Digital PCR (polymerase chain reaction) detection system for detecting proportion of donor-derived free DNA (deoxyribonucleic acid) in organ transplantation recipient and application of digital PCR detection system
The digital PCR detection method that uses a single probe to distinguish SNP sites solves the problems of high cost and complexity in existing technologies, and achieves efficient and highly sensitive allele detection and quantitative analysis, which is suitable for a variety of clinical scenarios.
Patent Information
- Application Number
- CN202511347186.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-19
- Publication Date
- 2025-11-14
AI Technical Summary
Existing SNP detection methods suffer from high cost, complexity, and low throughput in rapid clinical testing, making it difficult to efficiently and accurately distinguish between two alleles and achieve highly sensitive quantitative analysis.
This invention employs a digital PCR detection method based on a single probe to distinguish SNP sites. It uses a pair of universal primers and a specific probe across SNP sites to identify alleles at the amplification endpoint by the difference in fluorescence signal intensity. Combined with the absolute quantitative capability of digital PCR, it is suitable for various clinical scenarios such as fetal cell-free DNA ratio detection, transplantation monitoring, and tumor liquid biopsy.
It significantly reduces experimental costs and operational complexity, achieving highly sensitive target nucleic acid detection and accurate quantification, and is suitable for clinical diagnosis and disease monitoring in complex sample contexts.
Smart Images
Figure CN120945071A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of digital PCR, and in particular to a digital PCR detection system and its application for detecting the proportion of donor-derived cell-free DNA in organ transplant recipients. Background Technology
[0002] In recent years, with the rapid development of precision medicine, single nucleotide polymorphism (SNP) detection has played an increasingly important role in clinical diagnosis, disease prediction, and transplant monitoring. SNPs are the most common form of genetic variation in the genome, widely present in the human genome, and exhibit high polymorphism among different individuals. By detecting the two alleles at a specific SNP locus, quantitative analysis of specific nucleic acids in a sample can be achieved. Examples include detecting the proportion of fetal cell-free DNA in non-invasive prenatal testing (NIPT), monitoring the proportion of donor DNA in transplant recipients during transplant monitoring, and detecting circulating tumor DNA in liquid tumor biopsies.
[0003] In clinical practice, the core challenge of SNP detection lies in how to efficiently and accurately distinguish between two alleles and achieve highly sensitive quantitative analysis in complex sample backgrounds. Currently, the main SNP-based detection methods are: (1) Next-generation sequencing (NGS): using high-throughput sequencing technology to perform whole-genome scanning of SNP sites in samples. Although NGS has the advantage of high throughput, its data processing is complex, costly, and requires ultra-high sequencing depth to achieve high sensitivity, making it difficult to meet the needs of rapid clinical detection. (2) Digital PCR (dPCR): by dividing the sample into tens of thousands of independent droplets, each droplet may or may not contain the target nucleic acid molecule, absolute quantification of the target nucleic acid is achieved. Although dPCR has the advantages of high sensitivity, simple operation, and relatively low cost, traditional dPCR methods have low throughput and require the design of independent probes for each allele, increasing the complexity of experimental design and increasing costs.
[0004] To meet the needs of rapid clinical testing, this invention proposes a digital PCR detection method and kit for distinguishing two alleles at a SNP site based on a single probe. By designing a pair of universal primers and a probe that crosses the SNP site, the sequences of two alleles are amplified simultaneously. The difference in melting temperature (Tm) between the probe and different allele sequences is utilized to distinguish alleles at the amplification endpoint through differences in fluorescence signal intensity. This method has the following significant advantages: (1) Highly efficient differentiation of two alleles: The single-probe design significantly reduces experimental costs and operational complexity. (2) High-sensitivity detection: Combined with the absolute quantification capability of dPCR, it can achieve highly sensitive detection of the target nucleic acid. (3) Wide applicability: It is suitable for various clinical scenarios such as fetal cell-free DNA ratio detection, transplantation monitoring, and tumor liquid biopsy. Through the system of this invention, accurate quantification of the target nucleic acid can be achieved in complex sample backgrounds, providing powerful tool support for clinical diagnosis and disease monitoring. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a digital PCR detection system for detecting the proportion of donor-derived cell-free DNA in organ transplant recipients. The system is used to detect one or more of the following biselequential SNP loci: rs13031497, rs1109037, rs11714258, rs7441242, rs1979255, rs4868851, rs4709032, rs13218440, rs10100663, rs10776839, rs740598, rs1498553, rs2269355, rs11174191, rs7160304, rs910792, rs2016276, rs1898230, rs576261, and rs1000. 551; and the reagents used to detect each of the above-mentioned bis-allele SNP sites include a pair of universal primers and a specific probe spanning the bis-allele SNP site. The specific probe detects the bis-allele SNP site by means of a sequence that is completely complementary to a segment of the wild-type or mutant template of the bis-allele SNP site, and correspondingly, a segment of the other template that is not completely complementary to the other template, i.e., it does not match at the SNP site, but the sequences at other positions are completely complementary. When the probe binds to a completely matched sequence, it produces a relatively strong fluorescence signal; when the probe binds to a sequence with a single base mismatch, it produces a relatively weak fluorescence signal. Based on this difference in fluorescence signal intensity, the detection of the bis-allele SNP site is achieved in a single detection channel.
[0006] In one embodiment, the difference ΔTm between the Tm value when the probe binds to the perfectly matching sequence and the Tm value when the probe binds to the partially matching sequence is not less than 5°C.
[0007] In one embodiment, the difference ΔTm is 5-9°C.
[0008] In one embodiment, the Tm value at which the incompletely matched sequence binds is 3-7°C, which is the same as the ΔT value of the annealing temperature in the digital PCR amplification program.
[0009] In one embodiment, when detecting the SNP site rs13031497, primers SEQ ID NO.1 and SEQ ID NO.2 and probe SEQ ID NO.5 are used; when detecting the SNP site rs1109037, primers SEQ ID NO.9 and SEQ ID NO.10 and probe SEQ ID NO.11 are used; when detecting the SNP site rs11714258, primers SEQ ID NO.14 and SEQ ID NO.15 and probe SEQ ID NO.16 are used; when detecting the SNP site rs7441242, primers SEQ ID NO.17 and SEQ ID NO.18 and probe SEQ ID NO.19 are used; when detecting the SNP site rs1979255, primers SEQ ID NO.20 and SEQ ID NO.21 and probe SEQ ID NO.22 are used; when detecting the SNP site rs4868851, primers SEQ ID NO.1 are used. Primers NO. 23 and SEQ ID NO. 24, and probe NO. 25 were used; primers NO. 26 and SEQ ID NO. 27, and probe NO. 28 were used for detecting the SNP site rs4709032; primers NO. 29 and SEQ ID NO. 30, and probe NO. 31 were used for detecting the SNP site rs10100663; primers NO. 32 and SEQ ID NO. 33, and probe NO. 34 were used for detecting the SNP site rs10776839; primers NO. 35 and SEQ ID NO. 36, and probe NO. 37 were used for detecting the SNP site rs740598; primers NO. 38 and SEQ ID NO. 39, and probe NO. 25 were used for detecting the SNP site rs740598. For detecting the SNP site rs1498553, primers SEQ ID NO.41 and SEQ ID NO.42 and probe SEQ ID NO.43 were used; for detecting the SNP site rs2269355, primers SEQ ID NO.44 and SEQ ID NO.45 and probe SEQ ID NO.46 were used; for detecting the SNP site rs11174191, primers SEQ ID NO.47 and SEQ ID NO.48 and probe SEQ ID NO.49 were used; for detecting the SNP site rs7160304, primers SEQ ID NO.50 and SEQ ID NO.51. Primers and probe SEQ ID NO. 52 were used for detecting the SNP site rs910792; primers SEQ ID NO. 53 and SEQ ID NO. 54 were used, and probe SEQ ID NO. 55 was used; primers SEQ ID NO. 56 and SEQ ID NO. 57 were used, and probe SEQ ID NO. 58 was used; primers SEQ ID NO. 59 and SEQ ID NO. 60 were used, and probe SEQ ID NO. 61 was used; primers SEQ ID NO. 62 and SEQ ID NO. 63 were used, and probe SEQ ID NO. 64 was used; and primers SEQ ID NO. 65 and SEQ ID NO. 66 were used, and probe SEQ ID NO. 67 was used.
[0010] In one embodiment, the above-described detection system is provided for use in the preparation of reagents for detecting the proportion of donor-derived cell-free DNA in organ transplant recipients.
[0011] In one embodiment, the organ transplant is a kidney transplant, bone marrow transplant, liver transplant, heart transplant, lung transplant, and / or pancreas transplant.
[0012] To meet the needs of rapid clinical testing, this invention proposes a digital PCR detection system for detecting the proportion of donor-derived cell-free DNA in kidney transplant recipients. This system utilizes a digital PCR detection method and kit based on a single probe to distinguish two alleles at SNP sites. By designing a pair of universal primers and a probe that crosses SNP sites, the sequences of the two alleles are amplified simultaneously. The difference in melting temperature (Tm) between the probe and different allele sequences is used to distinguish alleles at the amplification endpoint through differences in fluorescence signal intensity. This method has the following significant advantages: (1) Highly efficient differentiation of two alleles: The single-probe design significantly reduces experimental costs and operational complexity. (2) High-sensitivity detection: Combined with the absolute quantification capability of dPCR, it enables highly sensitive detection of the target nucleic acid. Through this system, accurate quantification of the target nucleic acid can be achieved in complex sample environments. Attached Figure Description
[0013] Figure 1 This is a schematic diagram illustrating the principle of using a single probe to distinguish between biallelic SNPs. Figure 2 This is a one-dimensional result diagram of digital PCR screening using primers and probes at the rs13031497 site; Figure 3This is a one-dimensional plot of the annealing temperature screening results for the rs1109037 site using digital PCR. Figure 4 A schematic diagram of the principle for determining the proportion of dd-cfDNA (DF) based on specific SNPs. Detailed Implementation
[0014] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.
[0015] Example 1: Design and Screening of Primers and Probes for SNP Detection
[0016] I. Principle of Single Probe Differentiation of Bialelic SNPs This detection method uses a pair of universal primers and a specific probe spanning the SNP site to detect SNPs. Taking the rs1109037 site as an example, the allele type at this site is G / A. When the probe binds to a perfectly matched sequence, the hybridization stability is higher, exhibiting higher hydrolysis efficiency during PCR amplification, thus producing a stronger fluorescence signal. When the probe binds to a sequence with a single base mismatch, the hybridization stability decreases, leading to a decrease in probe hydrolysis efficiency and producing a relatively weaker fluorescence signal. Based on this difference in fluorescence signal intensity, the two alleles can be identified in a single detection channel. The specific technical principle is as follows: Figure 1 As shown. Digital PCR uses Poisson statistics to calculate the exact copy number of the two alleles in each sample based on the ratio of the number of strongly fluorescent positive droplets or the number of weakly fluorescent positive droplets to the total number of droplets.
[0017] like Figure 1 As shown, a pair of universal primers and a specific probe spanning the SNP site were used for SNP detection. Taking the rs1109037 site as an example, the allele type of this site is G / A. When the probe binds to a perfectly matched sequence, its hybridization stability is higher, exhibiting higher hydrolysis efficiency during PCR amplification, thus producing a stronger fluorescence signal. When the probe binds to a sequence with a single base mismatch, the hybridization stability decreases, leading to a decrease in probe hydrolysis efficiency and producing a relatively weaker fluorescence signal. Based on this difference in fluorescence signal intensity, the two alleles can be identified in a single detection channel.
[0018] II. SNP Primer and Probe Design Download the SNP site sequence from the NCBI database and design primers and probes using software such as Primer3, Oligo, and Beacon Designer. The designed primers and probes must meet the following conditions: (1) The probe binding region must contain the SNP site; (2) The amplicon is <= 100 bp; (3) The primer length is generally 18-25 bp; (4) The primer GC content is generally 40-60%; (5) The difference in Tm value between the upstream and downstream primers is <= 2°C; (6) Avoid dimers and self-complementarity.
[0019] To clarify the requirements of this method for probe complete and incomplete matching Tm values, this embodiment takes the rs13031497 site as an example (allele type A / T) and designs probes with different Tm value conditions to lock nucleic acid modification to regulate the complete and incomplete matching Tm values of the probe (Table 1).
[0020] Table 1. Primer, probe, and plasmid information for the rs13031497 site.
[0021] Note: The "+" in the probe sequence indicates locked nucleic acid modification. letter underscore Represents SNP sites.
[0022] III. SNP Primer and Probe Screening Digital PCR experiments were performed using pre-designed primers and probes. The digital PCR reaction system was prepared according to Table 2. Quantitative analysis of digital PCR was performed using the fully automated digital PCR platform D50 (NewYi Manufacturing Technology (Beijing) Co., Ltd., Beijing). 30 µL of the digital PCR reaction system was added to the sample wells of the integrated droplet chip, sealed with sealing oil, covered with a gasket, and then placed in the chip loading area of the instrument. Water-in-oil droplets were generated in the instrument and stored in the chip's reaction tube. Amplification was performed according to a PCR amplification program of 95°C for 10 min; 40 cycles of 94°C for 30 s and 56°C for 1 min. Droplet detection was performed after PCR amplification.
[0023] Table 2. Preparation of Digital PCR Reaction System
[0024] Digital PCR uses Poisson statistics to calculate the exact copy number of the target nucleic acid molecule in each sample based on the ratio of positive droplets to total droplets. Based on the principle of single-probe differentiation of two allele SNPs, the amplification signals of alleles that are perfectly matched and partially matched with the probe need to be clearly distinguishable; otherwise, the number of positive droplets cannot be accurately calculated and quantified. The results of digital PCR tests using four probes with different Tm differences (ΔTm) at the rs13031497 locus (perfectly matched and partially matched) are presented. Figure 2 When ΔTm is 4.2°C, the positive amplification signals of perfectly matched and partially matched alleles cannot be distinguished; when ΔTm >= 5°C, the positive amplification signals of perfectly matched and partially matched alleles can be distinguished, and the larger the ΔTm, the better the distinguishing effect. Therefore, the difference in Tm values between perfectly matched and partially matched templates for probe binding needs to be >= 5°C.
[0025] Example 2: Optimization of PCR Procedure for SNP Detection The annealing temperature setting has a significant impact on PCR amplification efficiency. In order to clarify the requirements of this method for annealing temperature, this example takes the rs1109037 locus (allele type G / A) as an example and designs primers and probes that meet the conditions of Example 1 (Table 3) to study the selection of annealing temperature.
[0026] Table 3 Primer and probe information for the rs1109037 site.
[0027] Digital PCR experiments were performed using pre-designed primers and probes. The digital PCR reaction system was prepared according to Table 4. Quantitative analysis of digital PCR was performed using the fully automated digital PCR platform D50 (NewYi Manufacturing Technology (Beijing) Co., Ltd., Beijing). 30 µL of the digital PCR reaction system was added to the sample wells of the integrated droplet chip, sealed with sealing oil, covered with a rubber gasket, and then placed in the chip loading area of the instrument. Water-in-oil droplets were generated in the instrument and stored in the chip's reaction tube. Amplification was performed according to a PCR amplification program of 95°C for 10 min; 40 cycles of 94°C for 30 s and X°C for 1 min (X = 50.7, 51.7, 52.7, 53.7, 54.7, 55.7, 56.7, 57.7°C). Droplet detection was performed after PCR amplification.
[0028] Table 4. Preparation of Digital PCR Reaction System
[0029] Digital PCR uses Poisson statistics to calculate the exact copy number of the target nucleic acid molecule in each sample based on the ratio of positive droplets to total droplets. Based on the principle of single-probe differentiation of two allele SNPs, the amplification signals of alleles that are perfectly matched and partially matched with the probe need to be clearly distinguishable; otherwise, the number of positive droplets cannot be accurately calculated and quantified. The results of digital PCR tests at different annealing temperatures at the rs1109037 site (…) Figure 3 When the difference between the incomplete match Tm value and the annealing temperature (ΔT) is 8 and 9°C (annealing temperatures of 51.7 and 50.7°C), the positive amplification signals of the complete match allele and the incomplete match allele cannot be distinguished; when ΔT is 3~7°C (annealing temperatures of 52.7~56.7°C), the positive amplification signals of the complete match allele and the incomplete match allele can be distinguished; when ΔT is 2°C (annealing temperature of 57.7°C), the incomplete match allele cannot effectively bind to the template due to the high annealing temperature, and there is no amplification signal. On the other hand, examining the relative deviation of the quantitative copy number of perfectly matched and partially matched alleles (calculated as |theoretical value - measured value| / theoretical value × 100%) reveals that when ΔT is 3°C (annealing temperature 56.7°C), although the amplification signals of the two alleles are still distinguishable, the amplification efficiency of the partially matched allele is affected by the excessively high annealing temperature, resulting in a lower quantitative copy number and a large deviation (>5%), which in turn affects the accuracy of subsequent applications (Table 5). In conclusion, the difference between the Tm value and the annealing temperature for partially matched alleles should be between 4°C and 7°C.
[0030] Table 5. Ratio of quantitative copy number of perfectly matched and partially matched alleles
[0031] Example 3: Detection of the proportion of donor-derived cell-free DNA in kidney transplant recipients Following kidney transplantation, monitoring the proportion of donor-derived cell-free DNA (dd-cfDNA) in the recipient's plasma or urine is an important non-invasive method for assessing transplant rejection or graft injury. Due to genetic differences between donor and recipient genomes, particularly SNPs, these loci can serve as specific markers to distinguish donor and recipient DNA, thus enabling the quantitative detection of dd-cfDNA. In this example, a peripheral blood sample was collected from one kidney transplant recipient on the first day post-surgery, and the condition of the transplanted kidney was monitored by detecting the proportion of dd-cfDNA.
[0032] I. Principle of determining the proportion of dd-cfDNA based on specific SNPs
[0033] When a transplanted kidney experiences rejection or damage, donor-derived cells (such as renal tubular epithelial cells and endothelial cells) release DNA fragments (dd-cfDNA) into the recipient's bloodstream through apoptosis or necrosis. SNPs are stable single-base variations in the genome, exhibiting high individual specificity. In the recipient's plasma, donor-derived cell-free DNA carries donor-specific SNP alleles, while the recipient's own DNA does not contain these sites. For example... Figure 4 As shown, by using digital PCR to target and detect these donor-specific SNPs and calculate the proportion of donor alleles in total cell-free DNA, the graft status can be non-invasively assessed.
[0034] II. Screening of Candidate SNP Sites
[0035] SNP loci were selected from the dbSNP database of the National Center for Biotechnology Information (NCBI) in the United States. The selected SNP loci had to meet the following conditions simultaneously: (1) biselequential SNP; (2) allele frequency between 0.35 and 0.65 (high-frequency polymorphic SNP); (3) no association between the SNP and the disease; and (4) SNP conforming to Hardy-Weinberg law. Twenty SNP loci were selected based on the above conditions, and the specific information is shown in Table 6.
[0036] Table 6 shows the selected SNP sites.
[0037] III. Primer and probe design for candidate SNP sites The designed primers and probes must meet the following conditions: (1) the probe binding region must contain SNP sites; (2) the amplicon <= 100 bp; (3) the primer length is generally 18-25 bp; (4) the primer GC content is generally 40-60%; (5) the difference in Tm values between upstream and downstream primers <= 2°C; (6) avoid dimers and self-complementarity; (7) the difference in Tm values between probes that bind to perfectly matched and partially matched templates is greater than 5°C; (8) the difference between the Tm value of the partially matched template and the annealing temperature must be between 4°C and 7°C. Under the condition that the annealing temperature is fixed at 55°C, the specific primer and probe sequences are shown in Tables 7-1 and 7-2. One tube system can detect 5 SNP sites simultaneously (each probe is labeled with a different fluorescent group for a 5-channel digital PCR detection system).
[0038] Table 7-1 Primer and probe sequence information for 20 SNP sites
[0039] Table 7-2 Primer and probe sequence information for 20 SNP sites
[0040] Note: The "+" in the probe sequence indicates locked nucleic acid modification. letter underscore Represents SNP sites.
[0041] IV. Recipient SNP Genotyping Determination 1. Sample collection and nucleic acid extraction A saliva sample was collected from one kidney transplant recipient. The saliva sample contained no donor-derived DNA and could be used to confirm the recipient's SNP genotype. Human genomic DNA was extracted from the saliva sample using the gDNA nucleic acid extraction reagent (NewYi Manufacturing Technology (Beijing) Co., Ltd., Beijing), following the manufacturer's instructions. The DNA concentration was measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) after extraction.
[0042] 2. Quantitative analysis of SNP sites based on digital PCR The digital PCR reaction system was prepared according to Table 8. There were 4 tubes in total, and each tube was used to detect 5 SNP sites (with 5 upstream primers, 5 downstream primers, and 5 probes, n=5, m=5). The primer and probe sequences used are shown in Table 7. The template was nucleic acid extracted from saliva.
[0043] Table 8. Preparation of Digital PCR Reaction System
[0044] Digital PCR quantification of SNP sites was performed using the fully automated digital PCR platform D50 (Newyi Manufacturing Technology (Beijing) Co., Ltd., Beijing). 30 µL of digital PCR reaction mixture was added to the sample wells of the integrated droplet chip, sealed with sealing oil, covered with a gasket, and then placed in the chip loading area of the instrument. Water-in-oil droplets were generated in the instrument and stored in the chip's reaction tube. Amplification was performed according to a PCR amplification program of 95°C for 10 min; 40 cycles of 94°C for 30 s and 55°C for 1 min. Droplet detection was performed after PCR amplification. By setting different fluorescence amplitude thresholds, positive droplets containing amplification products of different targets could be distinguished. Using Poisson statistics, the exact copy number of the target nucleic acid molecule in each sample was calculated based on the ratio of positive droplets to the total number of droplets.
[0045] 3. Determination of homozygous SNP loci in recipients After digital PCR testing, the copy numbers of the two alleles at each of the 20 SNP loci were obtained. Taking a single SNP locus as an example, if the copy numbers of both alleles at that locus are greater than 0, the recipient's SNP locus is heterozygous (Aa); if the copy number of one allele is equal to 0, the recipient's SNP locus is homozygous (AA or aa). Table 9 shows the genotypes of the 20 SNP loci in this kidney transplant recipient. Among them, SNP1, SNP5, SNP7, SNP9, SNP10, SNP12, SNP16, SNP17, and SNP18 are homozygous loci.
[0046] Table 9 Genotypes of recipients at 20 SNP loci
[0047] V. Donor Genotype Determination 1. Sample collection and nucleic acid extraction Peripheral blood samples were collected from one kidney transplant recipient on the first postoperative day. 10 mL of peripheral blood was collected and plasma separation was performed. Cell-free DNA was extracted from 4 mL of plasma using the cfDNA-B1 nucleic acid extraction reagent (NewYi Manufacturing Technology (Beijing) Co., Ltd., Beijing), following the manufacturer's instructions. The concentration of cell-free DNA was determined using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) after extraction.
[0048] 2. Quantitative analysis of SNP sites based on digital PCR The digital PCR reaction system was prepared according to Table 10. There were 4 tubes in total, and each tube was used to detect 5 SNP sites (with 5 upstream primers, 5 downstream primers, and 5 probes, n=5, m=5). The primer and probe sequences used are shown in Table 7. The template was cell-free nucleic acid extracted from plasma.
[0049] Table 10. Preparation of Digital PCR Reaction System
[0050] Digital PCR quantification of SNP sites was performed using the fully automated digital PCR platform D50 (Newyi Manufacturing Technology (Beijing) Co., Ltd., Beijing). 30 µL of digital PCR reaction mixture was added to the sample wells of the integrated droplet chip, sealed with sealing oil, covered with a gasket, and then placed in the chip loading area of the instrument. Water-in-oil droplets were generated in the instrument and stored in the chip's reaction tube. Amplification was performed according to a PCR amplification program of 95°C for 10 min; 40 cycles of 94°C for 30 s and 55°C for 1 min. Droplet detection was performed after PCR amplification. By setting different fluorescence amplitude thresholds, positive droplets containing amplification products of different targets could be distinguished. Using Poisson statistics, the exact copy number of the target nucleic acid molecule in each sample was calculated based on the ratio of positive droplets to the total number of droplets.
[0051] 3. Identify SNP loci containing donor-specific alleles (i.e., SNP loci in the donor whose genotype differs from that in the recipient; referred to as target SNP loci in this paper). After digital PCR detection, copy numbers of two alleles at each of the 20 SNP loci were obtained. Analysis was performed on SNP1, SNP5, SNP7, SNP9, SNP10, SNP12, SNP16, SNP17, and SNP18 (in recipient homozygous genotype). Taking an AA-type SNP locus as an example, if the copy number of the 'a' allele is greater than 0, then that locus is the target SNP locus (containing donor-specific alleles), and the results are shown in Table 11.
[0052] Table 11 Target SNP sites
[0053] 4. Genotyping of the target SNP locus from the donor. Taking the SNP locus with recipient AA as an example, for the target SNP locus, the donor's genotype may be Aa or aa. This method uses K-means cluster analysis to determine the genotype of the donor SNP locus.
[0054]
[0055] Table 12 Proportion of dd-cfDNA calculated assuming homozygous donors.
[0056] K-means cluster analysis was performed on each dd-cfDNA proportion. This method is based on the following steps: (1) Initialize the centroid: Randomly select two data points as the initial centroids c1 and c2, c1=3.19, c2=6.97.
[0057] (2) Calculate distance and assign category: For each data point, calculate the Euclidean distance d between it and the two centroids.
[0058] Data points are assigned to the category of the centroid that is closer to them (Table 13).
[0059] Table 13 Calculation process of K-means clustering analysis
[0060] (3) Update centroid: For each category, calculate the mean of all its members and use the mean as the new centroid.
[0061] Class 1 = [3.19] → New centroid = 3.19 Class 2 = [6.26, 5.80, 6.34, 6.97] → New centroid = (6.26+5.80+6.34+6.97) / 4 = 6.34 (4) Iteration: Repeat steps 2 and 3 until the classification results of each data point no longer change and the clustering process converges.
[0062] The clustering results automatically divided the dd-cfDNA proportions into two groups: one with lower values and the other with higher values, as shown in Table 9. Theoretically, the mean of group 2 should be a multiple of the mean of group 1 (2 / 1). This is because if some donors at certain loci are heterozygous, the accurate calculation formula should be... , The previous assumption that all loci were homozygous for the donor led to an underestimation of the proportion of dd-cfDNA at loci where the donor was heterozygous.
[0063] The mean of class 2 / the mean of class 1 = 6.34 / 3.19. A chi-square test was performed on 6.34 / 3.19 to determine whether it meets the 2 / 1 multiple relationship. The calculated result p = 0.998 (p > 0.05), indicating that it meets the 2 / 1 multiple relationship and supports the rationality of the classification.
[0064] Therefore, by cluster analysis of multiple target sites, the genotype of the donor can be determined: Class 1 corresponds to the donor being heterozygous (Aa), and Class 2 corresponds to the donor being homozygous (aa) (Table 13).
[0065] Table 13 Results of K-means clustering analysis
[0066] VI. Final Calculation of dd-cfDNA Ratio Once the genotypes of the recipient and donor at the target locus were determined, the dd-cfDNA ratio (DF) at the target locus could be calculated using the formula in Table 14. The final reported dd-cfDNA ratio was the average of the DF at multiple loci, which was 6.52% (Table 15).
[0067] Table 14 Formula for calculating the proportion of dd-cfDNA at a single SNP site
[0068] Table 15 Final dd-cfDNA ratio calculation results (peripheral blood sample on postoperative day 1)
[0069] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.
[0070] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.
Claims
1. A digital PCR detection system for detecting the proportion of donor-derived cell-free DNA in organ transplant recipients, characterized in that, The system is used to detect one or more of the following biselenate SNP loci: rs13031497, rs1109037, rs11714258, rs7441242, rs1979255, rs4868851, rs4709032, rs13218440, rs10100663, rs10776839, rs740598, rs1498553, rs2269355, rs11174191, rs7160304, rs910792, rs2016276, rs1898230, rs576261, and rs1000551; and when detecting each of the above biselenate SNP loci, the following is used: The reagent comprises a pair of universal primers and a specific probe spanning the biselenomeric SNP site. The specific probe detects the biselenomeric SNP site. The probe is completely complementary to a sequence in either the wild-type or mutant template of the biselenomeric SNP site, and correspondingly, the probe is not completely complementary to a sequence in another template, i.e., it does not match at the SNP site, but the sequences at other positions are completely complementary. When the probe binds to a completely matched sequence, it generates a relatively strong fluorescence signal; when the probe binds to a sequence with a single base mismatch, it generates a relatively weak fluorescence signal. Based on this difference in fluorescence signal intensity, the detection of the biselenomeric SNP site is achieved in a single detection channel.
2. The detection system according to claim 1, characterized in that, The difference ΔTm between the Tm value when the probe binds to the perfectly matching sequence and the Tm value when the probe binds to the partially matching sequence is not less than 5°C.
3. The detection system according to claim 2, characterized in that, The difference ΔTm is 5-9℃.
4. The detection system according to claim 1, characterized in that, The Tm value for binding to the incompletely matched sequence is 3-7℃, which is the same as the ΔT value of the annealing temperature in the digital PCR amplification program.
5. The detection system according to claim 1, characterized in that, When detecting the SNP site rs13031497, primers SEQ ID NO.1 and SEQ ID NO.2 and probe SEQ ID NO.5 were used; when detecting the SNP site rs1109037, primers SEQ ID NO.9 and SEQ ID NO.10 and probe SEQ ID NO.11 were used; when detecting the SNP site rs11714258, primers SEQ ID NO.14 and SEQ ID NO.15 and probe SEQ ID NO.16 were used; when detecting the SNP site rs7441242, primers SEQ ID NO.17 and SEQ ID NO.18 and probe SEQ ID NO.19 were used; when detecting the SNP site rs1979255, primers SEQ ID NO.20 and SEQ ID NO.21 and probe SEQ ID NO.22 were used; when detecting the SNP site rs4868851, primers SEQ ID NO.23 and SEQ ID NO.14 were used. Primers NO. 24 and probe NO. 25 were used for detecting the SNP site rs4709032; primers NO. 26 and NO. 27 and probe NO. 28 were used for detecting the SNP site rs13218440; primers NO. 29 and NO. 30 and probe NO. 31 were used for detecting the SNP site rs10100663; primers NO. 32 and NO. 33 and probe NO. 34 were used for detecting the SNP site rs10776839; primers NO. 35 and NO. 36 and probe NO. 37 were used for detecting the SNP site rs740598; primers NO. 38 and NO. 39 and probe NO. 31 were used for detecting the SNP site rs13218440; primers NO. 29 and NO. 30 and probe NO. 31 were used for detecting the SNP site rs10100663; primers NO. 32 and NO. 33 and probe NO. 34 were used for detecting the SNP site rs10776839; primers NO. 38 and NO. 39 and probe NO. 31 were used for detecting the SNP site rs740598. For detecting the SNP site rs1498553, primers SEQ ID NO.41 and SEQ ID NO.42 and probe SEQ ID NO.43 were used; for detecting the SNP site rs2269355, primers SEQ ID NO.44 and SEQ ID NO.45 and probe SEQ ID NO.46 were used; for detecting the SNP site rs11174191, primers SEQ ID NO.47 and SEQ ID NO.48 and probe SEQ ID NO.49 were used; for detecting the SNP site rs7160304, primers SEQ ID NO.50 and SEQ ID NO.
51. Primers and probe SEQ ID NO. 52 were used for detecting the SNP site rs910792; primers SEQ ID NO. 53 and SEQ ID NO. 54 were used, and probe SEQ ID NO. 55 was used; primers SEQ ID NO. 56 and SEQ ID NO. 57 were used, and probe SEQ ID NO. 58 was used; primers SEQ ID NO. 59 and SEQ ID NO. 60 were used, and probe SEQ ID NO. 61 was used; primers SEQ ID NO. 62 and SEQ ID NO. 63 were used, and probe SEQ ID NO. 64 was used; and primers SEQ ID NO. 65 and SEQ ID NO. 66 were used, and probe SEQ ID NO. 67 was used.
6. The use of the detection system according to any one of claims 1-5 in the preparation of reagents for detecting the proportion of donor-derived cell-free DNA in organ transplant recipients.
7. The application according to claim 6, wherein the organ transplant is a kidney transplant, liver transplant, heart transplant, lung transplant, bone marrow transplant, and / or pancreas transplant.