CN116445596A - Product and method for human genotyping and application thereof - Google Patents
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Abstract
Provided are SNP molecular markers for human genotyping, and primer sets, probe sets, kits and chips for detecting the same. The application also provides a method for genotyping in a human subject using the above SNP molecular markers and related uses of the above SNP molecular markers.
Description
Technical Field
The present application is in the field of molecular biology, in particular to products, methods for differentiating donor and recipient cells and uses thereof.
Background
Cell therapy has become one of the most important emerging medical therapies in the world. Treatment with stem cells, induced pluripotent stem cells, somatic cells and immune cells has been carried out in a number of clinical trials. To date, most studies are phase 1 and 2 trials assessing safety and feasibility, and evidence of therapeutic efficacy remains lacking, and stem cell-based therapeutic products remain unapproved by the U.S. food and drug administration. The lack of consistent and standardized methods to characterize the safety and efficacy of stem cell products is a major problem, and an important obstacle to achieving good efficacy is the lack of reliable in vivo Pharmacokinetic (PK) data for the cells, which greatly slows the progression of stem cell therapy to clinical use. In view of the great potential of stem cells in the treatment of various diseases, it is necessary to determine sensitive and effective detection means to quantify cells in vivo, and increasing knowledge of cell distribution can help evaluate PK, thereby determining the dosing regimen required to achieve therapeutic effects to facilitate our understanding of the safety and effectiveness of cell therapy for stem cells.
The biodistribution or PK of stem cells is affected by many different factors and an ideal quantification technique should have the following characteristics: high sensitivity and specificity; the quantitative stem cell detection method is suitable for most detection and long-term monitoring, is more common in treatment of Hematopoietic Stem Cell Transplantation (HSCT), and the lymphatic and hematopoietic systems of a receiver and a donor coexist in early stages after allogeneic hematopoietic stem cell transplantation. In most successful stem cell transplants, this period is transient, known as mixed chimeras, and genotyping or Fluorescence In Situ Hybridization (FISH) based on Short Tandem Repeat (STR)/Variable Number of Tandem Repeat (VNTR) -Polymerase Chain Reaction (PCR) has been considered a routine means of chimera analysis following allogeneic hematopoietic stem cell transplantation for the last decade. A significant advantage of the STR/VNTR-PCR STR-PCR method is that the use of several STR loci can in almost all cases (> 99%) distinguish between donor and recipient, which is necessary for any chimeric analysis. However, this method has a sensitivity of only 1-5%, depending on the gene type of each donor and recipient, FISH requires a longer time, requires more samples, and is relatively costly. Real-time polymerase chain reaction analysis of Single Nucleotide Polymorphisms (SNPs) -is more sensitive than STR-PCR methods. Since most SNPs have bi-alleles with alleles that differ from each other only on a single nucleotide, they are easier to genotype than STR markers, but not all SNP chimera analysis methods achieve the high sensitivity of real-time SNP-PCR analysis methods. Insertion/deletion polymorphism (Indel) analysis using real-time Quantitative Polymerase Chain Reaction (QPCR) is an alternative technique. It has been used for chromosome analysis after allogeneic hematopoietic stem cell transplantation and shows high sensitivity, but its inherent accuracy is of interest in hybrid chimeras. Overall, these techniques have good accuracy but limited sensitivity at low stem cell levels (3-5%). Failure to detect low levels of cells or DNA may limit interpretation of PK data.
Disclosure of Invention
In order to solve the technical problems, the application provides the following technical scheme:
in a first aspect, the present application provides SNP molecular markers for human genotyping comprising one or more of the following SNPs: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
In a second aspect, the present application provides a primer set for human genotyping, wherein the primer set comprises primers for amplifying one or more of the SNP molecular markers of claim 1.
In a third aspect, the present application provides a probe set for genotyping a human, wherein the probe set comprises probes for detecting one or more of the SNP molecular markers described in the first aspect.
In a fourth aspect, the present application provides a kit for genotyping a human, wherein the kit is for detecting the SNP molecular markers described in the first aspect.
In a fifth aspect, the present application provides a human genotyping chip, wherein the chip is used to detect the SNP molecular markers described in the first aspect.
In a sixth aspect, the present application provides a method for genotyping in a human subject comprising quantifying one or more of the SNP molecular markers selected from the group consisting of: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
In a seventh aspect, the present application provides the use of SNP molecular markers comprising one or more of the following for human genotyping: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
The application establishes a method for accurately, reliably and sensitively genotyping. The method can well meet the requirements on sensitivity, specificity, operation convenience and universality. Advantages of the present application may include at least one or more of the following:
1. the InDel/SNP combined ddPCR detection technology is adopted, so that the sensitivity is higher on the basis of ensuring the specificity;
2. 22 sets of universal InDel/SNP locus detection programs are designed, so that the screening of any donor/acceptor differential genes above 90% can be ensured, and the composition power of clinical trials is improved; and
3. the detection method can be rapidly implemented in a common laboratory, is not dependent on sequencing, is efficient and convenient, and has strong feasibility.
Drawings
FIGS. 1 to 9 show the specificity and sensitivity of the results of in vitro simulated allogeneic autologous mixed samples detected by the SNP-ddPCR quantification system of the present application.
Detailed Description
The definition of terms used in this application is as follows:
the term "allele" as used herein refers to a gene that controls different morphologies of the same trait at the same position on a pair of homologous chromosomes.
The terms "single nucleotide polymorphism", "SNP molecular marker", "SNP site" as used herein mainly refer to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. The change may occur within the coding region, non-coding region (e.g., the promoter region or vicinity thereof, or an intron) of the gene, in the intergenic region.
The term "SNP chip" or "chip" as used herein refers to a biological microchip capable of analyzing the presence of SNPs contained in a sample DNA by arranging and attaching several hundred to several hundred thousand biomolecules such as DNA, DNA fragments, cDNA, oligonucleotides, RNA or RNA fragments having a known sequence, which are immobilized at intervals on a small solid substrate formed of glass, silicon or nylon. Hybridization occurs between the nucleic acid contained in the sample and the immobilized surface according to the degree of complementarity. By detecting and judging hybridization, information about genetic material contained in a sample can be obtained at the same time.
As used herein, the term "molecular marker (Molecular Markers)" or "polymorphic site" refers to a genetic marker based on nucleotide sequence variation within genetic material between individuals, reflecting DNA level genetic polymorphism. The advantages of DNA molecular markers over several other genetic markers (morphological markers, biochemical markers, cytological markers) include, but are not limited to: the method is applicable to DNA samples of different tissues in different stages of biological development, and any tissue of an organism can be used for analysis in any development period; the change of the environment can change the transcription and expression of the gene without affecting the genome DNA sequence of the gene; the number of molecular markers is greatly expanded in space due to high variation; most of the genes are co-dominant, so that the homozygous and heterozygous genotypes can be accurately identified, and recessive character screening is facilitated; the detection means is simple and rapid and can realize automation. With the development of molecular biology technology, the DNA molecular marker technology has tens of kinds, and is widely applied to genetic breeding, genome mapping, gene positioning, species genetic relationship identification and other aspects. In the context of the present application, molecular markers are generally referred to as SNP molecular markers.
Digital PCR (ddPCR) is an absolute quantitative technique of nucleic acid that has been developed rapidly for several years following the first and second generation PCR techniques. As a brand new nucleic acid detection method, digital PCR solves the problem that qPCR needs to rely on standard curves for quantitative analysis. The number of target DNA molecules can be directly counted by an end-point detection method, so that absolute quantification of single-molecule template amplification is realized, and the detection sensitivity is greatly improved. It can be said that digital PCR is the current means for detecting nucleic acid with the highest detection sensitivity.
As a quantification method formed by combining the emerging absolute quantification technology of nucleic acid molecules and single nucleotide polymorphism (single nucleotide polymorphism, SNP), the digital PCR can obtain more sensitive and accurate quantitative analysis results, and the sensitivity can reach 0.01 percent. In order to improve sensitivity and specificity of allogeneic detection and stability of quantitative analysis, the application adopts a composite SNP typing system to genotype a donor and applies a ddPCR technology with high sensitivity to detect the continuous state of stem cells in a blood sample, explores feasibility and accuracy of the method in the clinical evaluation process of stem cell treatment products, and provides a new detection technology for the pharmacokinetic analysis of stem cell treatment drugs.
Specifically, the application provides the following technical scheme:
in a first aspect, the present application provides SNP molecular markers for human genotyping comprising one or more of the following SNPs: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
In a second aspect, the present application provides a primer set for human genotyping, wherein the primer set comprises primers for amplifying one or more of the SNP molecular markers of claim 1.
In some specific embodiments, the primer set is selected from one or more of the following:
primers shown in SEQ ID NOS.1 and 2 for amplifying rs 2634074;
primers shown in SEQ ID NOS.5 and 6 for amplifying rs 4377290;
primers shown in SEQ ID NOS.9 and 10 for amplifying rs 7193343;
primers shown in SEQ ID NOS.13 and 14 for amplifying rs 3071379;
primers shown in SEQ ID NOS.17 and 18 for amplifying rs 891700;
primers shown in SEQ ID NOS.21 and 22 for amplifying rs 1109037;
primers shown in SEQ ID NOS.25 and 26 for amplifying rs 1666130;
primers shown in SEQ ID NOS.29 and 30 for amplifying rs 9277534;
primers shown in SEQ ID NOS.33 and 34 for amplifying rs 7793828;
primers shown in SEQ ID NOS.37 and 38 for amplifying rs 449851;
primers shown in SEQ ID NOS.41 and 42 for amplifying rs 1775737;
primers shown in SEQ ID NOS.45 and 46 for amplifying rs 1498553;
primers shown in SEQ ID NOS.49 and 50 for amplifying rs 8053194;
primers shown in SEQ ID NOS.53 and 54 for amplifying rs 5911;
primers shown in SEQ ID NOS.57 and 58 for amplifying rs 987640;
primers shown in SEQ ID NOS.61 and 62 for amplifying rs 9781645;
primers shown in SEQ ID NOS.65 and 66 for amplifying rs 25487;
primers shown in SEQ ID NOS.69 and 70 for amplifying rs 203462;
primers shown in SEQ ID NOS.73 and 74 for amplifying rs 312470;
primers shown in SEQ ID NOS.77 and 78 for amplifying rs 715463;
primers shown in SEQ ID NOS.81 and 82 for amplifying rs 1946519; and
primers shown in SEQ ID NOS.85 and 86 for amplifying rs2488457.
In a third aspect, the present application provides a probe set for genotyping a human, wherein the probe set comprises probes for detecting one or more of the SNP molecular markers described in the first aspect.
In some specific embodiments, the set of probes is selected from one or more of the following:
probes shown in SEQ ID NOS.3 and 4 for detecting rs 2634074;
probes shown in SEQ ID NOS.7 and 8 for detecting rs 4377290;
probes shown in SEQ ID NOS.11 and 12 for detecting rs 7193343;
probes shown in SEQ ID NOS.15 and 16 for detecting rs 3071379;
probes shown in SEQ ID NOS.19 and 20 for detecting rs 891700;
probes shown in SEQ ID NOS.23 and 24 for detecting rs 1109037;
probes shown in SEQ ID NOS.27 and 28 for detecting rs 1666130;
probes shown in SEQ ID NOS.31 and 32 for detecting rs 9277534;
probes shown in SEQ ID NOS.35 and 36 for detecting rs 7793828;
probes shown in SEQ ID NOS.39 and 40 for detecting rs 449851;
probes shown in SEQ ID NOS.43 and 44 for detecting rs 1775737;
probes shown in SEQ ID NOS.47 and 48 for detecting rs 1498553;
probes shown in SEQ ID NOS.51 and 52 for detecting rs 8053194;
probes shown in SEQ ID NOS.55 and 56 for detecting rs 5911;
probes shown in SEQ ID NOS.59 and 60 for detecting rs 987640;
probes shown in SEQ ID NOS.63 and 64 for detecting rs 9781645;
probes shown in SEQ ID NOS.67 and 68 for detecting rs 25487;
probes shown in SEQ ID NOS.71 and 72 for detecting rs 203462;
probes shown in SEQ ID NOS.75 and 76 for detecting rs 312470;
probes shown in SEQ ID NOS.79 and 80 for detecting rs 715463;
probes shown in SEQ ID NOS.83 and 84 for detecting rs 1946519; and
probes shown in SEQ ID NOS.87 and 88 for detecting rs2488457.
In a fourth aspect, the present application provides a kit for genotyping a human, wherein the kit is for detecting the SNP molecular markers described in the first aspect.
In some specific embodiments, the kit comprises reagents for detecting the SNP molecular markers described in the first aspect.
In some specific embodiments, the kit comprises a primer set according to the second aspect.
In some specific embodiments, the kit further comprises a set of probes according to the third aspect.
In a fifth aspect, the present application provides a human genotyping chip, wherein the chip is used to detect the SNP molecular markers described in the first aspect.
In some specific embodiments, the chip comprises reagents for detecting the SNP molecular markers described in the first aspect.
In some specific embodiments, the chip comprises a set of probes according to the third aspect.
In a sixth aspect, the present application provides a method for genotyping in a human subject comprising quantifying one or more of the SNP molecular markers selected from the group consisting of: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
In a preferred embodiment, the human subject has undergone stem cell transplantation.
In some specific embodiments, the ddPCR utilizes one or more of the primer sets selected from the group consisting of:
primers shown in SEQ ID NOS.1 and 2 for amplifying rs 2634074;
primers shown in SEQ ID NOS.5 and 6 for amplifying rs 4377290;
primers shown in SEQ ID NOS.9 and 10 for amplifying rs 7193343;
primers shown in SEQ ID NOS.13 and 14 for amplifying rs 3071379;
primers shown in SEQ ID NOS.17 and 18 for amplifying rs 891700;
primers shown in SEQ ID NOS.21 and 22 for amplifying rs 1109037;
primers shown in SEQ ID NOS.25 and 26 for amplifying rs 1666130;
primers shown in SEQ ID NOS.29 and 30 for amplifying rs 9277534;
primers shown in SEQ ID NOS.33 and 34 for amplifying rs 7793828;
primers shown in SEQ ID NOS.37 and 38 for amplifying rs 449851;
primers shown in SEQ ID NOS.41 and 42 for amplifying rs 1775737;
primers shown in SEQ ID NOS.45 and 46 for amplifying rs 1498553;
primers shown in SEQ ID NOS.49 and 50 for amplifying rs 8053194;
primers shown in SEQ ID NOS.53 and 54 for amplifying rs 5911;
primers shown in SEQ ID NOS.57 and 58 for amplifying rs 987640;
primers shown in SEQ ID NOS.61 and 62 for amplifying rs 9781645;
primers shown in SEQ ID NOS.65 and 66 for amplifying rs 25487;
primers shown in SEQ ID NOS.69 and 70 for amplifying rs 203462;
primers shown in SEQ ID NOS.73 and 74 for amplifying rs 312470;
primers shown in SEQ ID NOS.77 and 78 for amplifying rs 715463;
primers shown in SEQ ID NOS.81 and 82 for amplifying rs 1946519; and
primers shown in SEQ ID NOS.85 and 86 for amplifying rs2488457.
In some specific embodiments, the ddPCR utilizes one or more of the probe sets selected from the group consisting of:
probes shown in SEQ ID NOS.3 and 4 for detecting rs 2634074;
probes shown in SEQ ID NOS.7 and 8 for detecting rs 4377290;
probes shown in SEQ ID NOS.11 and 12 for detecting rs 7193343;
probes shown in SEQ ID NOS.15 and 16 for detecting rs 3071379;
probes shown in SEQ ID NOS.19 and 20 for detecting rs 891700;
probes shown in SEQ ID NOS.23 and 24 for detecting rs 1109037;
probes shown in SEQ ID NOS.27 and 28 for detecting rs 1666130;
probes shown in SEQ ID NOS.31 and 32 for detecting rs 9277534;
probes shown in SEQ ID NOS.35 and 36 for detecting rs 7793828;
probes shown in SEQ ID NOS.39 and 40 for detecting rs 449851;
probes shown in SEQ ID NOS.43 and 44 for detecting rs 1775737;
probes shown in SEQ ID NOS.47 and 48 for detecting rs 1498553;
probes shown in SEQ ID NOS.51 and 52 for detecting rs 8053194;
probes shown in SEQ ID NOS.55 and 56 for detecting rs 5911;
probes shown in SEQ ID NOS.59 and 60 for detecting rs 987640;
probes shown in SEQ ID NOS.63 and 64 for detecting rs 9781645;
probes shown in SEQ ID NOS.67 and 68 for detecting rs 25487;
probes shown in SEQ ID NOS.71 and 72 for detecting rs 203462;
probes shown in SEQ ID NOS.75 and 76 for detecting rs 312470;
probes shown in SEQ ID NOS.79 and 80 for detecting rs 715463;
probes shown in SEQ ID NOS.83 and 84 for detecting rs 1946519; and
probes shown in SEQ ID NOS.87 and 88 for detecting rs2488457.
In a seventh aspect, the present application provides the use of SNP molecular markers comprising one or more of the following for human genotyping: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
In some specific embodiments, the human genotyping is used to distinguish recipient stem cells from donor stem cells in a human subject undergoing stem cell transplantation.
The technical scheme provided by the application establishes a strict test scheme, and achieves good effects meeting expectations in application practice through comparison, screening and testing.
The following examples are illustrative of the present application, but are not intended to limit the scope of the present application.
Examples
Example 1: construction of InDel/SNP-ddPCR universal composite typing screening system
The design of this example was performed according to the following scheme:
firstly, according to SNP distribution characteristics of people in China, proper InDel/SNP loci are screened out from db SNP and 1000genome Browser databases, and further screening is carried out by a TaqMan probe method, so that an InDel/SNP locus composite typing system suitable for genotype identification before and after reinfusion of more than 90% allogeneic stem cells is constructed; wherein the SNP screening principle is as follows:
a. the distribution frequency of InDel/SNP loci in Chinese population is in the range of 0.4-0.6, and the InDel/SNP loci have wide heterogeneity, so that good distinction between donor and acceptor is facilitated;
b. InDel/SNP sites are located on different chromosomes;
c. InDel/SNP sites all belong to the double allele mutations.
Secondly, designing specific primers and probes for applying ddPCR detection of all screening sites after confirming the specific sites, and optimizing a ddPCR detection system and a ddPCR detection program;
thirdly, carrying out InDel/SNP locus screening on a sample before treatment of the donor stem cells and the receptor stem cells by a qPCR technology after extracting nucleic acid; and
fourthly, selecting the specific genes representing the donor stem cells after screening to carry out ddPCR specific amplification detection, obtaining specific gene copy numbers, correcting and calculating back through the total DNA concentration of the genome to obtain the genome content of the donor cells in the blood sample corresponding to the sample time point, and drawing PK characteristics by taking time-copy numbers/uggDNA as coordinates.
According to the screening principle, 22 InDel/SNP loci are screened, taqMan probes and primers are independently designed through Primer Express software according to the 22 InDel/SNP loci, and the primers and probes are entrusted to be synthesized by Shanghai biological engineering Co., ltd. The sequences of these primers and probes and ddPCR annealing temperatures are shown in Table 1 below.
Table 1: 22 InDel/SNP loci screened and primer probe information thereof
Example 2: use of InDel/SNP-ddPCR universal composite typing screening system
The specific operation flow of this embodiment is described as follows:
1. sample DNA extraction
The Sample is subjected to nucleic acid extraction by using a MagMAX ™ DNA M mu Lti-Sample mu M Ltra 2.0 kit and a KingFisher Apex full-automatic nucleic acid extractor, and the Sample and a buffer solution are balanced to room temperature before use, and the operation steps are as follows:
table 2: magMAX ™ DNA mu Lti-Sample mu M Ltra 2.0 kit operation flow
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2. Screening of donor/acceptor specific genes
Table 3: preparation system for qPCR detection of single SNP locus
The extracted recipient blood gDNA and donor stem cell DNA samples were diluted to 5 ng/. Mu.L, and 3. Mu.L each was taken for reaction. And (3) subpackaging 17 [ mu ] L of the Mix vortex after uniform mixing to 8 continuous tube bottoms (Thermofisher), adding 3 [ mu ] L of samples and reference substances (volunteer DNA samples confirmed by qPCR typing or standard substance plasmids synthesized by the entrusted Bai Lig biotechnology Co., ltd.) into each of the sample holes and the reference substance holes, namely, covering the 8 continuous tube bottoms, centrifuging transiently, and centrifuging the reaction liquid to the tube bottoms. Placing 8 rows of tubes into an instrument (ABI, T100) TM Thermal Cycler), information of the corresponding well is set to the instrument program, and a reaction program is set as follows.
Table 4: reaction program settings
After the machine is started, the genotype of the single SNP locus is judged according to the detection result.
3. ddPCR detection of the content of the Donor Stem cell specific Gene
Table 5: preparation system of ddPCR detection sample
The system except the template DNA can be prepared into Mix according to the usage amount, 17.04 mu L of the system except the template DNA is subpackaged to the bottom of a pipe after vortex mixing, and 6.96 mu L of the template DNA is added into a sample hole. Fully vibrating, uniformly mixing and centrifuging for a short time to ensure that all liquid is at the bottom of the pipe; transfer samples into ddPCR dedicated 96-well plates (Bio-Rad); placing the 96-well plate into a droplet generator (Bio-Rad, automated Droplet Genderator) to generate droplets; sealing film: 180℃for 6sec.
And (3) PCR amplification: samples were taken into a PCR instrument (ABI, T100) according to the PCR procedure in the following table TM Thermal Cycler).
Table 6: PCR program set-up
The amplified samples were taken to a ddPCR reader (Bio-rad, QX200 TM Droplet Reader), and data processing is performed by QuantaSoft software.
Detection result
The InDel/SNP complex system was tested in DNA extracted from whole blood of 10 volunteer individuals (extracted according to the DNA extraction method described above), the genotype of each site of each individual was tested by the qPCR described above, 45 combinations were combined for every two of 10 individuals (formula n×1)/2), genotypes of homozygous differences of every two individuals of 45 combinations were compared, effective individual identification was calculated for homozygous differences of at least 1 site, and individual identification rate=number of effective individual identification combinations/total number of combinations was 100%. The results were: the 45 pairwise combination pairs have the homozygous difference that the 43 pairs of combinations at least contain 1 SNP locus, so that the effective recognition is realized; there were 2 pairs of SNP sites combined without homozygous differences, which were not recognized. The individual recognition rate is as follows: 43/45 x 100 = 95.6%. Referring to tables 7 and 8, it can be seen that the feasibility of using the InDel/SNP composite system to effectively and specifically detect the specific gene of the donor cell in the recipient is more than 95%.
Table 7: typing of the InDel/SNP described above was detected by qPCR in 10 volunteer individuals
Table 8: genotyping of homozygous differences between each two individuals of 45 combinations
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The sensitivity of the method was evaluated by randomly selecting 2 samples of DNA extracted from whole blood samples of volunteers and mixing them in different mass ratios to simulate the preparation of autologous mixed samples (i.e., DNA extracted from blood samples of subjects after they had been infused with allogeneic stem cells). Specifically, the allogeneic sample and the autologous sample are diluted to the same concentration and mixed according to different proportions, so that the allogeneic sample accounts for the proportion of (ng/ng) in the mixed sample: 0%, 0.1%, 0.5%, 5%, 50%, 95%, 99.5%, 99.9%. These in vitro simulated allogeneic autologous mixed samples were tested by the SNP-ddPCR quantification system described above. The specificity and sensitivity of the results are shown in Table 9 and FIGS. 1-9.
These results show that the calculated ratio (average value D/(d+r)) of the acceptor-specific gene copy number (original data report acceptor cp) and the donor-specific gene copy number (original data report donor cp) detected by the method described in this example for samples mixed according to different mass ratios (normal MT%) of acceptor and donor DNA is smaller than ±20% of the deviation range conventionally required by bioanalytical methods, and that the 22 InDel/SNP sites obtained by screening by the present inventors can be used for precise typing of genes in humans, such as asians, especially chinese, and in the context of cell transplantation, the content of donor DNA can be accurately quantified even in the case that donor DNA only accounts for 0.1 wt% of acceptor, providing reliable support for pharmacokinetic data of cell transplantation in vivo.
Table 9: detection result of in vitro allogeneic autologous mixed sample
The present application has been described in detail with reference to general descriptions and specific embodiments thereof, and such modifications and improvements can be made without departing from the spirit of the application, and are intended to be within the scope of the present application.
Claims (10)
1. A SNP molecular marker for use in human genotyping, the molecular marker comprising one or more of the following SNPs: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
2. A primer set for human genotyping, wherein the primer set comprises primers for amplifying one or more of the SNP molecular markers of claim 1, optionally wherein the primer set is selected from one or more of the following:
primers shown in SEQ ID NOS.1 and 2 for amplifying rs 2634074;
primers shown in SEQ ID NOS.5 and 6 for amplifying rs 4377290;
primers shown in SEQ ID NOS.9 and 10 for amplifying rs 7193343;
primers shown in SEQ ID NOS.13 and 14 for amplifying rs 3071379;
primers shown in SEQ ID NOS.17 and 18 for amplifying rs 891700;
primers shown in SEQ ID NOS.21 and 22 for amplifying rs 1109037;
primers shown in SEQ ID NOS.25 and 26 for amplifying rs 1666130;
primers shown in SEQ ID NOS.29 and 30 for amplifying rs 9277534;
primers shown in SEQ ID NOS.33 and 34 for amplifying rs 7793828;
primers shown in SEQ ID NOS.37 and 38 for amplifying rs 449851;
primers shown in SEQ ID NOS.41 and 42 for amplifying rs 1775737;
primers shown in SEQ ID NOS.45 and 46 for amplifying rs 1498553;
primers shown in SEQ ID NOS.49 and 50 for amplifying rs 8053194;
primers shown in SEQ ID NOS.53 and 54 for amplifying rs 5911;
primers shown in SEQ ID NOS.57 and 58 for amplifying rs 987640;
primers shown in SEQ ID NOS.61 and 62 for amplifying rs 9781645;
primers shown in SEQ ID NOS.65 and 66 for amplifying rs 25487;
primers shown in SEQ ID NOS.69 and 70 for amplifying rs 203462;
primers shown in SEQ ID NOS.73 and 74 for amplifying rs 312470;
primers shown in SEQ ID NOS.77 and 78 for amplifying rs 715463;
primers shown in SEQ ID NOS.81 and 82 for amplifying rs 1946519; and
primers shown in SEQ ID NOS.85 and 86 for amplifying rs2488457.
3. A set of probes for human genotyping, wherein the set of probes comprises probes for detecting one or more of the SNP molecular markers of claim 1, optionally wherein the set of probes is selected from one or more of the following:
probes shown in SEQ ID NOS.3 and 4 for detecting rs 2634074;
probes shown in SEQ ID NOS.7 and 8 for detecting rs 4377290;
probes shown in SEQ ID NOS.11 and 12 for detecting rs 7193343;
probes shown in SEQ ID NOS.15 and 16 for detecting rs 3071379;
probes shown in SEQ ID NOS.19 and 20 for detecting rs 891700;
probes shown in SEQ ID NOS.23 and 24 for detecting rs 1109037;
probes shown in SEQ ID NOS.27 and 28 for detecting rs 1666130;
probes shown in SEQ ID NOS.31 and 32 for detecting rs 9277534;
probes shown in SEQ ID NOS.35 and 36 for detecting rs 7793828;
probes shown in SEQ ID NOS.39 and 40 for detecting rs 449851;
probes shown in SEQ ID NOS.43 and 44 for detecting rs 1775737;
probes shown in SEQ ID NOS.47 and 48 for detecting rs 1498553;
probes shown in SEQ ID NOS.51 and 52 for detecting rs 8053194;
probes shown in SEQ ID NOS.55 and 56 for detecting rs 5911;
probes shown in SEQ ID NOS.59 and 60 for detecting rs 987640;
probes shown in SEQ ID NOS.63 and 64 for detecting rs 9781645;
probes shown in SEQ ID NOS.67 and 68 for detecting rs 25487;
probes shown in SEQ ID NOS.71 and 72 for detecting rs 203462;
probes shown in SEQ ID NOS.75 and 76 for detecting rs 312470;
probes shown in SEQ ID NOS.79 and 80 for detecting rs 715463;
probes shown in SEQ ID NOS.83 and 84 for detecting rs 1946519; and
probes shown in SEQ ID NOS.87 and 88 for detecting rs2488457.
4. A kit for genotyping a human, wherein the kit is for detecting the SNP molecular marker of claim 1, optionally the kit comprises reagents for detecting the SNP molecular marker of claim 1, optionally the kit comprises the primer set of claim 2, optionally the kit comprises the probe set of claim 3.
5. A human genotyping chip, wherein the chip is for detecting the SNP molecular markers of claim 1, optionally the chip comprises reagents for detecting the SNP molecular markers of claim 1, optionally the chip comprises the probe set of claim 3.
6. A method for genotyping in a human subject comprising quantifying one or more of the SNP molecular markers selected from the group consisting of: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519 and rs2488457, optionally, the human subject has undergone stem cell transplantation.
7. The method of claim 6, wherein the ddPCR utilizes one or more sets of primer sets selected from the group consisting of:
primers shown in SEQ ID NOS.1 and 2 for amplifying rs 2634074;
primers shown in SEQ ID NOS.5 and 6 for amplifying rs 4377290;
primers shown in SEQ ID NOS.9 and 10 for amplifying rs 7193343;
primers shown in SEQ ID NOS.13 and 14 for amplifying rs 3071379;
primers shown in SEQ ID NOS.17 and 18 for amplifying rs 891700;
primers shown in SEQ ID NOS.21 and 22 for amplifying rs 1109037;
primers shown in SEQ ID NOS.25 and 26 for amplifying rs 1666130;
primers shown in SEQ ID NOS.29 and 30 for amplifying rs 9277534;
primers shown in SEQ ID NOS.33 and 34 for amplifying rs 7793828;
primers shown in SEQ ID NOS.37 and 38 for amplifying rs 449851;
primers shown in SEQ ID NOS.41 and 42 for amplifying rs 1775737;
primers shown in SEQ ID NOS.45 and 46 for amplifying rs 1498553;
primers shown in SEQ ID NOS.49 and 50 for amplifying rs 8053194;
primers shown in SEQ ID NOS.53 and 54 for amplifying rs 5911;
primers shown in SEQ ID NOS.57 and 58 for amplifying rs 987640;
primers shown in SEQ ID NOS.61 and 62 for amplifying rs 9781645;
primers shown in SEQ ID NOS.65 and 66 for amplifying rs 25487;
primers shown in SEQ ID NOS.69 and 70 for amplifying rs 203462;
primers shown in SEQ ID NOS.73 and 74 for amplifying rs 312470;
primers shown in SEQ ID NOS.77 and 78 for amplifying rs 715463;
primers shown in SEQ ID NOS.81 and 82 for amplifying rs 1946519; and
primers shown in SEQ ID NOS.85 and 86 for amplifying rs2488457.
8. The method of claim 6 or 7, wherein the ddPCR utilizes one or more of the probe sets selected from the group consisting of:
probes shown in SEQ ID NOS.3 and 4 for detecting rs 2634074;
probes shown in SEQ ID NOS.7 and 8 for detecting rs 4377290;
probes shown in SEQ ID NOS.11 and 12 for detecting rs 7193343;
probes shown in SEQ ID NOS.15 and 16 for detecting rs 3071379;
probes shown in SEQ ID NOS.19 and 20 for detecting rs 891700;
probes shown in SEQ ID NOS.23 and 24 for detecting rs 1109037;
probes shown in SEQ ID NOS.27 and 28 for detecting rs 1666130;
probes shown in SEQ ID NOS.31 and 32 for detecting rs 9277534;
probes shown in SEQ ID NOS.35 and 36 for detecting rs 7793828;
probes shown in SEQ ID NOS.39 and 40 for detecting rs 449851;
probes shown in SEQ ID NOS.43 and 44 for detecting rs 1775737;
probes shown in SEQ ID NOS.47 and 48 for detecting rs 1498553;
probes shown in SEQ ID NOS.51 and 52 for detecting rs 8053194;
probes shown in SEQ ID NOS.55 and 56 for detecting rs 5911;
probes shown in SEQ ID NOS.59 and 60 for detecting rs 987640;
probes shown in SEQ ID NOS.63 and 64 for detecting rs 9781645;
probes shown in SEQ ID NOS.67 and 68 for detecting rs 25487;
probes shown in SEQ ID NOS.71 and 72 for detecting rs 203462;
probes shown in SEQ ID NOS.75 and 76 for detecting rs 312470;
probes shown in SEQ ID NOS.79 and 80 for detecting rs 715463;
probes shown in SEQ ID NOS.83 and 84 for detecting rs 1946519; and
probes shown in SEQ ID NOS.87 and 88 for detecting rs2488457.
9. Use of SNP molecular markers comprising one or more of the following for human genotyping: rs2634074, rs4377290, rs7193343, rs3071379, rs891700, rs1109037, rs1666130, rs9277534, rs7793828, rs449851, rs1775737, rs1498553, rs8053194, rs5911, rs987640, rs9781645, rs25487, rs203462, rs312470, rs715463, rs1946519, and rs2488457.
10. The use of claim 9, wherein the human genotyping is used to distinguish between recipient cells and donor stem cells in a human subject undergoing stem cell transplantation.
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