A SNP molecular marker for detecting donor cfDNA of a pregnant organ transplant patient, a detection method and device

Abstract

The present application relates to the technical field of gene detection, in particular to a SNP molecular marker for detecting donor cfDNA of a pregnant organ transplant patient, a detection method and device. The SNP molecular marker for detecting donor cfDNA of a pregnant organ transplant patient is shown in Table 10. The present application uses free DNA in peripheral blood of a pregnant patient and genomic DNA in peripheral blood of a biological father of a fetus as detection objects, adopts liquid phase hybridization capture and high-throughput sequencing technology, uses thousands of SNP sites, obtains the genotype of the fetus through the genotype of the couple, eliminates the influence of cfDNA from the fetus on the content of cfDNA from the transplanted organ, accurately determines the concentration of cfDNA from the transplanted organ, has higher detection sensitivity, can be detected multiple times only by using peripheral blood of the patient, and achieves the purpose of long-term dynamic monitoring.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to a SNP molecular marker, detection method, and device for detecting cfDNA in donors from pregnant organ transplant patients. Background Technology

[0002] Organ transplantation is the preferred clinical treatment for end-stage organ disease. However, postoperative rejection is a significant factor affecting the survival rate of transplanted organs and represents the greatest threat faced by organ transplant patients. Early diagnosis and timely treatment of rejection can effectively reduce the degree of pathological damage to transplanted organs and prolong their survival time.

[0003] Percutaneous biopsy has long been the gold standard for clinically assessing transplant organ damage. However, the sampling sites are limited, it cannot fully reflect the pathological condition of the transplanted organ, and it often involves invasive complications. Therefore, the search for a non-invasive testing technique has become a priority. Currently, routine non-invasive testing methods in clinical practice include imaging and biochemical indicators (alanine aminotransferase, total bilirubin, alkaline phosphatase and gamma-glutamyl transferase, creatinine, or cardiac enzyme profiles), but these methods have low sensitivity and specificity. For example, serum creatinine levels can rise not only due to renal rejection but also due to acute renal failure caused by rejection drug toxicity in kidney transplant patients. Currently, one of the main clinical problems faced by organ transplant patients is the lack of highly sensitive, highly specific, and non-invasive tests for early diagnosis and continuous monitoring of graft rejection risk.

[0004] Cell-free DNA (cfDNA) refers to partially degraded endogenous DNA that exists outside of cells, primarily originating from cell apoptosis or necrosis. Both heterologous and autologous somatic mutant DNA can be detected in the patient's blood using non-invasive methods and are widely used in early cancer screening, targeted cancer therapy, and non-invasive prenatal diagnosis. In the field of organ transplantation, Dennis Lo was the first to detect cell-free DNA from male donor tissue in the plasma of female liver and kidney transplant patients. This was believed to be cfDNA released during donor organ apoptosis or necrosis in the patient, and cell-free DNA can serve as a marker of organ transplant rejection.

[0005] Pregnant women undergoing organ transplantation are a special group. Their blood contains not only their own cell-derived DNA (cfDNA), but also cfDNA released from the transplanted organ and cfDNA from the fetus. Both of these latter types of cfDNA are allogeneic. Simply detecting the level of allogeneic cfDNA cannot accurately reflect the level of cfDNA released from the transplanted organ, thus failing to accurately assess the degree of organ damage in these patients. This poses a major challenge for non-invasive testing in pregnant women undergoing organ transplantation and is a significant pain point in clinical management. Therefore, a suitable non-invasive testing method for pregnant women undergoing organ transplantation is needed in clinical practice. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide an SNP molecular marker, detection method and device for detecting cfDNA of donors in pregnant organ transplant patients.

[0007] Therefore, the present invention provides the following technical solution:

[0008] A SNP molecular marker for detecting cfDNA in pregnant organ transplant patients, the SNP molecular markers are shown in Table 10.

[0009] A probe for detecting SNP molecular markers in cfDNA from pregnant organ transplant patients, the probe being designed based on the aforementioned SNP molecular markers.

[0010] Optionally, each SNP molecular marker's probe includes an upstream probe and a downstream probe. The upstream probe is designed with the base sequence extending 80 bp from the SNP site towards the 5' end as the sequence information, and the downstream probe is designed with the base sequence extending 80 bp from the SNP site towards the 3' end as the sequence information.

[0011] A kit or detection chip for detecting cfDNA from donors in pregnant organ transplant patients, comprising a probe for detecting SNP molecular markers of cfDNA from donors in pregnant organ transplant patients.

[0012] A method for detecting cfDNA in pregnant organ transplant patients includes the following steps:

[0013] S1. Obtain the cfDNA of the sample to be tested and the genomic DNA of the biological father of the fetus;

[0014] S2. Library construction, obtaining cfDNA library and genomic DNA library;

[0015] S3. Using the probe described above for detecting SNP molecular markers of cfDNA from pregnant organ transplant patients, the constructed library is used to capture the target region, amplify and enrich the target region sequence, and then sequence it.

[0016] S4. Extract the SNP molecular marker site information from the sequencing data of the cfDNA library and the genomic DNA library respectively, perform genotyping, and screen SNP sites with the same genotype and homozygous in the cfDNA library sequencing data and the genomic DNA library sequencing data as valid SNP sites.

[0017] S5. Detect the genotype at the effective SNP sites in the cfDNA library. Divide the signal that is inconsistent with the genotype of the effective SNP sites in step S4 by the total sequencing signal at the effective SNP sites. The ratio obtained is the concentration of donor-derived cfDNA.

[0018] S6. Correct the concentration of donor-derived cfDNA to ddcfDNA. The correction calculation formula is as follows:

[0019] adj_ddcfDNA=ddcfDNA / (ddcfDNA+(1-ddcfDNA)*(1-ffcfDNA));

[0020] The ffcfDNA in the formula is obtained from Table 11;

[0021] Substitute the ddcfDNA obtained in step S5 into the correction calculation formula.

[0022] Optionally, the sample to be tested is a blood sample;

[0023] Optional, peripheral blood sample.

[0024] A device for detecting cfDNA in pregnant organ transplant patients, comprising:

[0025] Sample acquisition unit: used to acquire the cfDNA of the sample to be tested and the genomic DNA of the biological father of the fetus.

[0026] Library construction unit: used to construct libraries from the cfDNA of the sample to be tested and the genomic DNA of the biological father of the fetus;

[0027] Target region capture and sequencing unit: The constructed library is targeted by using the probes described above to detect SNP molecular markers of cfDNA from pregnant organ transplant patients, the target region sequences are amplified and enriched, and then sequenced.

[0028] Effective SNP site extraction unit: used to extract the SNP molecular marker site information from the sequencing data of cfDNA library and genomic DNA library, perform genotyping, and screen SNP sites with the same genotype and homozygous in the cfDNA library sequencing data and genomic DNA library sequencing data as effective SNP sites.

[0029] The donor-derived cfDNA concentration ddcfDNA calculation unit is used to detect the genotype at the effective SNP sites in the cfDNA library. The signal that is inconsistent between the genotype of the effective SNP site and the genotype of the effective SNP site in the effective SNP site extraction unit is divided by the total sequencing signal at the effective SNP site. The ratio obtained is the donor-derived cfDNA concentration ddcfDNA.

[0030] Correction Unit: Corrects the concentration of donor-derived cfDNA (ddcfDNA). The correction calculation formula is as follows:

[0031] adj_ddcfDNA=ddcfDNA / (ddcfDNA+(1-ddcfDNA)*(1-ffcfDNA));

[0032] The ffcfDNA in the formula is obtained from Table 11;

[0033] Substitute the ddcfDNA obtained from the donor-sourced cfDNA concentration ddcfDNA calculation unit into the correction calculation formula to obtain the corrected donor-sourced cfDNA concentration adj_ddcfDNA.

[0034] Optional, including:

[0035] The sample to be tested is a blood sample;

[0036] Optional, peripheral blood sample.

[0037] The technical solution of this invention has the following advantages:

[0038] 1. This invention provides a SNP molecular marker for detecting cfDNA from donors in pregnant organ transplant patients, as shown in Table 10. This invention uses cell-free DNA from the peripheral blood of the pregnant patient and genomic DNA from the peripheral blood of the biological father of the fetus as the detection targets. It employs liquid-phase hybridization capture and high-throughput sequencing technology, using thousands of the aforementioned SNP molecular markers. By analyzing the genotypes of both parents, the genotype of the fetus is obtained, eliminating the influence of fetal-derived cfDNA on the content of cfDNA from the transplanted organ, accurately determining the concentration of cfDNA from the transplanted organ, and exhibiting high detection sensitivity. Furthermore, multiple tests can be performed using only the patient's peripheral blood, achieving the purpose of long-term dynamic monitoring.

[0039] 2. This invention provides a method for detecting cfDNA from donors in pregnant organ transplant patients. It uses cell-free DNA from the peripheral blood of the pregnant patient and genomic DNA from the peripheral blood of the biological father of the fetus as the detection targets. Using thousands of SNP molecular markers, the genotype of the fetus is obtained through the genotypes of both parents, eliminating the influence of fetal-derived cfDNA on the content of cfDNA from the transplanted organ. The concentration of cfDNA from the transplanted organ is calculated using a correction formula. The method has high detection sensitivity and accuracy. Detailed Implementation

[0040] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0041] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0042] Example 1

[0043] 1. Acquisition of cfDNA and genomic DNA

[0044] 1.1 Sample collection and plasma separation

[0045] (1) Sample collection: Approximately 8 ml of peripheral blood was drawn from the patient and the biological father of the fetus, and stored and transported in a special cfDNA blood collection tube.

[0046] (2) Plasma separation: Obtain fresh whole blood sample, 1600g, centrifuge at 4℃ for 10min, separate the supernatant into a new centrifuge tube, continue to centrifuge the supernatant at 16000g, 4℃ for 10min, transfer the supernatant after centrifugation to a new centrifuge tube to obtain the separated plasma, which can be stored at -20℃;

[0047] (3) Blood cell separation: The lower sediment after centrifugation of whole blood sample is blood cells.

[0048] 1.2. Genomic DNA was obtained from the separated blood cells using the Laifeng Genomic DNA Extraction Kit (DK603); cfDNA was obtained from the separated plasma using the Qiagen Cell-Free DNA Extraction Kit (DK607).

[0049] 2. Library Construction

[0050] 2.1 Take 1 μl of cfDNA and genomic DNA respectively for Qubit3.0 quantification.

[0051] 2.2 Using genomic DNA and cfDNA as samples, prepare genomic DNA libraries and cfDNA libraries respectively using the KAPA LTP Library Preparation Kit. The specific steps are as follows:

[0052] (1) End leveling

[0053] a. Add the end-filling mixture shown in Table 1 to the marked centrifuge tubes and mix thoroughly by pipetting.

[0054] Table 1 End-Filling Mixture

[0055] Reagent Name volume water 8μl KAPA End Repair Buffer (10X) 7μl KAPA End Repair Enzyme Mix 5μl Total volume 20μl

[0056] b. Take 50 μl of the quantified sample from step 2.1, add 20 μl of the end-filling mixture shown in Table 1 to obtain a total volume of 70 μl of sample reaction solution, and mix it by pipetting.

[0057] c. Run the following program on the PCR instrument:

[0058] Keep at 20℃ for 30 minutes.

[0059] Let stand at 10℃.

[0060] (2) Purification after compensation

[0061] Resuspend (purified with Agencourt AMpure XP reagent), and incubate the magnetic beads at room temperature for 30 minutes, as follows:

[0062] a. Add 120 μL of magnetic beads and the sample reaction solution after end leveling in step (1) to the sample tube, mix thoroughly by blowing and stirring, and let stand at room temperature for 5 min;

[0063] b. Place the sample tube on the magnetic rack and wait for the supernatant to become clear before discarding it;

[0064] c. Keep the sample tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to clean the magnetic beads, and discard the supernatant;

[0065] d. Keep the sample tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to clean the magnetic beads, and discard the supernatant;

[0066] e. Centrifuge the sample tube briefly, place it on a magnetic rack to remove residual ethanol, and let it air dry at room temperature until there is no bright reflection on the surface of the magnetic beads. Then, remove the sample tube from the magnetic rack and add 42ul of Nuclease-Free water to resuspend the magnetic beads.

[0067] (3) Add A tail

[0068] a. Calculate the required amount of reagents based on the number of samples, add the mixture shown in Table 2 to the marked centrifuge tubes, and mix well by pipetting.

[0069] Table 2 Mixture with A-tail Addition

[0070] Reagent Name 1 sample 8 samples 48 samples KAPA A-Tailing Buffer (10X) 5μl 40μl 240μl KAPA A-Tailing Enzyme 3μl 24μl 144μl Total volume 8μl 64μl 384μl

[0071] b. Take 42 μl of the purified sample from step (2), add 8 μl of the mixture shown in Table 2, for a total volume of 50 μl, and mix thoroughly by pipetting.

[0072] c. Run the following program on the PCR instrument:

[0073] Keep at 30℃ for 30 minutes.

[0074] Let stand at 10℃.

[0075] (4) Purification after adding A tail

[0076] a. Add 90 μl of KAPA PEG / NaCl SPRI Solution and the sample reaction solution with A tail added in step (3) to the sample tube, mix thoroughly by pipetting, and let stand at room temperature for 5 min;

[0077] b. Place the sample tube on the magnetic rack and wait for the supernatant to become clear before discarding it;

[0078] c. Keep the sample tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to clean the magnetic beads, and discard the supernatant;

[0079] d. Keep the sample tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol, incubate at room temperature for at least 30 seconds, rotate the centrifuge tube to clean the magnetic beads, and discard the supernatant;

[0080] e. Centrifuge the sample tube briefly, place it on a magnetic rack to remove residual ethanol, and let it air dry at room temperature until there is no bright reflection on the surface of the magnetic beads. Then, remove the sample tube from the magnetic rack and add 32ul of Nuclease-Free water to resuspend the magnetic beads.

[0081] (5) Add monomolecular marker linkers

[0082] a. Calculate the required reagent volume based on the number of samples, and add the adapter connection mixture shown in Table 3 below and the purified sample (Beads with DNA) obtained in step (4) to the labeled centrifuge tubes. Mix well by pipetting.

[0083] Table 3 Connector Connection Mixture

[0084] Reagent Name 1 sample Single-molecule labeled adapters (synthesized by Anhui General Biotechnology) 5μl 5X KAPA Ligation Buffer 10μl KAPA T4 DNA Ligase 5μl Beads with DNA 30μl Total volume 18μl

[0085] c. Connection: Keep at 20℃ for 15 minutes.

[0086] (6) Purification of magnetic beads after adding a connector

[0087] (7) Library expansion

[0088] a. Thaw 2x KAPA HiFi HotStart ReadyMix at room temperature, add the library amplification system shown in Table 4 to the labeled centrifuge tubes, and mix well by pipetting.

[0089] Table 4 Library amplification system

[0090]

[0091]

[0092] b. Take 23 μl of the purified sample from step (6) and add it to 27 μl of the PCR amplification system shown in Table 4. The total volume is 50 μl. Mix gently with a pipette, centrifuge briefly for 2 seconds, and run the following program on the PCR instrument: denaturation at 98℃ for 45 seconds, 8 cycles (denaturation at 98℃ for 45 seconds, annealing at 65℃ for 30 seconds, extension at 72℃ for 30 seconds), extension at 72℃ for 1 minute, and cool and stand at 4℃.

[0093] (8) Document Authentication

[0094] Take 2 μl of the PCR product obtained in step (7) and perform electrophoresis on a 2% agarose gel to determine that the fragment is distributed between 250-500 bp.

[0095] (9) Library purification

[0096] a. Add 50 μl of Agencourt AMpure XP reagent to a sample tube containing the amplified library sample from step (7). Mix thoroughly by pipetting and let stand at room temperature for 5 min.

[0097] b. Centrifuge the sample tube briefly for 2 seconds, place it on a magnetic rack for 5 minutes, and discard the supernatant.

[0098] c. Keep the sample tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol, quickly rotate the sample tube to clean the magnetic beads, and discard the supernatant.

[0099] d. Briefly centrifuge the sample tube, place it on a magnetic rack to remove residual ethanol, and air dry at room temperature until the surface of the magnetic beads no longer has a bright reflection. Add 22 μl of Nuclease-free water and mix well by pipetting. Let it stand at room temperature for 2 min, then place it on a magnetic rack again and transfer 20 μl of the supernatant to a new centrifuge tube.

[0100] e. Take a 1μl sample and use QuantiFluor TM -ST (Promega) provides precise quantification, yielding a sample library. The sample library can then be stored at -20°C or used for further hybridization capture.

[0101] 3. Hybridization capture and sequencing of target regions in the library

[0102] 3.1. According to the concentration of the purified library sample in step 2, take a total of 500 ng of sample into a new 1.5 ml centrifuge tube, add the reagents shown in Table 5, and concentrate in a concentrator until completely dry. If you are not going to perform the next experiment immediately, you can leave it at room temperature (15-25℃) overnight.

[0103] Table 5 Concentration System

[0104]

[0105]

[0106] 3.2 Preparation of hybridization systems and denaturation of libraries

[0107] (1) Dissolve xGen 2X Hybridization Buffer at room temperature, prepare the hybridization mixture shown in Table 6 according to the number of library samples, and mix well by pipetting.

[0108] Table 6 Hybrid Mixture

[0109] Reagent Name 1 reaction volume xGen 2X Hybridization Buffer 8.5μL xGen Hybridization Buffer Enhancer 2.7μL Nuclease-Free Water 1.8μL Total volume 13μL

[0110] (2) Add 13 μl of the hybridization mixture shown in Table 6 to each tube of sample after concentration in step 1, and let stand at room temperature for 5 min;

[0111] (3) Mix the sample by pipetting and transfer it to a low-bind 0.2ml PCR tube. Place the sample in the PCR instrument and run the following program:

[0112] Maintain at 95℃ for 10 minutes.

[0113] Let stand at 65℃;

[0114] (4) When the 95℃ run is finished, keep the sample on the PCR instrument and immediately add 4μl of xGen Lockdown Probepool (IDT (Integrated DNA Technologies, Inc.)). Mix well with a pipette to avoid generating bubbles. The total reaction volume at this time is 17μl.

[0115] (5) Record the start time of hybridization. Depending on the progress of the experiment, choose a hybridization time of 4h or 16h.

[0116] 3.3 Preparation of washing buffer solution

[0117] (1) Prepare 1X working solution by mixing xGen 2X Bead Wash Buffer, xGen 10X Wash Buffer I, xGen 10X Wash Buffer II, xGen 10X Wash Buffer III and xGen 10X Stringent Wash Buffer according to Table 7;

[0118] Table 7 Working Fluid System

[0119]

[0120] (2) Prepare diluted 1×Wash Buffer I and 1×Stringent Wash Buffer, store them according to the conditions in Table 8, and store other reagents at room temperature (ensure that the incubation time at 65℃ is not less than 2h);

[0121] Table 8 Storage Conditions

[0122]

[0123] (3) Prepare M-270 magnetic beads

[0124] Take the M-270 magnetic beads out of the 4℃ refrigerator, confirm that the magnetic beads have been placed at room temperature for 30 minutes, and vortex mix them to resuspend the magnetic beads.

[0125] ① Prepare 100 μl of magnetic beads for each sample in a 1.7 ml low-bind tube;

[0126] ② Place the low-bind tube on a magnetic rack and let it stand until the liquid inside the tube is clear, then discard the supernatant;

[0127] ③ Add 200 μl of 1X Bead Wash Buffer, vortex for 10 seconds, let stand on a magnetic rack until the liquid in the tube is clear, and discard the supernatant;

[0128] ④ Repeat step ③ once;

[0129] ⑤ Add 100 μl of 1X Bead Wash Buffer and mix by pipetting.

[0130] ⑥ Transfer 100 μl of suspended magnetic beads to a new 0.2 ml low-bind tube, place it on a magnetic rack, and let it stand until the liquid in the tube is clear. Discard the supernatant.

[0131] 3.4. Capture

[0132] (1) Confirm that the hybridization reaction in step 3.2 has been completed for 4 hours. Keep the sample and magnetic beads on the PCR instrument, transfer the sample to the prepared magnetic bead tube, and mix by pipetting to avoid generating air bubbles.

[0133] (2) Incubate at 65℃ for 45 min, and mix the magnetic beads every 12 min during this period (keep the sample on the PCR instrument) to avoid generating bubbles.

[0134] 3.5 Washing

[0135] Note that the following steps need to be performed quickly at 65℃:

[0136] (1) Add 100 μl of 1X Wash Buffer I (preheated at 65℃) to each sample and mix quickly;

[0137] (2) Transfer the sample to a new 1.7 ml tube (preheated to 65°C) and mix quickly;

[0138] (3) Place the sample tube on the magnetic rack and let it stand until the liquid in the tube is clear, then discard the supernatant.

[0139] (4) Add 200 μl of 1X Stringent Wash Buffer (preheated at 65°C), mix gently, and incubate in a water bath at 65°C for 5 min. Place the sample tube on a magnetic rack and let it stand until the liquid in the tube becomes clear. Discard the supernatant.

[0140] (5) Repeat step (4) once;

[0141] (6) Add 200 μl of 1X Wash Buffer I at room temperature and vortex for 2 min. Place the sample tube on a magnetic rack and let it stand until the liquid in the tube is clear. Discard the supernatant.

[0142] (7) Add 100 μl of 1X Wash Buffer II at room temperature and vortex for 1 min. Place the sample tube on a magnetic rack and let it stand until the liquid in the tube is clear. Discard the supernatant.

[0143] (8) Add 200 μl of 1X Wash Buffer Ⅲ at room temperature, vortex mix for 30 seconds, place the sample tube on a magnetic rack, let stand until the liquid in the tube is clear, and discard the supernatant.

[0144] (9) Remove the sample tube from the magnetic rack, add 20 μl of Nuclease-Free Water, pipette and mix well, ensuring that all magnetic beads are in suspension and transfer them all to a 0.2 ml PCR tube.

[0145] 3.6 PCR enrichment

[0146] (1) Thaw 2X KAPA HiFi HotStart ReadyMix at room temperature, prepare the PCR amplification system shown in Table 9, and mix quickly:

[0147] Table 9 PCR Amplification System

[0148] Reagent Name Genome library 2X KAPA HiFi HotStart ReadyMix 25μL 10uM P5 / P7 Primer 2μL Total volume 27μL

[0149] (2) Add 27 μL of PCR mix to each sample (23 μL), for a total volume of 50 μL. Mix gently with a pipette, centrifuge briefly for 2 seconds, and run the following program on the PCR instrument: denaturation at 98℃ for 45 seconds, 12 cycles (denaturation at 98℃ for 15 seconds, annealing at 60℃ for 30 seconds, extension at 72℃ for 30 seconds), extension at 72℃ for 1 minute, and cool and stand at 4℃.

[0150] 3.7 Purification

[0151] Resuspend the Agencourt AMpure XP reagent, confirm that the magnetic beads have been incubated at room temperature for 30 min, purify each sample with 70 μl of beads, and elute with 20 μl.

[0152] 3.8 Quantitative Library Analysis

[0153] a. Take 1 μl of the purified sample from step 3.7 and quantify it using a Qubit Fluorometer 3.0;

[0154] b. Take 2 μl of PCR product for 2% agarose gel electrophoresis, and store the remaining sample at -20℃.

[0155] 3.9. Submission for testing

[0156] The sample data volume was 10M Reads, and the insert fragment was 178 (the sequencing platform was Illumine X-Ten or other high-throughput sequencing platforms).

[0157] 4. Quantitative analysis of donor-derived cfDNA (ddcfDNA)

[0158] 4.1. After high-throughput sequencing of the fetal biological father's genomic DNA library and the patient's cfDNA library, callSNP (samtool) to extract 5754 SNP loci information, as shown in Table 10 below;

[0159] Table 10. SNP sites

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] 4.2 Genotyping was performed on the 5754 SNP sites listed in Table 10 above in the patient's cfDNA library and the fetal biological father's genomic DNA library. Genotyping can be obtained when the sequencing depth is 50X. SNP sites that are homozygous in both the pregnant woman and the fetal biological father and have the same genotype were screened out as valid SNP sites.

[0185] 4.3. Analyze the patient's cfDNA library based on the effective SNP sites in step 4.2, and detect the proportion of signals at the effective sites of cfDNA sequencing results that are inconsistent with the effective SNP site typing in step 4.2 to the total sequencing signal of cfDNA at the effective sites, that is, obtain the concentration of donor-derived cfDNA (ddcfDNA).

[0186] 4.4. Based on the calculation in step 4.3, the ddcfDNA concentration = donor-derived cfDNA / total sample cfDNA, where total sample cfDNA includes donor-derived cfDNA, patient-derived cfDNA, and fetal-derived cfDNA. Therefore, the ddcfDNA concentration needs to be corrected to eliminate the influence of fetal-derived cfDNA. The corrected ddcfDNA concentration (adj_ddcfDNA) is calculated using the following formula:

[0187] Formula 1: adj_ddcfDNA = Total donor-sourced cfDNA / (Total donor-sourced cfDNA + Total maternal-sourced cfDNA).

[0188] The calibration model is as follows:

[0189] In the blood of pregnant women undergoing organ transplantation, let the total amount of cell-free DNA (cfDNA) from the mother, fetus, and donor be N. The concentration of heterologous cfDNA calculated in step 4.3 is the uncorrected donor cfDNA concentration (ddcfDNA). Using data published in "Analysis of cell-free fetal DNA in 16,843 pregnant women from a single center in China using a targeted sequencing approach" (see Table 11 below), the concentration of fetal free DNA (ffcfDNA) at the corresponding gestational week can be obtained. The total amount of maternal cfDNA can then be calculated using Formula 2, as follows:

[0190] Formula 2: Total maternal cfDNA = (1 - ddcfDNA) * (1 - ffcfDNA) * N;

[0191] Formula 3: Total cfDNA from donors = ddcfDNA * N;

[0192] Substituting Equations 2 and 3 into Equation 1, we obtain the corrected ddcfDNA concentration (adj_ddcfDNA):

[0193] That is, adj_ddcfDNA=ddcfDNA / (ddcfDNA+(1-ddcfDNA)*(1-ffcfDNA)).

[0194] Table 11. Concentration of fetal cell-free DNA (ffcfDNA) at corresponding gestational weeks

[0195] Pregnancy week ffcfDNA (%) Pregnancy week ffcfDNA (%) 1 0.45％ 21 9.71％ 2 0.78％ 22 10.29％ 3 0.89％ 23 10.69％ 4 1.64％ 24 11.75％ 5 1.75％ 25 12.51％ 6 2.55％ 26 13.16％ 7 4.32％ 27 14.39％ 8 6.33％ 28 15.48％ 9 7.72％ 29 16.19％ 10 8.57％ 30 18.23％ 11 8.91％ 31 18.01％ 12 8.64％ 32 19.56％ 13 9.30％ 33 21.61％ 14 9.07％ 34 22.22％ 15 8.54％ 35 21.44％ 16 8.69％ 36 21.69％ 17 9.23％ 37 22.59％ 18 8.89％ 38 33.77％ 19 8.99％ 39 35.91％ 20 9.35％ 40 38.34％

[0196] Experimental example: Validation of cfDNA (ddcfDNA) concentration from pregnant women as donors

[0197] Blood samples from three pregnant women who underwent kidney transplantation and had known gestational age and pathological findings are shown in the table below. Blood samples from these individuals were processed according to Example 1, and the results are shown in the table below.

[0198] Table 12

[0199]

[0200] Note: 1. "+" indicates that the pathological diagnosis shows a rejection reaction, and "-" indicates that the pathological diagnosis shows no rejection reaction.

[0201] 2. A ddcfDNA concentration >1% indicates a rejection reaction.

[0202] The results above show that, in the blood samples of three pregnant women undergoing kidney transplantation in Examples 1-4 of this invention, the consistency rate between the corrected ddcfDNA concentration and the pathological diagnosis was 100%, while the consistency rate between the uncorrected ddcfDNA concentration and the pathological diagnosis was 33.3%. This indicates that the method of this invention can effectively improve the accuracy of ddcfDNA detection in assessing the risk of rejection in pregnant women undergoing organ transplantation.

[0203] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An apparatus for detecting donor cfDNA in a pregnant organ transplant patient, comprising: The method comprises the following steps: a sample acquisition unit for acquiring cfDNA of a sample to be tested and genomic DNA of a fetal biological father; a library construction unit for constructing a library of the cfDNA of the sample to be tested and the genomic DNA of the fetal biological father; a target region capture and sequencing unit for performing target region capture on the constructed library using probes for detecting SNP molecular markers of donor cfDNA of a patient with a transplanted organ, amplifying and enriching sequences of a target region, and sequencing; the probes are designed based on the SNP molecular markers shown in Table 10; an effective SNP site extraction unit for extracting information of the SNP molecular marker sites in sequencing data of the cfDNA library and the genomic DNA library, performing genotyping, and screening SNP sites with the same genotype and being homozygous in both the cfDNA library sequencing data and the genomic DNA library sequencing data as effective SNP sites; a donor-derived cfDNA concentration ddcfDNA calculation unit for detecting genotyping at the effective SNP sites in the cfDNA library, dividing signals of the effective SNP sites with different genotypes from the effective SNP site genotyping in the effective SNP site extraction unit by total sequencing signals at the effective SNP sites, and obtaining a ratio as the donor-derived cfDNA concentration ddcfDNA; a correction unit for correcting the donor-derived cfDNA concentration ddcfDNA, and a correction calculation formula is as follows: adj_ddcfDNA = ddcfDNA / (ddcfDNA + (1-ddcfDNA)*(1-ffcfDNA)); ffcfDNA is obtained through Table 11 in the formula; ddcfDNA obtained in the donor-derived cfDNA concentration ddcfDNA calculation unit is substituted into the correction calculation formula to obtain the corrected donor-derived cfDNA concentration adj_ddcfDNA; the sample to be tested is a peripheral blood sample.

2. The device for detecting donor cfDNA in a pregnant organ transplant patient of claim 1, wherein, The probe of each SNP molecular marker comprises an upstream probe and a downstream probe, the upstream probe is designed with a base sequence of 80 bp extending from the SNP site to the 5' end as sequence information, and the downstream probe is designed with a base sequence of 80 bp extending from the SNP site to the 3' end as sequence information.

Images