Molecular marker and kit for monitoring pig xenograft rejection

Abstract

The invention relates to a molecular marker and a kit for monitoring pig xenograft rejection, and belongs to the technical field of molecular biology. The invention provides a molecular marker for monitoring transplant rejection. The molecular marker comprises at least one of an IL-15L gene, a CBR2 gene, an MYH2 gene or an MBL1 gene. Researches show that specific amplification of corresponding sites of pig ddcfDNA can be completed by taking at least one of IL-15L gene, CBR2 gene, MYH2 gene or MBL1 gene as the molecular marker, and the positive detection condition in a ddPCR detection method is consistent with the detection result of a specific antibody. Therefore, the molecular marker for monitoring the xenogenic organ transplant rejection reaction by taking the pig as the donor provided by the invention has an extremely high correlation degree with the transplant rejection reaction, and can be used for monitoring the xenogenic organ transplant rejection reaction by taking the pig as the donor.

Description

Technical Field

[0001] The invention relates to a molecular marker and a kit for monitoring pig xenotransplant rejection reaction, belonging to the technical field of molecular biology. Background Art

[0002] Transplant rejection refers to a series of immune responses caused by the interaction between the recipient's immune system and the transplant due to the different genetic backgrounds of the recipient and the transplant after the transplant surgery. It is the most important factor affecting the survival and function of the transplant. Transplant rejection is mainly manifested as the gradual decline or even complete loss of graft function, which can endanger the life of the recipient in severe cases. Most transplant recipients will experience varying degrees of rejection at different times after surgery, and generally require lifelong immunosuppressant therapy for prevention and treatment. At present, the main method for assessing the risk of transplant rejection is to perform specific antibody testing on organ transplant recipients through live puncture. However, as an invasive test, live puncture will cause trauma risks during the test process.

[0003] Donor-derived cell-free DNA (ddcfDNA) refers to free DNA from apoptotic or necrotic donor cells in the recipient's circulating body fluids after organ transplantation, which carries the cell information of the donor tissue. As a transplantation liquid biopsy technology, dd-cfDNA technology can predict the damage of the transplant and evaluate the transplant rejection of the recipient by detecting the content of ddcfDNA in the recipient's blood and urine. Compared with biopsy, dd-cfDNA technology has the advantages of non-invasive, safe, and real-time monitoring. However, at this stage, dd-cfDNA quantification technology needs to first distinguish donor- and recipient-derived cfDNA through differences in human genome SNP / indel sites, and then quantify the relative content of ddcfDNA through the relative ratio of donor gene reads to recipient gene reads, thereby calculating the dd-cfDNA content. Since the ddcfDNA quantification of the second-generation sequencing method is limited to the relative quantification of the allogeneic transplant genome, this method can only complete the monitoring of allogeneic transplant rejection, but cannot achieve transplant rejection monitoring in xenotransplantation. The current main idea for quantifying xenogeneic ddcfDNA is to directly quantify the ddcfDNA copy number based on the non-homologous regional differences between the human and donor species genomes, and then determine the ddcfDNA content by the proportion of the ddcfDNA copy number in the total cfDNA copy number. This ddcfDNA quantification method has the advantages of accurate, rapid, and low-cost detection. However, it lacks the screening of effective non-homologous regional targets and the establishment of systematic detection methods. It is urgent to screen effective non-homologous regional targets and establish dd-cfDNA technology that can detect transplant rejection reactions in xenogeneic organ transplantation. Summary of the invention

[0004] To solve the above problems, the present invention provides a molecular marker for monitoring transplant rejection reaction, wherein the molecular marker comprises at least one of IL-15L gene, CBR2 gene, MBL1 gene or MYH2 gene.

[0005] In one embodiment of the present invention, the molecular marker further comprises at least one of the BSP1 gene, the CYM gene, the PGA5 gene, the WAP gene, the VGF gene, the GH1 gene, the SYN1 gene or the SPRP gene.

[0006] In one embodiment of the present invention, the xenotransplant rejection reaction comprises a xenotransplant rejection reaction using pigs as donors.

[0007] The present invention also provides the use of a reagent for detecting the level of the above-mentioned molecular marker in a sample to be tested in the preparation of a product for monitoring transplant rejection.

[0008] In one embodiment of the present invention, the reagent comprises at least one of a primer set targeting IL-15L gene, a primer set targeting CBR2 gene, a primer set targeting MBL1 gene or a primer set targeting MYH2 gene.

[0009] In one embodiment of the present invention, the primer set targeting the IL-15L gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4; the primer set targeting the CBR2 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6; the primer set targeting the MBL1 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.25 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.26; the primer set targeting the MYH2 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.119 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.120.

[0010] In one embodiment of the present invention, the reagent further comprises at least one of a fluorescent probe targeting the IL-15L gene, a fluorescent probe targeting the CBR2 gene, a fluorescent probe targeting the MYH2 gene, or a fluorescent probe targeting the MBL1 gene.

[0011] In one embodiment of the present invention, the reagent also includes at least one of a primer set targeting the BSP1 gene, a primer set targeting the CYM gene, a primer set targeting the PGA5 gene, a primer set targeting the WAP gene, a primer set targeting the VGF gene, a primer set targeting the GH1 gene, a primer set targeting the SYN1 gene, or a primer set targeting the SPRP gene.

[0012] In one embodiment of the present invention, the reagent also includes at least one of a fluorescent probe targeting the BSP1 gene, a fluorescent probe targeting the CYM gene, a fluorescent probe targeting the PGA5 gene, a fluorescent probe targeting the WAP gene, a fluorescent probe targeting the VGF gene, a fluorescent probe targeting the GH1 gene, a fluorescent probe targeting the SYN1 gene, or a fluorescent probe targeting the SPRP gene.

[0013] In one embodiment of the present invention, the xenotransplant rejection reaction comprises a xenotransplant rejection reaction using pigs as donors.

[0014] The present invention also provides a monitoring kit for monitoring transplant rejection reaction, wherein the monitoring kit comprises a reagent for detecting the level of the above-mentioned molecular marker in the sample to be tested.

[0015] In one embodiment of the present invention, the reagent comprises at least one of a primer set targeting IL-15L gene, a primer set targeting CBR2 gene, a primer set targeting MBL1 gene or a primer set targeting MYH2 gene.

[0016] In one embodiment of the present invention, the primer set targeting the IL-15L gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4; the primer set targeting the CBR2 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6; the primer set targeting the MBL1 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.25 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.26; the primer set targeting the MYH2 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.119 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.120.

[0017] In one embodiment of the present invention, the reagent further comprises at least one of a fluorescent probe targeting the IL-15L gene, a fluorescent probe targeting the CBR2 gene, a fluorescent probe targeting the MYH2 gene, or a fluorescent probe targeting the MBL1 gene.

[0018] In one embodiment of the present invention, the reagent also includes at least one of a primer set targeting the BSP1 gene, a primer set targeting the CYM gene, a primer set targeting the PGA5 gene, a primer set targeting the WAP gene, a primer set targeting the VGF gene, a primer set targeting the GH1 gene, a primer set targeting the SYN1 gene, or a primer set targeting the SPRP gene.

[0019] In one embodiment of the present invention, the reagent also includes at least one of a fluorescent probe targeting the BSP1 gene, a fluorescent probe targeting the CYM gene, a fluorescent probe targeting the PGA5 gene, a fluorescent probe targeting the WAP gene, a fluorescent probe targeting the VGF gene, a fluorescent probe targeting the GH1 gene, a fluorescent probe targeting the SYN1 gene, or a fluorescent probe targeting the SPRP gene.

[0020] In one embodiment of the present invention, the xenotransplant rejection reaction comprises a xenotransplant rejection reaction using pigs as donors.

[0021] The present invention also provides a ddPCR detection method for detecting the ddcfDNA content in a sample to be tested. The ddPCR detection method is not a disease diagnosis and treatment method. The ddPCR detection method comprises: using the above-mentioned monitoring kit to detect the sample to be tested.

[0022] In one embodiment of the present invention, the ddPCR detection method includes: extracting cfDNA from the sample to be tested; and performing ddPCR detection on the extracted cfDNA using the above-mentioned monitoring kit.

[0023] In one embodiment of the present invention, the method for extracting cfDNA includes: using a free DNA extraction kit to extract cfDNA from a sample to be tested.

[0024] In one embodiment of the present invention, the cfDNA extraction method includes: checking the sample information with the extraction order information; adding 720 μL ACL to the bottom of each 5 mL centrifuge tube, adding 5 μL carrier RNA to the bottom of each centrifuge tube, aspirating 90 μL Proteinase K to the corresponding 5 mL centrifuge tube, and aspirating 900 μL blood to the corresponding marked 5 mL centrifuge tube according to the sample name; shaking the centrifuge tube vigorously for 10 seconds, centrifuging at 8000 rpm for 5 seconds, incubating at 58°C for 30 minutes, and shaking every 15 minutes during the incubation period; preparing a corresponding number of 1.5 mL centrifuge tubes and writing the complete extraction number, preparing a corresponding number of QIAamp Mini column filter columns, writing the number, installing the adapter on the VacConnector, and The column is installed on the adapter joint; after the incubation is completed, wipe the centrifuge tube dry, add 1000μL of isopropanol precooled to 4℃ in the order of the samples, and mix them by inversion 10 times; place the samples in a -20℃ refrigerator for 10 minutes; after the placement, take the samples out of the refrigerator, transfer them to the filter column accordingly, turn on the vacuum pump, and control the negative pressure at 50kPa for filtration; after all the filtration is completed, add 500μLAW1 to the filter column for elution, and be careful not to wet the edge of the adsorption column; after the elution is completed, add 500μLAW2 to the filter column for elution, and be careful not to wet the edge of the adsorption column; after the elution is completed, add 500μL of anhydrous ethanol to the filter column for elution, and be careful not to wet the edge of the adsorption column; after the elution is completed, turn off the vacuum pump, unplug the QIAamp Mini column and put it into the collection tube, and centrifuge it at 14000rpm for 3 minutes; Place the column in a prepared 1.5 mL centrifuge tube, open the lid, and dry at room temperature (25°C) for 3 min; add 50 μL AVE / H 2O, after being placed at room temperature (25°C) for 3 minutes, the DNA was eluted by centrifugation at 1200 rpm for 1 minute, and the cfDNA sample was extracted.

[0025] The technical solution of the present invention has the following advantages:

[0026] 1. The present invention provides a molecular marker for monitoring transplant rejection, wherein the molecular marker includes at least one of the IL-15L gene, CBR2 gene, MYH2 gene or MBL1 gene. Studies have shown that using at least one of the IL-15L gene, CBR2 gene, MYH2 gene or MBL1 gene as a molecular marker can complete the specific amplification of the corresponding site of pig ddcfDNA, and the positive detection in the ddPCR detection method is consistent with the specific antibody detection result. At the same time, studies have shown that the cut-off threshold of the monitoring method for monitoring allogeneic pig organ transplant rejection based on these molecular markers is 5% of the relative content of ddcfDNA. Therefore, the molecular marker for monitoring transplant rejection provided by the present invention has a very high correlation with the occurrence of transplant rejection and can be effectively used for risk assessment of xenotransplant rejection.

[0027] 2. The present invention also provides a ddPCR detection method for detecting the ddcfDNA content in a sample to be tested. The ddPCR detection method is not a disease diagnosis and treatment method. The ddPCR detection method comprises: using a monitoring kit to detect the sample to be tested; the monitoring kit comprises a reagent for detecting the level of molecular markers in the sample to be tested; the molecular markers comprise at least one of IL-15L gene, CBR2 gene, MYH2 gene or MBL1 gene. First, the study showed that using at least one of the IL-15L gene, CBR2 gene, MYH2 gene or MBL1 gene as a molecular marker, the specific amplification of the corresponding sites of pig ddcfDNA can be completed, and the positive detection situation in the ddPCR detection method (ddcfDNA relative content > 5%) is consistent with the specific antibody detection result. At the same time, the study showed that the cut-off threshold of the monitoring method for monitoring rejection of allogeneic pig organ transplantation established based on these molecular markers is 5% relative content of ddcfDNA. Therefore, the ddPCR detection method can reflect the rejection reaction of allogeneic pig organ transplantation in the human body by quantitatively calculating the ddcfDNA content of the donor pig, and according to the rejection reaction situation, the purpose of accurate, rapid, real-time, non-invasive and non-invasive detection of the risk of postoperative rejection in allogeneic pig transplant patients can be achieved. Secondly, the ddPCR detection method is based on digital droplet PCR (ddPCR), which can complete the absolute quantitative detection of ddcfDNA. Therefore, the ddPCR detection method can amplify the pig ddcfDNA-specific gene sequence through ddPCR without amplifying human cfDNA, thereby achieving absolute quantitative calculation of the ddcfDNA copy number of allogeneic pig transplant recipient samples, thereby making ddcfDNA quantitative detection more convenient and less costly.

[0028] Further, the reagent includes at least one of a primer set targeting IL-15L gene, a primer set targeting CBR2 gene, a primer set targeting MBL1 gene or a primer set targeting MYH2 gene; the primer set targeting IL-15L gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4; the primer set targeting CBR2 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6; the primer set targeting MBL1 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.25 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.26; the primer set targeting MYH2 gene includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.119 and an upstream primer having a nucleotide sequence as shown in SEQ ID NO.120. At present, in the design of cfDNA amplification primers, in order to consider the influence of the genomic nucleosome assembly map on the amplification efficiency, the quantitative amplification results of different genomes are greatly different when designing primers. The ddPCR detection method is based on the current porcine FPKM reference gene expression data and analyzes the template abundance of sequences at different sites of cfDNA, thereby improving the stability, uniformity and reliability of designed primers for ddPCR detection results.

[0029] Furthermore, the cfDNA extraction method of the ddPCR detection method includes: extracting cfDNA from the sample to be tested; using the above-mentioned monitoring kit to perform ddPCR detection on the extracted cfDNA; the cfDNA extraction method includes: using a free DNA extraction kit to extract cfDNA from the sample to be tested. The clinical sample acquisition amount for xenotransplantation patients is scarce, and the test samples are relatively precious. The ddPCR detection method establishes an efficient cfDNA extraction method with ultra-low sample volume. Compared with the current general cfDNA method using a sample volume (blood) of 1.8mL, the ddPCR detection method uses a cfDNA extraction method using a 300μL blood sample to obtain a cfDNA concentration that meets the detection requirements, significantly reducing the demand for the use of test samples, so that a single sample collection can meet the needs of multiple types of tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1 to 4 : Electropherogram of primer specificity verification for ddPCR amplification. Figures 1 to 4 In the figure, different lanes (1-20) represent the amplification conditions of the corresponding primer pairs (see Table 2).

[0031] Figure 5 : Amplification results of FPKM low-expression gene loci.

[0032] Figure 6 : Amplification results of FPKM high expression sites.

[0033] Figure 7 :ddPCR positive test results (ddPCR detection of donor cfDNA positive signal droplet number through pig-specific amplification sites IL-15L, CBR2, MYH2, and MBL1).

[0034] Figure 8 : Results of positive detection of donors by specific antibodies (logarithmic radial plot of the average intensity values ​​of two metal-labeled antibodies detected by mass spectrometry flow cytometry analysis; left, metal labeling is Cd113; right, metal labeling is Lu162).

[0035] Fig. 9 : The test results of quantitative detection of pig ddcfDNA by ddPCR and NGS methods.

[0036] Figures 10-12 :The results of quantitative detection of pig ddcfDNA by ddPCR method.

[0037] Fig.13 : Quantitative results of conventional cfDNA extraction method and cfDNA extraction method in Experimental Example 1.

[0038] Fig.14 :ddcfDNA quantitative detection clinical cut off value determines the results. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0040] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0041] Experimental Example 1: Acquisition of Molecular Markers

[0042] 1. Screening of molecular markers

[0043] The orthologous gene sequences of the pig (Sus scrofa) and human (GRCh38) reference genomes were obtained from the NCBI database. The orthologous non-homologous regions were aligned by Blast to obtain the highly specific genomic sequence regions of the donor pig (the identity of the target region was less than 5%) as target sites. A total of 131 target sites (i.e., molecular markers) were screened and obtained, as shown in Table 1.

[0044] 2. Primer design

[0045] Based on the highly specific genomic sequence regions (i.e., molecular markers) of the donor pigs screened in Table 1, a pig genome-specific amplification primer pair was designed.

[0046] 3. Primer screening

[0047] Extract the human genome and the pig genome and mix them at a molar ratio of human: pig = 9:1 to obtain a mixed amplification sample; use the pig genome-specific amplification primer pair designed in step 2 to perform PCR amplification on the mixed amplification sample through a PCR instrument (Bori PCR amplification instrument); after the amplification, verify the amplification specificity of the pig genome-specific amplification primer pair based on the migration of the amplified product's 2% (w / v, g / 100mL) agarose gel electrophoresis band. Screen the pig genome-specific amplification primer pair with clear, single bands, and fragment amplification size that meets the target amplification length.

[0048] The PCR reaction system (50 μL) consisted of 0.25 μL of EX Taq (Takara, 5 U / μL), 10×Ex Taq buffer (Mg 2+ ) 5 μL, dNTP mixture (each 2.5 mM) 4 μL, DNA template 1 μL (~50 ng), upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL and ultrapure water 37.75 μL.

[0049] Amplification results are shown in Figures 1 to 4 .from Figures 1 to 4 It can be seen that the primer pairs obtained by screening have clear and single bands in the amplification of the mixed amplification template, the band size is consistent with the designed amplification length, there is no non-specific amplification, and the primer pairs obtained by screening have good specificity and amplification efficiency.

[0050] 4. Optimization of primers for free DNA amplification

[0051] By analyzing the fragmentation pattern of pig cfDNA and the abundance of the target sites (i.e., molecular markers) screened in step 1, the amplification performance of the primers designed for different sites is ensured, thereby ensuring the stability and uniformity of the reference sites selected in PCR amplification.

[0052] With the help of the relative expression of FPKM pig reference genes, the high-abundance template sites of cfDNA were screened. Table 3 shows that the nucleosome assembly sites are positively correlated with the relative expression of the gene. The gene sequence was amplified by screening the high-abundance sites of cfDNA by this method. The amplification results are shown in Figures 5-6 .

[0053] Figures 5-6 The results showed that the FPKM reference gene expression was negatively correlated with the template abundance (copy number concentration) of the site in cfDNA. Figure 5 ) and high expression sites ( Figure 6 ) was amplified, and the results showed that the low expression region had good amplification efficiency.

[0054] By screening the primer set for this cfDNA fragmentation pattern, the test results are shown in Table 4 compared with the random selection of amplification sites. The results in Table 4 show that the uniformity of the detection stability is significantly improved (coefficient of variation before screening CV = 0.129773038, after screening CV = 0.652026466). Therefore, low expression sites were selected for primer pair design, and the designed primer pairs are shown in Table 2, with an expression multiple lower than 0.5.

[0055] 5. Preliminary validation of molecular markers

[0056] 5.1 Extraction of cfDNA

[0057] Blood from patients with pig kidney transplantation was used as the test sample and a free DNA extraction kit ( Circulating Nucleic Acid, QIAGEN) was used to extract free DNA from the sample to be tested. The specific operation is as follows:

[0058] Check the sample information with the extraction sheet information; add 720 μL ACL to the bottom of each 5 mL centrifuge tube, add 5 μL carrier RNA to the bottom of each centrifuge tube, pipette 90 μL Proteinase K into the corresponding 5 mL centrifuge tube, and pipette 900 μL blood into the corresponding labeled 5 mL centrifuge tube according to the sample name; shake the centrifuge tube vigorously for 10 seconds, centrifuge at 8000 rpm for 5 seconds, incubate at 58°C for 30 minutes, and shake every 15 minutes during the incubation period; prepare the corresponding number of 1.5 mL centrifuge tubes and write the complete extraction number, prepare the corresponding number of QIAamp Mini column filter columns, write the number, install the adapter on the VacConnector, and install the QIAamp Mini The column was installed on the adapter; after the incubation, the centrifuge tube was wiped dry, and 1000 μL of isopropanol precooled to 4°C was added according to the order of the samples, and mixed by inversion 10 times; the samples were placed in a -20°C refrigerator for 10 minutes; after the placement, the samples were taken out of the refrigerator and transferred to the filter column, the vacuum pump was turned on, and the negative pressure was controlled to filter at 50 kPa (the centrifugal parameters during filtration were: 12000 rpm, 1 min); after all the filtration was completed, 500 μL of AW1 was added to the filter column for elution, and the injection Be careful not to wet the edge of the adsorption column (the centrifugal parameters during elution are: 8000rpm, 1min); after the elution is completed, add 500μL AW2 to the filter column for elution, and be careful not to wet the edge of the adsorption column (the centrifugal parameters during elution are: 14000rpm, 3min); after the elution is completed, add 500μL of anhydrous ethanol to the filter column for elution, and be careful not to wet the edge of the adsorption column (the centrifugal parameters during elution are: 14000rpm, 3min); after the elution is completed, turn off the vacuum pump, unplug the QIAamp Minicolumn and put it into the collection tube, and centrifuge it at 14000rpm for 3min; put the centrifuged QIAamp Mini column into the prepared 1.5mL centrifuge tube, open the lid, and dry it at room temperature (25℃) for 3min; add 50μL AVE / H 2 O, after being placed at room temperature (25°C) for 3 minutes, the DNA was eluted by centrifugation at 1200 rpm for 1 minute to extract the cfDNA sample; according to the instructions of the QubitdsDNA HS Assay Kit DNA concentration quantification kit, the concentration of the extracted cfDNA sample was determined using the Qubit 2.0 Fluorometer quantifier (Thermo Fisher).

[0059] 5.2. ddPCR absolute quantitative cfDNA copy number concentration calculation

[0060] The level of Cd4-specific donor antibodies in the serum of porcine kidney transplant patients was detected by flow cytometry (for specific antibody positive detection method, see the literature: Hou Jun et al., 2012, Application of C4d detection in acute rejection after renal transplantation), and the ddcfDNA content in the serum and plasma of porcine kidney transplant patients was detected by whole genome sequencing NGS (for the second-generation sequencing method, see the literature: K. Getchell, Translational Sciences, eGenesis, Cambridge, MA. Longitudinal Monitoring of Donor Cell-Free DNA in Porcine Kidney Xenotransplantations, eGenesis, 2023.), with specific antibody positive detection and second-generation sequencing (NGS) as controls, ddPCR detection was performed through pig-specific amplification sites (i.e., molecular markers) to obtain the number of positive signal droplets of donor cfDNA in the blood of porcine kidney transplant patients, thereby monitoring the xenotransplant rejection reaction of porcine kidney transplant patients. The specific steps of ddPCR are as follows:

[0061] Use the free DNA extracted from the sample to be tested to prepare the droplet digital PCR reaction solution; take the droplet generation card of the droplet digital PCR instrument (MicroDrop digital PCR system, including MicroDrop-100 digital PCR instrument, S100 sealing instrument) and place it in the card holder to fix it, add 20μL of reaction solution to the 8 holes in the middle row of the droplet generation card, use an 8-row 20μL pipette and a 20μL dropper, when adding samples, the gun tip is close to the bottom of one side of the hole, at an angle of 15° to the side wall, and the liquid is slowly pumped out. After pumping out a part, slowly raise the position of the pipette head and then pump out the remaining liquid. Do not press the pipette to exceed the first gear position to cause the introduction of bubbles; add 70μL of droplet generation oil to the 8 holes in the bottom row of the droplet generation card; gently and steadily place the above card holder in the droplet generation instrument, start generating droplets, pay attention to the status of the indicator light on the instrument, which is generally completed within 2 minutes; droplets are generated at the top of the droplet generation card In a row of wells, use an 8-channel pipette and a 200μL dropper to carefully and slowly aspirate, adjust the aspirated volume to 40μL, lay the card holder flat, put the gun tip in at a 45° angle to the well wall, touch the bottom of the well lightly, aspirate 40μL after 5 seconds, and then slowly inject it into the well at the corresponding position of the 96-well plate (5s), with the dropper close to the well wall and close to the bottom of the well. Pay attention to sealing the cover to prevent oil evaporation, and discard the used droplet generation card and rubber pad each time; after transferring the oil droplets into the 96-well plate, use a preheated (preheated to 175°C) heat sealer to seal the film (the bright side of the film faces up and the dark side faces down), and the operating procedure for sealing the film is: 180°C, 10s, generally there is no need to reverse the direction for secondary sealing; after sealing the film, perform a droplet digital PCR reaction within 30 minutes; perform an absolute quantitative result of the donor free DNA copy number based on the droplet digital PCR, and determine the total free DNA copy number based on the actual input amount of the free DNA template in the droplet digital PCR reaction;

[0062] The droplet digital PCR (ddPCR) reaction solution (20 μL) consisted of One-step ddPCR supermix 10 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, probe (10 μM) 0.5 μL, RNase Free ddH 2 O 3.5 μL and free DNA template 4 μL;

[0063] The amplification reaction conditions of the droplet digital PCR instrument are as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 sec, annealing at 60°C for 60 sec, for a total of 40 cycles; end the reaction at 98°C for 10 min; and perform droplet digital PCR reaction detection after the reaction ends.

[0064] The matching probe sequences used for detection are as follows:

[0065] Probe 1 (IL-15L): 5'-FAM-TAACCCACTGAGTGAG-VIC-3' (SEQ ID NO. 263);

[0066] Probe 2 (CBR2): 5'-FAM-GAGCCAGAGTGGTGGC-VIC-3' (SEQ ID NO. 264);

[0067] Probe 3 (MYH2): 5'-FAM-GAACGATGCACACAG-VIC-3'CHU (SEQ ID NO. 265);

[0068] Probe 4 (MBL1): 5'-FAM-ATCACATGGTAGAGA-VICCH-3' (SEQ ID NO. 266);

[0069] The free DNA content of pig donors was calculated according to the following formula:

[0070] Relative content of donor free DNA (%) = donor free DNA copy number (copies / μL) / total free DNA copy number (copies / μL).

[0071] The validation results of the four molecular markers IL-15L, CBR2, MYH2 and MBL1 for the positive detection of pig ddcfDNA in the tested samples are shown in Figure 7 Specific antibody test results are shown in Figure 8 The quantitative detection results of the next generation sequencing (NGS) and ddPCR methods are shown in Fig. 9 . Figures 7-8 The number of positive droplets detected showed similar results to that of specific antibodies. Fig. 9 In the study, the same samples were used to detect the ddcfDNA content by next-generation sequencing (NGS) and ddPCR, and the comparison results showed that there was no significant difference between the ddPCR quantitative results and the NGS quantitative results. The above shows that IL-15L, CBR2, MYH2 and MBL1 can be used as molecular markers to predict the rejection of xenotransplantation with pigs as donors (after ddPCR with molecular markers as targets for the samples to be tested, the percentage of ddcfDNA copies is calculated, and the higher the content, the stronger the rejection reaction).

[0072] In addition to the above four genes that can be used as ddPCR detection molecular markers, this experimental example also verified the actual effect of other molecular markers BSP1, CYM, PGA5, WAP, VGF, GH1, SYN1, SPRP, AKR1C1, NPY4R, FBP2, and ANXA8 in Table 1 for the ddPCR detection method. The verification results are as follows Figures 10-12and as shown in Table 5; the probes used for verification are as follows:

[0073] Probe 1 (BSP1): 5'-FAM-GGCAGATGTTGATCT-VIC-3' (SEQ ID NO. 267);

[0074] Probe 2 (CYM): 5'-FAM-AGTCCGGCTCCTCC-VIC-3' (SEQ ID NO. 268);

[0075] Probe 3 (PGA5): 5'-FAM-AAAGTCGCCGGGGCCT-VIC-3' (SEQ ID NO. 269);

[0076] Probe 4 (WAP): 5'-FAM-GGGCCAGGGCCAGACT-VIC-3' (SEQ ID NO. 270);

[0077] Probe 5 (VGF): 5'-FAM-GGGATTATCAGTCCT-VIC-3' (SEQ ID NO. 271);

[0078] Probe 6 (GH1): 5'-FAM-ACGAATTCACCCCTCT-VIC-3' (SEQ ID NO. 272);

[0079] Probe 7 (SYN1): 5'-FAM-GCAGTGGCGGTCCCA-VIC-3' (SEQ ID NO. 273);

[0080] Probe 8 (SPRP): 5'-FAM-GTACACACAGGGACAA-VIC-3' (SEQ ID NO. 274);

[0081] Probe 9 (AKR1C1): 5'-FAM-CATGGAATTCCTGTGT-VIC-3' (SEQ ID NO. 275);

[0082] Probe 10 (NPY4R): 5'-FAM-GCTGAAATTTCAGGAA-VIC-3' (SEQ ID NO. 276);

[0083] Probe 11 (FBP2): 5'-FAM-CCAGCTGCCACGTAT-VIC-3' (SEQ ID NO. 277);

[0084] Probe 12 (ANXA8): 5'-FAM-GTGGCTCAGTAGGAAC-VIC-3' (SEQ ID NO. 278).

[0085] from Figures 10-12 As shown in Table 5, the molecular markers BSP1, CYM, PGA5, WAP, VGF, GH1, SYN1 and SPRP have a stable number of positive droplets for the detection of pig ddcfDNA. In comparison, the molecular markers AKR1C1, NPY4R, FBP2 and ANXA8 have poor detection effects in ddPCR.

[0086] Table 1 Screening of high specificity sites in the pig reference genome

[0087]

[0088]

[0089]

[0090] Table 2 Primers designed for pig cfDNA loci

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Table 3 Relative expression folds of reference genes

[0100]

[0101]

[0102]

[0103] Table 4 Comparison of the results before and after screening based on the nucleosome map

[0104]

[0105] Table 5 ddPCR results of different molecular markers

[0106] Site name Number of positive droplets Negative droplet count Total droplet number Copy number (copies / μL) BSP1 492 32430 32669 201.5151 CYM 424 34670 34891 198.2412 PGA5 473 32709 32963 250.2142 WAP 373 30816 30991 180.2145 VGF 428 31876 32081 208.6322 GH1 327 31106 31273 178.3261 SYN1 268 36225 36371 150.2156 SPRP 285 32145 32296 160.1251 IL-15L 478 32430 32908 212.1252 CBR2 452 34670 35122 205.5642 MYH2 498 32709 33207 208.6587 MBL1 487 30816 31303 219.1287

[0107] Experimental Example 2: Effect of cfDNA extraction method on test results

[0108] The blood of 6 porcine kidney transplant patients was used as the test samples, and the conventional cfDNA extraction method (i.e., operating according to the instructions of the free DNA extraction kit) was used as a control. The conventional cfDNA extraction method was used to extract cfDNA from the conventional sample amount (900 μL) and the low sample amount (300 μL), respectively. The cfDNA extraction method in Experimental Example 1 was used to extract cfDNA from the low sample amount (300 μL). After the extraction, the cfDNA concentration was quantitatively extracted by qubit.

[0109] The quantitative results of the cfDNA extraction method in Experimental Example 1 are shown in Tables 5 to 6 and Fig.13 The quantitative results of the conventional cfDNA extraction method are shown in Tables 6 and Fig.13 From Table 5 and Fig.13 It can be seen that the ultra-low sample volume (300 μL) cfDNA extraction method used in Experimental Example 1 obtains a significantly higher concentration of cfDNA in a 300 μL sample volume than the general cfDNA extraction method, which can meet the cfDNA detection concentration requirements. As can be seen from Table 6, there is no significant difference between the ddcfDNA copy number quantification results of the samples extracted by the ultra-low cfDNA extraction method and the ddcfDNA copy number quantification results of the cfDNA samples extracted by the conventional method with a general dosage (900 μL).

[0110] Table 5 Quantification of blood free DNA extraction concentration

[0111]

[0112] Table 6 Quantitative results of ddcfDNA copy number in pig kidney transplantation blood samples

[0113]

[0114] Experimental Example 3: Validation of ddPCR Detection Method

[0115] Using the puncture test results (for the puncture test method, see the literature: CHEN CC, LIN WC, LEE CY, et al. Two-year protocol biopsy after kidney transplantation in clinically stable recipients-a retrospective study [J]. Transpl Int, 2021, 34 (1): 185-193.) as a control, the method of Experimental Example 1 was used to perform quantitative detection of ddcfDNA in the blood of 6 clinical samples (2 patients with no rejection reaction and 4 patients with rejection reaction). The comparison results of clinical rejection symptoms measured by the two are shown in Fig.14 .

[0116] from Fig.14 It can be seen that the ddcfDNA quantitative detection in Experimental Example 1 determined that the cutoff threshold of ddcfDNA content in clinical rejection reaction was 5% of the relative content of ddcfDNA. Based on this, the risk of xenotransplant rejection reaction can be evaluated by the ddcfDNA content.

[0117] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A molecular marker for monitoring transplant rejection, characterized in that: The molecular markers include at least one of IL-15L gene, CBR2 gene, MBL1 gene or MYH2 gene.

2. The molecular marker according to claim 1, characterized in that The molecular markers further include at least one of the BSP1 gene, the CYM gene, the PGA5 gene, the WAP gene, the VGF gene, the GH1 gene, the SYN1 gene or the SPRP gene.

3. The molecular marker according to claim 1 or 2, characterized in that The transplant rejection reaction includes the xenotransplant rejection reaction of xenogeneic organs using pigs as donors.

4. Use of a reagent for detecting the level of the molecular marker according to any one of claims 1 to 3 in a sample to be tested in the preparation of a product for monitoring transplant rejection.

5. The use according to claim 4, characterized in that The reagents include at least one of a primer set targeting the IL-15L gene, a primer set targeting the CBR2 gene, a primer set targeting the MBL1 gene, or a primer set targeting the MYH2 gene.

6. The use according to claim 4 or 5, characterized in that The reagents also include at least one of a primer set targeting the BSP1 gene, a primer set targeting the CYM gene, a primer set targeting the PGA5 gene, a primer set targeting the WAP gene, a primer set targeting the VGF gene, a primer set targeting the GH1 gene, a primer set targeting the SYN1 gene, or a primer set targeting the SPRP gene.

7. The use according to claim 5, characterized in that The primer set targeting the IL-15L gene includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and an upstream primer with a nucleotide sequence as shown in SEQ ID NO.4; the primer set targeting the CBR2 gene includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and an upstream primer with a nucleotide sequence as shown in SEQ ID NO.6; the primer set targeting the MBL1 gene includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.25 and an upstream primer with a nucleotide sequence as shown in SEQ ID NO.26; the primer set targeting the MYH2 gene includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.119 and an upstream primer with a nucleotide sequence as shown in SEQ ID NO.

120.

8. A monitoring kit for monitoring transplant rejection, characterized in that: The monitoring kit comprises a reagent for detecting the level of the molecular marker according to any one of claims 1 to 3 in a sample to be tested.

9. The monitoring kit according to claim 8, characterized in that The reagents include at least one of a primer set targeting the IL-15L gene, a primer set targeting the CBR2 gene, a primer set targeting the MBL1 gene, or a primer set targeting the MYH2 gene.

10. A ddPCR detection method for detecting the content of ddcfDNA in a sample to be tested, characterized in that: The ddPCR detection method is a non-disease diagnosis and treatment method, and the ddPCR detection method comprises: using the monitoring kit described in claim 8 or 9 to detect the sample to be tested.

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