WO2018082037A1 - Method for monitoring xenotransplantation immune rejection by using porcine specific free dna, and primers - Google Patents

Abstract

A method for monitoring xenotransplantation immune rejection by using porcine specific free DNA, and primers, wherein the method is used for monitoring xenotransplantation immune rejection in a recipient subject using pig as donor. The method includes: a recipient plasma DNA sample is subject to quantitative fluorescence PCR amplification by using primers having specific amplification for porcine specific free DNA, so as to quantify the amount of porcine specific free DNA in the plasma of the recipient. Quantifying porcine specific free DNA in plasma by using quantitative fluorescence PCR amplification has advantages such as non-invasiveness, high specificity, high sensitivity, simplicity and convenience, low detection cost and capability for continuous monitoring, thus having a broad prospect for market applications.

Description

Method and primer for monitoring xenograft immune rejection using porcine specific free DNA Technical field

The invention relates to the technical field of organ transplantation, in particular to the technical field of xenotransplantation, in particular to a method and a primer for monitoring xenograft immune rejection using pig-specific free DNA.

Background technique

Cell, tissue and organ transplantation are effective treatment strategies for treating major diseases such as organ failure and cancer. However, China and the world are facing a serious shortage of donors. Xenografts are currently one of the most effective strategies for addressing human donor shortages, saving a large number of patients who die from organ failure. At present, the genetically modified pig heart can be transplanted into the sputum for up to 945 days; transplanting the pig kidney into the sputum can survive for up to 10 months (TTS 2016 latest report data); transplanting porcine islets into diabetic patients can be maintained Blood sugar is stable for more than 804 days. The survival of xenogeneic organs or tissue transplants is expected to be further extended by multiple gene modifications. Nevertheless, immune rejection is still a difficult research problem in the field of xenotransplantation, so real-time monitoring of host immune rejection is particularly important.

The clinical diagnosis of rejection mainly depends on the patient's symptoms and laboratory test data (such as tissue biopsy, blood biochemistry, immunoassay) and other indicators, each with its own limitations. The patient's clinical symptoms are non-specific, subjective, and relatively lagging. Secondly, although tissue biopsy is the "gold standard" for judging immune rejection, it is invasive and has high detection costs. Blood biochemistry such as blood creatinine level, urinary albumin, etc. can reflect the impaired function of the kidney, but it has the disadvantage of low sensitivity. Immunological tests such as T/B cell counts, cellular inflammatory factors, etc. can reflect the host's immune status, but have the disadvantage of poor specificity. For example, the Clinical Trials in Organ Transplantation (CTOT)-05 study showed that immune-related biomarkers were not significantly associated with immunorejection gold standard-tissue biopsy results, including activated T cells, anti-human leukocyte antigen (HLA) class II, Anti-donor specific antibodies and the like. At present, the detection methods for xenograft immune rejection are particularly lacking, and most of them are transplanted to allogeneic transplants. law. Therefore, the development of biomarkers with high specificity, high sensitivity (early), non-invasiveness, rapidity and low cost for xenotransplantation and its detection methods have practical urgency and broad market application prospects.

There is a certain amount of free DNA in human blood, mainly derived from apoptosis and cell necrosis. Due to the immune stabilization and immune surveillance of the body, senescent cells, tumor cells, and allogeneic cells undergo apoptosis or necrosis under the action of immune effector cells and cytokines, releasing DNA fragments into the blood and becoming free DNA.

Cancer-related DNA fragments are present in the blood, and information such as cancer mutation sequences and mutant DNA content is captured by microarray and high-throughput sequencing to provide a personalized solution for early diagnosis and treatment of cancer. Recently, it was reported that kidney-injury was evaluated in a male-to-women kidney transplant using a sex-specific single-copy Y-chromosome SRY site in allogeneic organ transplantation, and it was found that this site can be used as a kidney injury. A fast, non-invasive biomarker, but the biomarker has a relatively limited range of applications. Using SNP differences between the donor genome and the receptor genome, quantification of donor-specific SNPs in plasma by gene chip and high-throughput sequencing can be used to determine whether a patient has rejection. However, this type of method has the disadvantages of high detection cost and long time consumption.

Summary of the invention

The invention provides a method and a primer for monitoring xenograft immune rejection by using pig-specific free DNA, which has the advantages of high specificity, high sensitivity, non-invasiveness, rapidity, low cost and convenient real-time monitoring.

According to a first aspect of the present invention, the present invention provides a primer for use in a method for monitoring pig-to-human or non-human primate xenograft immunological rejection using porcine-specific free DNA, the sequence of which is as follows:

Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7);

Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8).

According to a first aspect of the present invention, the present invention also provides a primer for use in a method for monitoring a mouse-to-mouse xenograft immune rejection using porcine-specific free DNA, the sequence of which is as follows:

Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21);

Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22).

According to a second aspect of the present invention, the present invention provides a method for monitoring xenograft immune rejection using porcine-specific free DNA for monitoring xenograft immunorejection in a recipient object of a pig as a donor, The method comprises performing quantitative PCR amplification on a receptor plasma DNA sample using primers specifically amplified for porcine-specific free DNA to quantify the amount of porcine-specific free DNA in the recipient plasma.

Further, the above method further comprises designing and screening the above primers, wherein the design of the primers comprises: obtaining a donor pig-specific genomic sequence by aligning the receptor genome with the donor pig genome; and capturing by high-throughput sequencing The sequence and abundance information of free DNA in the body and donor plasma, according to the fragmentation pattern of free DNA, avoid primer cross-fragment design, and obtain candidate primers according to the specificity, abundance and design principle of fluorescent quantitative PCR primers; screening of the above primers Including: verifying the specificity of the above candidate primers in the receptor and the donor DNA by PCR, and selecting a primer which specifically expands in the donor DNA without specific amplification in the recipient DNA.

Further, the sequences of the above primers are as follows:

Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7);

Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8).

Further, the sequences of the above primers are as follows:

Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21);

Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22).

Further, the above receptor object is a human, a non-human primate or all mammals except pigs. Among them, non-human primates include, but are not limited to, cynomolgus monkeys, baboons, orangutans, etc. All mammals include, but are not limited to, mice and the like.

According to a third aspect of the present invention, there is provided a kit for use in a method for monitoring xenograft immune rejection using porcine-specific free DNA, the kit comprising a primer, the primer being obtained by the following method: The genome of the genome and the donor pig genome, obtain the donor-specific genomic sequence; and capture the sequence and abundance information of the free DNA in the receptor and donor plasma by high-throughput sequencing, avoiding the primer crossover according to the fragmentation pattern of the free DNA Fragment design, candidate primers are obtained according to the specificity, abundance and design principles of fluorescent quantitative PCR primers; screening of the above primers includes: verifying the specificity of the above candidate primers in the receptor and donor DNA by PCR, and selecting the donor DNA There are primers that specifically expand but do not specifically expand in the recipient DNA.

Further, the sequences of the above primers are as follows:

Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7);

Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8); or

Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21);

Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22).

Further, the kit further includes: a reagent component for performing quantitative PCR amplification.

Considering that the human genomic DNA has less than 70% homology with the donor pig genomic DNA sequence, there is a large sequence difference, which can distinguish the donor (porcine) and the receptor (human)-derived free DNA, and can be used for real-time PCR. qPCR) and high-throughput sequencing accurately quantify pig-specific free DNA. The invention utilizes pig specific free DNA to monitor graft survival and immune rejection, and quantifies to quantify porcine specific free DNA in plasma by qPCR amplification. The method of the invention has non-invasiveness, specificity, high sensitivity, simplicity, and low detection cost. It can be continuously monitored and has broad market application prospects.

DRAWINGS

Figure 1 shows the conditions for pig-specific amplification primers by gradient PCR. The asterisk (*) indicates that there is specific PCR amplification in the porcine DNA without a band.

Figure 2 shows the specific identification of primer species. Primer PCR identified primer species specificity (A), asterisk (*) indicates no PCR amplification in monkey or human genomic DNA, well number (#) indicates no PCR amplification in mouse genomic DNA; qPCR for The primer No. 4 (B) and the No. 11 primer (C) establish a standard curve, Y value is the Ct value of the instrument reading, X is the copy number of the sample, and R = 0.997 of the standard curve shown in Fig. 2 (B), Fig. 2 ( C) The standard curve shown has R = 0.999.

Figure 3 is a graph showing the correlation between pig-specific free DNA and complement killing in vitro. Morphological changes in human serum or monkey serum co-incubated with PIEC cells (A), human serum treated cells (B) or monkey serum treated cells (C), cpsDNA and cell viability loss. Hrs: hour, CDC: complement dependent killing, cpsDNA: pig specific free DNA, r: correlation coefficient, scale: 135 μm.

Figure 4 shows the correlation between pig-specific free DNA and immune rejection in vivo (porcine-mouse cell transplantation and in vivo imaging). Cell injection, in vivo imaging, experimental design of blood sampling time (A), one of the mice was injected with cells at different time points after cell injection (B), injected with PIEC ^luc group (C) or injected with cell-free PBS group ( D) Comparative analysis of relative luciferase activity and cpsDNA, comparative analysis of cpsDNA with anti-porcine IgG antibody (E) and anti-porcine IgM antibody (F). PIEC ^luc : Porcine vascular endothelial cells stably expressing luciferase, PBS: phosphate buffer, m1/m2/m3: mouse number, IgG/IgM: immunoglobulin G/M.

Figure 5 is a graph showing whether pig-specific free DNA can reflect the immunosuppressive effect of rapamycin in vivo (porcine-mouse cell transplantation and in vivo imaging). The fluorescence intensity of luciferase (A) was observed by in vivo imaging at different time points (A), and the level of cpsDNA was detected by qPCR at different time points (B). pAEC ^luc : primary porcine aortic endothelial cells stably expressing luciferase , cpsDNA: pig-specific free DNA, Rapa: rapamycin.

Figure 6 is a comparison of the evaluation of pig-specific free DNA with conventional immunological indicators (IgG/IgM) in vivo (porcine-cynomolgus monkey arterial patch transplantation model). A and B are transplanted in the first case, and C and D are the second case. Planting, E and F were the third case of transplantation. cpsDNA: pig-specific free DNA, IgG/IgM: immunoglobulin G/M.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

First, the experimental method

1. High-throughput sequencing and bioinformatics analysis

Bioinformatics methods were used to compare BLAST to human genome, monkey genome and pig genome, and obtain highly specific genomic sequences from donor pigs for qPCR amplification primer design. Human plasma, monkey plasma, porcine-monkey arterial patch plasma, porcine plasma DNA were extracted, and sequence and abundance information of free DNA in plasma was captured by Illumina HiSeqX (PE150) high-throughput sequencing. On the one hand, the fracture patterns of free DNA in human, monkey and pig plasma were compared to avoid primer design cross-fragment design; on the other hand, the candidate target sequences with specific, high abundance and qPCR primer design principles were screened by statistical analysis. 19 pairs of candidate primers are shown in Table 1.

Table 1 Quantitative primers for pig-specific DNA

2. Primer specific verification and establishment of quantitative methods

(1) In vitro verification of primer amplification specificity

The genomic DNA and the supernatant of the culture supernatant of HepG2, monkey Vero and pig PIEC cells were extracted by genomic DNA extraction kit (Tiangen, Beijing, China); 19 pairs were analyzed by gradient PCR (annealing temperature gradient) Candidate primers were specifically verified to screen for primers with high specificity. The reaction system is as follows:

(2) Verification of species specificity

Using blood genomic DNA extraction kit (Tiangen, Beijing, China) to extract human blood, monkey blood, pig-monkey (arterial patch transplanted monkey) blood and genomic DNA in pig blood, using the specificity screened in (1) Primers were tested for free heterologous DNA to further verify primer specificity. The reaction system is shown in 2(1).

(3) Establishment of an absolute quantitative method for free heterologous DNA

1) Specific amplification primers for pig-specific free DNA were used for subsequent experimental validation:

Use No. 4 primers in cynomolgus monkey models or humans:

Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7);

Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8).

Primer No. 11 used in the mouse model:

Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21);

Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22).

2) Using Ex Taq (TAKARA, Dalian, China) and specific amplification primers to amplify the pig-specific DNA fragment by ordinary PCR, and ligating it into the T vector (pMD19-T, TAKARA, Dalian, China) to construct a free heterologous A plasmid for the DNA fragment.

3) Calculate the molar molecular mass of the plasmid or calculate it by using 1 base pair approximately equal to 650 Daltons, calculate the plasmid copy number by Avogadro's constant (6.02×10 ²³ /mol) and prepare a standard for different copy numbers. Product.

4) The reaction system with plasmid copy numbers of 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ was prepared by conditional exploration as follows:

The qPCR program was as follows: pre-denaturation at 95 ° C for 5 min; denaturation at 95 ° C for 15 s; annealing at 60 ° C for 45 s. A dissolution profile (annealing temperature 60 ° C) was obtained. The amplification efficiency of fluorescence quantification = 100% ± 5%, and R ≈ 1 is achieved within a large concentration gradient range (concentration gradient number ≥ 5) (the closer R is to 1, the higher the degree of coincidence).

3. Evaluation of the correlation between free xenogenic DNA and complement killing ability by CDC in vitro

(1) Resuscitation of the porcine hip arterial endothelial cell line (PIEC), cultured at 37 ° C, 5% CO _{2 for} 24 hours.

(2) The cells were trypsinized, and the cells were seeded into a 96-well plate at a cell density of 30%; the cell density was about 50% to 70% after incubation at 37 ° C, 5% CO _{2 for} 12 hours.

(3) DMEM medium (Gibco, Grand Island, USA) was added with 20% human serum, monkey serum and respective inactivated serum (inactivated at 56 ° C water bath for 30 min), which was divided into 4 treatment groups. Mix well.

(4) Add the culture medium containing different serum to the cell culture wells, 6 replicates in each group, labeled as "3 hours treatment", and culture in a 37 ° C, 5% CO ₂ incubator.

(5) was added to the new cell culture wells in serum medium containing different groups of 6 replicates, labeled "2 hour treatment", placed in 37 ℃, 5% CO ₂ incubator.

(6) A culture medium containing different sera was added to the new cell culture wells, and each group was repeated 6 times and labeled as "1 hour treatment", and cultured in a 37 ° C, 5% CO ₂ incubator.

(7) A culture medium containing different sera was added to the new cell culture wells, and each group was repeated 6 times and labeled as "0 hour treatment".

(8) Collect the culture supernatant separately, add the apoptosis and proliferation reagent CCK8 (YESEN, Shanghai, China), incubate at 37 ° C, 5% CO _{2 for} 1-4 hours, and measure the OD value of the sample at 450 nm using a microplate reader. Variety.

(9) After culturing the supernatant at 3000 × g for 5 min, the DNA was extracted using a serum/circulating DNA extraction kit (Tiangen, Beijing, China).

4. Evaluation of pig-specific free DNA monitoring for graft rejection in a porcine-mouse cell transplantation in vivo imaging model

(1) Construction and identification of porcine cells stably expressing luciferase

1) Add the lentivirus (YESEN, Shanghai, China) containing the luciferase reporter gene to the porcine hip artery endothelial cell line (PIEC) or pig arterial endothelial cells (pAEC), and add 5 μg/ml polybrane (polybrane). , Sigma, Shanghai, China), after 12 hours of infection, change the fresh medium.

2) The cells were cultured for 48 hours, and 2 μg/ml of puromycin (puromycin, Invivogen, San Diego, USA) was added for screening for 48 hours.

3) Record cell survival by phase contrast microscopy and utilize luciferase substrate fluorescein (luciferin) (YESEN, Shanghai, China) confirmed whether the cells have luciferase activity.

(2) Pig-mouse cell transplantation, serum collection and in vivo imaging

1) Three days before transplantation, about 100 μL of whole blood was obtained by mandibular vein blood sampling, and the back and abdomen hair of C57BL/6 mice was removed and the body weight of the mice was recorded.

2) On the day of transplantation, the cells were subcutaneously transplanted into C57BL/6 mice or nude mice at a cell volume of at least 5 × 10 ⁶ /cell; after about 2 hours, the mice were intraperitoneally injected with 15 mg/ml of fluorescein. (10 μl per 1 g mouse body weight), the luciferase signal intensity in the mouse was recorded by a living imager, and finally about 100 μL was taken through the mandibular vein under anesthesia.

3) Serum collection and in vivo imaging were performed on the 3rd, 6th, 9th, 12th, and 15th day after transplantation, respectively.

4) All collected whole blood was centrifuged at 5000 x g for 5 min for two times, and mouse serum was isolated.

5) 20～40μl mouse serum, free DNA was extracted by serum/circulating DNA extraction kit (Tiangen, Beijing, China), and the specific DNA of pigs in serum was detected by the reaction system and method in 2(3) Copy number.

6) Porcine-specific antibody (IgG/IgM) levels were quantified by flow cytometry using porcine erythrocyte antibody binding assays.

5. Porcine-mouse cell transplantation in vivo imaging model to evaluate whether pig-specific free DNA reacts with rapamycin immunosuppressive effect

(1) Configuration of rapamycin injection: rapamycin (Selleck, Shanghai, China) was dissolved in phosphate buffer (PBS) containing 5% Tween 80 and 5% polyethylene glycol 400.

Among them, the PBS configuration method is: potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.2 g, disodium hydrogen phosphate (Na ₂ HPO ₄ · 12H ₂ O) 2.9 g, sodium chloride (NaCl) 8.0 g, potassium chloride ( KCl) 0.2g, add 800mL deionized water, adjust pH 7.4, add deionized water to 1000mL.

(2) 3 days before cell transplantation, different doses of rapamycin (rapamycin mass / mouse body weight: 0.5 Mg/kg, 0.1 mg/kg, 0 mg/kg) was injected intraperitoneally into C57/BL6 mice once a day.

(3) Cell transplantation, serum collection, and in vivo imaging are shown in 4(2).

(4) 20～40μl mouse serum, free DNA was extracted by serum/circulating DNA extraction kit (Tiangen, Beijing, China), and the specific DNA of pigs in serum was detected by the reaction system and method in 2(3) Copy number.

6. Specificity comparison between pig-specific free DNA and immunological parameters in a pig-monkey arterial patch transplantation model

(1) A total of 3 cases were performed in the transplantation experiment, that is, the carotid artery of Wuzhishan pig was transplanted into the abdominal aorta of cynomolgus monkey by vascular suture.

(2) Blood samples (anticoagulation tube collection) before transplantation, at the first week, the second week, the fourth week, and the seventh week after transplantation were collected, and plasma and blood cells were separated by centrifugation at 1700 rpm and 4 ° C for 5 minutes.

(3) T/B cell count using FACS.

(4) Transfer the plasma in step (2) to a new centrifuge tube, centrifuge at 3000 × g for 5 min at 4 ° C (two times to completely remove nucleated cells), and store in duplicate.

(5) Isolation of porcine erythrocytes, and transplantation of monkey inactivated plasma (inactivated at 56 ° C water bath for 30 min) at various time points for antibody (IgG/IgM) binding assay, detection of antibody binding to porcine erythrocytes in transplanted monkey plasma by flow cytometry On the level.

(6) 400 μL mouse serum, free DNA was extracted by serum/circulating DNA extraction kit (Tiangen, Beijing, China), and the copy of pig-specific free DNA in serum was detected by the reaction system and method in 2(3). number.

Second, the experimental results

According to the above experimental method, the following experimental results were obtained:

1. Designed qPCR primers, 11 pairs of specific amplification of porcine DNA, as shown in Figure 1. Further, a primer (No. 4 primer) capable of specifically amplifying porcine DNA in a cynomolgus monkey or a human, and a primer capable of specifically amplifying porcine DNA in a mouse model (No. 11 primer) were obtained, and the result is shown in Fig. 2A. Shown. Separate needle A standard curve was constructed for primers No. 4 and No. 11, and the results are shown in Fig. 2B and Fig. 2C.

2. In the complement-dependent killing experiment, the No. 4 primer was used to quantify the specific free DNA level of the pig in the culture supernatant. The results showed that the pig-specific free DNA was proportional to the killing time of the complement, and the correlation coefficient r was greater than 95%. . The results are shown in Figure 3, indicating that pig-specific free DNA can be used for the detection of complement killing in vitro.

3. In porcine-mouse cell transplantation and in vivo imaging experiments, luciferase signal decreased sharply between the 3rd and 6th day, and was not detected at the 9th day and beyond, while the pig-specific free DNA level It started to rise from 0 to 3 days, rose sharply on 3 to 6 days, and peaked on the 6th or 9th day, indicating that the pig-specific free DNA level can accurately reflect the rejection of the graft; compared with the levels of anti-porcine IgG and IgM. Pig-specific free DNA rises earlier, declines faster, and fits the luciferase signal more, indicating that pig-specific free DNA is an earlier, specific biomarker. The result is shown in Figure 4.

In pig-mouse cell transplantation and in vivo imaging experiments, pig-specific free DNA dose-dependently reflects the immunosuppressive effect of rapamycin, indicating that pig-specific free DNA can be used for optimization of immunosuppressants. The result is shown in Figure 5.

5. In the porcine-monkey arterial patch transplantation model, pig-specific free DNA more specifically reflects the rejection of the graft compared to the levels of anti-porcine IgG and IgM. The result is shown in Figure 6.

In summary, the present invention can be applied to the field of transplantation in which pigs are donors, and can accurately reflect the immune rejection of the graft. Compared with the detection method of the conventional immune rejection reaction, the invention utilizes qPCR amplification to quantify the pig-specific free DNA in the plasma, and has the advantages of non-invasiveness, high specificity, high sensitivity, simplicity, low detection cost, continuous monitoring, and the like. Has a broad market application prospects.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (10)

A primer for use in a method for monitoring pig-to-human or non-human primate xenograft immunological rejection using porcine-specific free DNA, characterized in that the sequence of the primer is as follows: Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7); Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8). A primer for use in a method for monitoring pig-to-mouse xenograft immune rejection using porcine-specific free DNA, characterized in that the sequence of the primer is as follows: Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21); Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22). A method for monitoring xenograft immune rejection using porcine-specific free DNA, characterized in that the method is used for monitoring xenograft immunorejection in a recipient object of a pig as a donor, the method comprising using a pair of pigs The specific free DNA has a specific amplification primer, and the receptor plasma DNA sample is subjected to real-time PCR amplification to quantify the amount of pig-specific free DNA in the recipient plasma. The method of claim 3, further comprising designing and screening said primers, wherein said primer design comprises: obtaining donor pigs by comparing a receptor genome to a donor pig genome Specific genomic sequences; and high-throughput sequencing to capture sequence and abundance information of free DNA in the receptor and donor plasma, avoiding primer cross-fragment design based on fragmentation patterns of free DNA, based on specificity, abundance, and fluorescence quantification The design principle of PCR primers gives candidate primers; the screening of the primers includes: verifying the specificity of the candidate primers in the receptor and donor DNA by PCR, and selecting specific amplification in the donor DNA at the receptor Primers without specific amplification in DNA. The method of claim 3 wherein the sequence of said primers is as follows: Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7); Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8). The method of claim 3 wherein the sequence of said primers is as follows: Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21); Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22). The method according to claim 3, wherein the recipient subject is a human, a non-human primate or all mammals except pigs. A kit for use in a method for monitoring xenograft immune rejection using porcine-specific free DNA, characterized in that the kit comprises a primer obtained by aligning a receptor genome with a donor Pig genome, obtaining donor-specific genomic sequences; and capturing sequence and abundance information of free DNA in receptor and donor plasma by high-throughput sequencing, avoiding primer cross-fragment design based on fragmentation pattern of free DNA, according to specificity The design principles of sex, abundance and fluorescent quantitative PCR primers lead to candidate primers; the screening of the primers includes: verifying the specificity of the candidate primers in the receptor and donor DNA by PCR, and selecting specificity in the donor DNA Primers that are amplified but not specifically amplified in the recipient DNA. The kit according to claim 8, wherein the sequence of the primers is as follows: Forward primer: 5'-TTCAATCCCACTTCTTCCACCTAA-3' (SEQ ID NO: 7); Reverse primer: 5'-CTTCATTCCATCTTCATAATAACCCTGT-3' (SEQ ID NO: 8); or Forward primer: 5'-TGCCGTGGTTTCCGTTGCTTG-3' (SEQ ID NO: 21); Reverse primer: 5'-TCACATTTGATGGTCGTCTTGTCGTCT-3' (SEQ ID NO: 22). The kit according to claim 8, wherein the kit further comprises: a reagent component for performing quantitative PCR amplification.

Images