1) quality control of the PCR process: the corresponding amplification product peaks appear in SID1, SID2, SID3, SID4, Ref19 and C1401L, and the AMEL locus presents XY. The PCR process was normal.
(2) The Box locus is IN a downstream Box monomodal type, which indicates that the RhD gene is completely deleted and homozygous, and pEx01, IN01, IN02, Ex03, IN03, IN07 and IN08 are IN RhCE specific fragment monomodal types, which are consistent with the Box locus and support the RhD gene to be completely deleted and homozygous.
(3) TAMRA channel: 1227Ga, wD15_ aG, wD2_ Gc, wD3_ Cg, wD4_ Cg, wD1_ gT and Ex10 have no amplification product peak at each site, and a Dc site RhD specific peak disappears, so that the Box site RhD complete deletion homozygote is supported. The c-specific peaks for the Dc site RhCE were all present. The Ee site is heterozygous. The C site has no peak of amplification product.
The carrier typing map of the wD15 mutation (typed as CcDdEE: RhD 845A) is shown in FIG. 4. 12 gene loci marked by FAM and 11 gene loci marked by TAMRA are all clearly typed.
And (3) analysis:
(1) quality control of the PCR process: the corresponding amplification product peaks appear in SID1, SID2, SID3, SID4, Ref19 and C1401L, and the AMEL site is XX. The PCR process was normal.
(2) Box is approximate to 1:1 peak type, which indicates that the RhD gene has no complete deletion, pEx01, IN01, Ex03, IN03, IN07 and IN08 are approximate to 1:1 peaks and are consistent with Box sites. IN02 was an approximate 3:1 peak (RhCE was type Cc), consistent with the Box site.
(3) TAMRA channel: wD15_ aG sites are heterozygote, each site of 1227Ga, wD2_ Gc, wD3_ Cg, wD4_ Cg and wD1_ gT is wild homozygote, Ex09 and Ex10 amplification peaks exist, and a Dc site RhD specificity peak and a c specificity peak both exist. E site short fragment E homozygous. The peak of the C site amplification product exists.
The gene typing detection kit for 23 gene loci of human Rh blood type can be stably implemented in a laboratory with a PCR instrument and genetic analysis, and the detection time is about 3-4 hours. Meanwhile, the reagent provided by the invention can be conveniently produced in biotechnology companies and used for detection in biomedical detection institutions, and has the conditions of industrialization, popularization and application.
The invention has been described with reference to specific embodiments thereof. Various modifications or alterations of this invention will become apparent to those skilled in the art in light of the foregoing description, including (but not limited to): changing different groups of fluorescein labels, changing the primer of the fluorescein label (such as changing the labeled upstream primer into the labeled downstream primer), changing the grouping arrangement of the genome according to the allele range of each gene locus, optimizing the PCR amplification condition and the primer reaction concentration according to other PCR reaction mother liquor, changing the recommended reaction system and the like. It will be apparent to those skilled in the art that the above modifications and variations are possible without departing from the spirit and scope of the invention.
Sequence listing
<110> Shanghai wusezite medical research Co., Ltd
<120> genotyping detection kit for 23 gene loci of human Rh blood type
<130> Z001039180031CN
<160> 53
<170> PatentIn version 3.5
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<400> 4
agaagaggtt ctgggtgttg gt 22
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
acatttctgc attcctctag tgaca 25
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
gccactggcc actgagtgta 20
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
ttacaagggc attggcactt aatag 25
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
gaggtttagc agggtcttag aagg 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
ataagtgggt gttcctagtg atgg 24
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
attcgtattt ccctttcgtg gt 22
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
ctgtcggcat gtggctggta 20
<210> 12
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
gaaacttgtt cttgcggtga tt 22
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
caacatagtc aaatgctgtg gaga 24
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
attgttctac cgaaccctgg tc 22
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
tggctcactc acagagctga ca 22
<210> 16
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
gcacttccat gcatctgtat tagc 24
<210> 17
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
attaggtgcc caacagtgtt tg 22
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
aaccccacca aatggagctt 20
<210> 19
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
gtttagccac cctggattta gtt 23
<210> 20
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
tgtgccctca cttggatgac c 21
<210> 21
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
ggctgctggt gccatttac 19
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
gtctcctttg ctggctctta ca 22
<210> 23
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
caggtaattg gtatgtgaaa aagtgt 26
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
gaatggaggg aaagtgggaa g 21
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
ccctgggctc tgtaaagaat agtg 24
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
atcagagctt aaactgggaa gctg 24
<210> 27
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
atttgatgac caagttttct gcaag 25
<210> 28
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
attattttga tgaccaagtt ttctggtaa 29
<210> 29
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
actcataaac agcaagtcaa catatattct 30
<210> 30
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
ggagaaaaag gatttctgtt gacat 25
<210> 31
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
attccatatt ttaagattta ggagcagac 29
<210> 32
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
attctttcca tattttaaga tttaggagca tag 33
<210> 33
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
attcaggagg cgtggctgta gg 22
<210> 34
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
caggaggcgt ggctgttga 19
<210> 35
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
gttgtctagt ttcttaccgg cacgt 25
<210> 36
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
attgcagtag tgagctggca catca 25
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
attgccaaca ccgcactgta ca 22
<210> 38
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
gccaacaccg cactgttcc 19
<210> 39
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
tgataactta gcaaatggct attgga 26
<210> 40
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
attcgagacc aacctgacgc ac 22
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
gagacggaca caggatgagg tc 22
<210> 42
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
attgagacgg acacaggatg acctg 25
<210> 43
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
tgctatttgc tcctgtgacc agtt 24
<210> 44
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
aatccaagga ctatcagggc gtg 23
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
gacaaactgg gtatcgttgg tg 22
<210> 46
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
attcagacaa actgggtatc gttgttc 27
<210> 47
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
attgtaatga gacatttagg ctgtttcaa 29
<210> 48
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
aatggtgaga ttctcctcaa agagt 25
<210> 49
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
agcatttgac catcacggag c 21
<210> 50
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
attgattgga cttctcagca cagc 24
<210> 51
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
gattggactt ctcagcagcg g 21
<210> 52
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
gcttagagct tcctttgcca tct 23
<210> 53
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 53
tgtcgttgag gctacccagt c 21
Claims (4)
1. A composite PCR amplification system for simultaneously genotyping 23 loci of human 5, 19, 21 and X/Y chromosomes, wherein the 23 loci are divided into two groups, the first group is 12 loci: box, IN07, pEx01, IN02, IN08, Ref19, SID1, SID2, Ex03, IN01, IN03, SID 3; the second set is 11 loci: AMEL, 1227Ga, wD2_ Gc, wD15_ aG, wD1_ gT, Dc, wD3_ Cg, wD4_ Cg, Ex10, Ee, SID 4; the composite PCR amplification system comprises a first group and a second group of PCR amplification primers, wherein the first group comprises the PCR amplification primers corresponding to the following groups of sites:
the corresponding primer sequences of the site Box are shown as SEQ ID No.1 and SEQ ID No. 2;
the sequences of corresponding primers of the IN07 site are shown as SEQ ID No.3 and SEQ ID No. 4;
at the position pEx01, the sequences of the corresponding primers are shown as SEQ ID No.5 and SEQ ID No. 6;
the sequences of corresponding primers of the IN02 site are shown as SEQ ID No.7 and SEQ ID No. 8;
the sequences of corresponding primers of the IN08 site are shown as SEQ ID No.9 and SEQ ID No. 10;
the primer sequence is shown as SEQ ID No.11 and SEQ ID No.12 at the position Ref 19;
the site SID1, the corresponding primer sequence is shown as SEQ ID No.13, SEQ ID No. 14;
the site SID2, the corresponding primer sequence is shown as SEQ ID No.15, SEQ ID No. 16;
the corresponding primer sequences of the Ex03 site are shown as SEQ ID No.17 and SEQ ID No. 18;
the sequences of corresponding primers of the IN01 site are shown as SEQ ID No.19 and SEQ ID No. 20;
the sequences of corresponding primers of the IN03 site are shown as SEQ ID No.21 and SEQ ID No. 22;
the site SID3, the corresponding primer sequence is shown as SEQ ID No.23 and SEQ ID No. 24;
wherein, the upstream primers corresponding to the gene loci Box, pEx01, IN02, IN08, Ref19, SID1, SID2 and SID3 are all marked by FAM fluorescein; downstream primers corresponding to gene sites IN07, Ex03, IN01 and IN03 are all marked by FAM fluorescein;
the second group includes PCR amplification primers corresponding to the following group of sites:
the corresponding primer sequences of the site AMEL are shown as SEQ ID No.25 and SEQ ID No. 26;
a site 1227Ga, the corresponding primer sequences are shown as SEQ ID No.27, SEQ ID No.28 and SEQ ID No. 29;
the corresponding primer sequences of the site wD2_ Gc are shown as SEQ ID No.30, SEQ ID No.31 and SEQ ID No. 32;
position wD15_ aG, the corresponding primer sequence is shown as SEQ ID No.33, SEQ ID No.34 and ID No. 35;
position wD1_ gT, the corresponding primer sequence is shown as SEQ ID No.36, SEQ ID No.37, SEQ ID No. 38;
the sequences of the corresponding primers of the site Dc are shown as SEQ ID No.39 and SEQ ID No. 40;
position wD3_ Cg, the corresponding primer sequence is shown as SEQ ID No.41, SEQ ID No.42, SEQ ID No. 43;
position wD4_ Cg, the corresponding primer sequence is shown as SEQ ID No.44, SEQ ID No.45, SEQ ID No. 46;
the corresponding primer sequences of the Ex10 site are shown as SEQ ID No.47 and SEQ ID No. 48;
the corresponding primer sequences of the site Ee are shown as SEQ ID No.49, SEQ ID No.50 and SEQ ID No. 51;
the site SID4, the corresponding primer sequence is shown as SEQ ID No.52 and SEQ ID No. 53;
wherein downstream primers corresponding to gene loci AMEL, 1227Ga, wD2_ Gc, wD1_ gT, wD3_ Cg, Ex10 and SID4 are marked by TAMRA; the upstream primers corresponding to gene positions wD15_ aG, Dc, wD4_ Cg, Ee were labeled with TAMRA.
2. The multiplex PCR amplification system of claim 1, wherein the working concentration of the PCR amplification primers corresponding to the first and second sets of loci is 80-900 nmol/L.
3. The multiplex PCR amplification system of claim 1, wherein the primers consist of a dry powder or solution of PCR amplification primers of the oligonucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 53.
4. A kit for performing Rh blood group genotyping detection on 23 gene loci on human chromosomes 5, 19, 21 and X/Y, the kit comprising a container containing a primer composition of the PCR amplification system of claim 1 and a container of PCR reaction mother liquor, wherein,
the primer composition container contains a storage solution of primers with sequences from SEQ ID No.1 to SEQ ID No.53, and the concentration of each primer in the storage solution is 4 times of that of the corresponding primer working solution;
the PCR reaction mother liquor container contains 2 times of reaction concentration of PCR reaction mother liquor, and the PCR reaction mother liquor contains DNA polymerase with enzyme activity of 0.5-5U, magnesium ions with concentration of 1-5 mmol/L, dNTP with concentration of 40-400 mu mol/L and Tris-HCl buffer solution with concentration of 20-100 mmol/L.
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| Title |
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| Genotyping approach for non-invasive foetal RHD detection in an admixed population;Carolina Trucco Boggione等;《Blood Transfus》;20170131;第15卷(第1期);66-73 * |
| Non-invasive fetal RHD genotyping for RhD negative women stratified into RHD gene deletion or variant groups: comparative accuracy using two blood collection tube types;Catherine A Hyland等;《Pathology》;20171231;第49卷(第7期);757-764 * |
| 新乡地区48例ＲhD阴性汉人ＲHD基因多态性研究;孙学兰等;《现代预防医学》;20161231;第43卷(第10期);1855-1858 * |
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