A multi-index joint non-invasive detection kit for graft injury

Abstract

The application discloses a kind of graft injury multi-index combined noninvasive detection kit, by optimized multiplex real-time fluorescent quantitative polymerase chain reaction technology, realize the quantitative detection of six categories with closely related DNA virus after transplantation Complications, the present application relates to the field of in vitro detection technology of clinical sample.The graft injury multi-index combined noninvasive detection kit, the present application first realizes the synchronous detection of BK virus, JC virus, B19 virus, CMV virus, adenovirus and six kinds of closely related viruses of circovirus (TTV) with graft injury.Test data see example 7, from the detection results of 100 clinical kidney transplant patients, it can be seen that the kit of the present application can simultaneously accurately detect the load level of six viruses, to provide comprehensive infection risk assessment for clinicians.Compared with the prior art single virus detection method, the present application avoids the cumbersome process of multiple sampling and detection, significantly improves the clinical work efficiency.

Description

Technical Field

[0001] This invention relates to the field of in vitro clinical sample testing technology, specifically a non-invasive test kit for multiple indicators of graft injury. Background Technology

[0002] Since the first successful human kidney transplant in 1954, organ transplantation has become the preferred treatment for end-stage organ failure, significantly improving the quality of life and long-term prognosis for tens of thousands of patients. However, transplant recipients need to take immunosuppressive drugs (such as tacrolimus) for life to prevent immune rejection. While these drugs effectively reduce the risk of rejection, they also significantly weaken the body's ability to fight infection, making recipients highly susceptible to opportunistic infections, especially viral infections.

[0003] Viral infections have become a significant cause of graft injury, functional impairment, and even organ failure in solid organ transplant recipients, seriously threatening their long-term survival. Therefore, donors for solid organ transplants must undergo rigorous screening to prove they are free from key viruses such as BKV, cytomegalovirus (CMV), JCV, human parvovirus B19 (B19V), adenovirus (AdV), and transcircovirus (TTV) to reduce the risk of viral reactivation and transmission after transplantation. These six DNA viruses are easily activated or continuously replicate under immunosuppressive conditions, directly damaging transplanted organs (e.g., BKV-related nephropathy) and indirectly inducing or exacerbating rejection reactions through immunomodulatory effects. It is noteworthy that viral infections and acute rejection reactions often present with similar clinical manifestations (such as fever, fatigue, dyspnea, and abnormal renal function), and routine laboratory tests often fail to effectively differentiate them, easily leading to misdiagnosis or delayed treatment.

[0004] Currently, while tissue biopsy is considered the "gold standard" for diagnosing rejection and certain infections, its invasive nature can damage transplanted organs and makes dynamic, repeatable monitoring difficult, failing to meet clinical needs for early, non-invasive, and continuous assessment. Therefore, establishing a non-invasive, accurate, and simultaneous technique for monitoring multiple transplant-related viral loads is of crucial clinical significance for early identification of viral damage, differentiation between infection and rejection, guidance of individualized immunosuppression adjustments and antiviral interventions, and ultimately, improving long-term survival rates in transplant recipients.

[0005] BK virus (BK polyomavirus) is a common human polyomavirus with an infection rate exceeding 80%. In immunocompetent individuals, it typically exists as a latent infection, but can be reactivated when the body's immune function declines. In kidney transplant recipients, BK virus-associated nephropathy (BKVN) is the second leading cause of graft failure, with an incidence of approximately 1-10%. Once BKVN develops, about 50% of cases result in transplant failure.

[0006] JC virus (JC polyomavirus) belongs to the same family as BK virus, the Polyomaviridae, and primarily infects the central nervous system. In solid organ transplant recipients, JC virus can cause progressive multifocal leukoencephalopathy (PML), a fatal neurological complication with an incidence of approximately 0.1-0.7% and a mortality rate as high as 50-80%.

[0007] Parvovirus B19 is a single-stranded DNA virus that can cause pure red cell aplastic anemia (PRCA). The infection rate in solid organ transplant recipients is about 5-10%, and it can cause graft dysfunction and anemia.

[0008] Cytomegalovirus (CMV) is one of the most common viral infections after organ transplantation, with an infection rate as high as 30-75%. CMV infection can not only directly cause tissue and organ damage, but also indirectly promote acute rejection and chronic graft failure through immunomodulation.

[0009] Adenovirus can cause severe respiratory, gastrointestinal, and urinary tract infections in immunocompromised individuals. The infection rate in organ transplant recipients is approximately 5-15%, which can lead to graft dysfunction.

[0010] Torque teno virus (TTV) is a common, asymptomatic infectious virus with an infection rate exceeding 90% in healthy individuals. Recent studies have found a close correlation between TTV viral load and the degree of immunosuppression, making it a potential biomarker for immunosuppressive states. In kidney transplant recipients, TTV viral load is significantly associated with acute rejection and infection risk.

[0011] Currently, clinical detection of these viruses primarily employs single-virus qPCR methods, with a few capable of detecting two viruses. Chinese patent CN202011120074.0 discloses a qPCR method for simultaneously detecting BK virus and human cytomegalovirus, Chinese patent CN119876488A discloses a qPCR method for detecting B19 virus, and Chinese patent CN111793832A discloses a qPCR method for detecting adenovirus. However, these methods have the following limitations: single-virus detection requires multiple sampling and testing, increasing patient discomfort and medical costs; inconsistent testing methods and reagents used by different laboratories lead to poor comparability of results; most qPCR methods have limited sensitivity, making it difficult to detect low viral loads; the lack of standardized multi-virus combined detection procedures makes it impossible to comprehensively assess the infection risk of transplant patients; existing qPCR methods lack specificity in high-background DNA samples, easily resulting in false positives or false negatives; and a unified threshold for the association between viral load and graft injury risk has not been established.

[0012] Problems with existing technology: 1. It cannot simultaneously detect multiple viruses closely related to graft injury (BK, JC, B19, CMV, adenovirus, TTV), requiring multiple sampling and testing, which increases the burden on patients and medical costs.

[0013] 2. Existing qPCR detection methods are not sensitive enough to detect samples with low viral load (<100 copies / mL).

[0014] 3. The testing methods and reagents used by different laboratories are not uniform, resulting in poor comparability of results and a lack of standardized multi-virus joint detection procedures.

[0015] 4. Existing qPCR methods lack specificity in high background DNA samples, and are prone to false positive or false negative results.

[0016] 5. No unified threshold for the association between viral load and graft injury risk has been established, making clinical interpretation difficult.

[0017] 6. The lack of optimized protocols for viral DNA extraction from blood samples of organ transplant patients results in low viral DNA recovery rates.

[0018] 7. Existing methods do not take into account the interactions between different viruses and their combined effects on graft damage.

[0019] 8. No correlation model has been established between dynamic changes in viral load and the progression of graft injury, making early warning difficult. Summary of the Invention

[0020] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a multi-indicator combined non-invasive detection kit for graft injury, solving key problems such as the inability to simultaneously monitor multiple graft injury-related viruses, insufficient detection sensitivity, high detection costs, and the lack of unified risk assessment standards. Through rigorous scientific experimental verification and clinical sample testing, this invention demonstrates significant technical advantages and clinical application value.

[0021] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation. This method utilizes optimized multiplex real-time quantitative polymerase chain reaction (qPCR) technology to achieve highly sensitive and specific quantitative detection of six types of DNA viruses closely related to post-transplant complications. Specifically, these DNA viruses include BK virus, JC virus, human parvovirus B19, CMV virus, adenovirus, and circovirus. The quantitative detection of these six types of DNA viruses closely related to post-transplant complications includes: design and screening of six virus-specific primers and probes, optimization of the multiplex qPCR reaction system, improvement of viral DNA extraction methods, and quantification of viral load.

[0022] Preferably, the design and screening of the six virus-specific primers and probes are as follows: S1. Using a set of primers for detecting pathogens in organ transplantation, the genomes of BK virus, JC virus, B19 virus, CMV virus, adenovirus and TTV virus were compared and analyzed based on the NCBI database and a self-built virus sequence database, and conserved regions of each virus were screened as detection targets. S2. Design specific primers and probes for conserved regions of DNA viruses. Design multiple pairs of primers and probes, and screen out the best primer-probe combination through computer simulation and experimental verification. Each pathogen contains 1-3 pairs of specific primers, and the amplicon length is 70-100bp. S3. This primer and probe combination can be synthesized in one tube to achieve multiplex PCR detection in a single tube. The primer combination has high sensitivity and good specificity, which can reduce the types of tests, reduce the amount of blood samples collected, reduce testing costs, shorten the time for clinicians to diagnose patients' conditions, and speed up the implementation of treatment measures for patients.

[0023] Preferably, in step S2, multiple pairs of primers and probes are designed using TaqMan hydrolysis probes, with a fluorescent group labeled at the 5' end and a quenching group labeled at the 3' end. The probe annealing temperature needs to be higher than that of the primers to preferentially bind to the template.

[0024] Table 1 Target gene selection and conservation verification

[0025] Preferably, the optimization of the multiplex qPCR reaction system is as follows: T1. The test employs a standardized sample processing procedure to ensure high efficiency in viral DNA extraction and reliability of test results. First, 8-10 mL of peripheral blood is collected using a cell-free DNA sample preservation tube and must be delivered to the laboratory for processing within 3 days. If EDTA anticoagulant tubes are used, plasma separation must be completed within 8 hours. T2. The plasma separation process adopts a two-step centrifugation method: first, centrifuge at 1600×g for 10 minutes, and aspirate the upper plasma layer to a new centrifuge tube, avoiding aspirating the middle layer of white blood cells; then, centrifuge at 12000×g at 4℃ for 10 minutes, and transfer the supernatant to a new centrifuge tube. The separated plasma can be stored at -20℃ for later use. T3. Viral DNA extraction was performed using a modified magnetic bead method. 200 μL of plasma sample was taken and 400 μL of lysis buffer was added. The sample was then incubated at 56°C for 30 minutes for complete lysis. T4. Then add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), centrifuge at 12,000×g for 10 minutes, take the supernatant, add 0.8 times the volume of binding buffer and 50 μL of magnetic beads, mix at room temperature for 10 minutes, then place on a magnetic rack for separation, discard the supernatant, wash twice with 70% ethanol, air dry for 5 minutes, add 50 μL of elution buffer, incubate at 56℃ for 3 minutes, and collect the supernatant as template DNA. T5. The concentration of the extracted DNA was detected by a Qubit 4.0 fluorescence quantitative instrument, and the purity was determined by a spectrophotometer. For T6, in terms of standard curve preparation, each virus-specific fragment was cloned into the pUC57 vector, and the copy number was calculated after concentration determination. - Starting at a concentration of copies / μL, the solution was serially diluted 10-fold to prepare 10¹- Standards in copies / μL; T7. Plot a logarithmic standard curve of Ct value versus copy number using qPCR amplification to verify the linear range, ensure the accuracy and sensitivity of the detection system, and provide a reliable basis for the accurate quantification of BK virus, JC virus, CMV virus, B19 virus, adenovirus, and circovirus in subsequent clinical samples.

[0026] Preferably, the lysis buffer in step T3 contains 1 mg / mL proteinase K.

[0027] Preferably, in step T5, the A260 / A280 ratio in the purity is determined to be 1.8-2 using a spectrophotometer.

[0028] Preferably, in step T7, the linear range is verified to be R²≥0.99, and the amplification efficiency is 90%-110%.

[0029] Reaction system configuration: Premixed solution: contains hot-start DNA polymerase, dNTPs, Stabilizers and fluorescent dyes. The final concentration is generally 50% of the reaction system.

[0030] Probe and primer concentrations: Optimize to 0.4-0.8 μM (primers) and 0.2-0.5 μM (probes) to avoid non-specific amplification.

[0031] Amplification and detection employed an optimized six-fold qPCR thermal cycling program: First, pre-denaturation at 95℃ for 5 minutes thoroughly activated the hot-start Taq enzyme and completely dissociated the DNA double strands. This was followed by 45 amplification cycles, each consisting of 95℃ denaturation for 15 seconds (to fully lyse the DNA double strands) and annealing / extension at 60℃ for 45 seconds (this temperature was validated as optimal by gradient PCR to ensure specific binding and efficient extension of the six viral primer-probe combinations). At the end of the annealing / extension step, signals from each fluorescence channel were automatically acquired, and the Ct value (i.e., the number of cycles at which the fluorescence signal reached the set threshold) was recorded. Fluorescence signal changes were strictly monitored throughout the amplification process to ensure no cross-interference in the acquisition of signals from the FAM, HEX, and CY5 fluorescence channels, and that the virus-specific amplification curves exhibited typical S-shaped growth characteristics. During the data analysis phase, the Ct values ​​of each virus were converted into precise viral DNA copy numbers (copies / mL) using a standard curve, and risk assessment was performed based on clinical thresholds. Simultaneously, rigorous quality control was implemented: the negative control (using nuclease-free water instead of template DNA) should have no Ct value or a Ct > 40 and no typical amplification curve to exclude contamination and false positives; the positive control (a mixture of viral DNA standards at known concentrations) should exhibit a typical S-shaped amplification curve and a Ct value within the expected range (usually ≤ 30) to verify reagent effectiveness and detection sensitivity; the internal control was used to monitor sample quality and extraction efficiency, ensuring intra-batch CV < 5% and inter-batch CV < 10%. Through this complete standardized process, this kit can achieve accurate quantitative detection of BK virus, JC virus, CMV virus, B19 virus, adenovirus, and circovirus, providing a reliable basis for assessing graft injury risk in organ transplant patients. The detection limit reaches 10¹ copies / mL, and the specificity is > 99.5%, significantly superior to existing single-virus detection methods.

[0032] Required reagents: This kit employs a complete detection system design, including the following key reagent components: The viral DNA extraction system utilizes a professionally optimized magnetic bead reagent combination, including high-efficiency viral lysis buffer (containing specific proteinase K, final concentration 1 mg / mL), high-affinity nucleic acid binding magnetic beads, and a series of washing buffers, ensuring efficient extraction of low-abundance viral DNA from 200 μL plasma samples with a recovery rate ≥85%; The specific primer and probe system targets BK virus (BKV), JC virus (JCV), human parvovirus B19 (B19V), and cytomegalovirus. The design incorporates six key pathogens: CMV, adenovirus (AdV), and triclovirus (TTV). Each virus contains a pair of specific primers (final concentration 0.2-0.4 μM) and a fluorescently labeled TaqMan probe (final concentration 0.1-0.2 μM). The 5' end of the probe is labeled with FAM, HEX, or CY5 fluorescent groups, and the 3' end is labeled with BHQ1 or BHQ2 quenching groups, targeting highly conserved regions of each virus. The amplicon length is strictly controlled within the range of 70-110 bp. The premixed fluorescent reagent uses an optimized formulation, containing hot-start Taq DNA polymerase (5 U / μL) and a dNTP mixture (10 mM each). (25mM), stabilizer, and ROX reference dye ensure the high efficiency and specificity of the six-fold qPCR reaction; the standard system is constructed by cloning six virus-specific fragments into the pUC57 vector, and after precise concentration determination, it is serially diluted 10-fold (10¹-10⁻¹⁰). The enzyme and excipients include UNG enzyme (1 U / μL) for establishing standard curves and quantitative analysis; the enzyme and excipients effectively prevent amplification product contamination; the quality control system is equipped with negative control (nuclease-free water) and positive control (a mixture of six virus standards, concentration...). (copies / mL) to ensure the accuracy and reliability of each test.

[0033] This invention also provides a multi-indicator combined non-invasive detection kit for graft injury, which is made using the multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation. It is used for continuous monitoring of viral infection after clinical lung transplantation, heart transplantation, kidney transplantation, and liver transplantation, to promptly detect changes in the patient's physical condition after organ transplantation and to assist doctors in targeted treatment.

[0034] (III) Beneficial Effects This invention provides a non-invasive detection kit for multiple indicators of graft injury. Compared with existing technologies, it has the following advantages: (1) This multi-indicator non-invasive detection kit for graft injury is the first to achieve simultaneous detection of six viruses closely related to graft injury: BK virus, JC virus, B19 virus, CMV virus, adenovirus, and transviral circovirus (TTV). Test data are shown in Example 7. The results from 100 clinical kidney transplant patients demonstrate that the kit can accurately detect the viral load levels of all six viruses simultaneously, providing clinicians with a comprehensive assessment of infection risk. Compared to existing single-virus detection methods, this invention avoids the cumbersome process of multiple sampling and testing, significantly improving clinical efficiency.

[0035] (2) This multi-index combined non-invasive detection kit for graft injury, with high sensitivity and wide linear range, provides test data as shown in Example 6: [Data on the prepared 10¹-] The kit was tested using a series of diluted standards at copies / mL. The results showed that the detection limit for all six viruses using the kit was 10¹ copies / mL, and the detection limit was within 10¹-... It exhibits good linearity within the range of copies / mL (R²>0.995). - Within the range of copies / mL, the CV value for repeated detections is <5%, demonstrating excellent repeatability and stability. Particularly noteworthy is that even in high background DNA samples (human genomic DNA concentrations up to 100 ng / μL), this invention can still accurately detect concentrations as low as [missing value]. The target virus copies / mL is significantly superior to existing detection methods.

[0036] (3) This multi-indicator non-invasive detection kit for graft injury demonstrates high specificity and no cross-reactivity. Test data is shown in Example 6: Specificity testing showed that the kit's detection specificity for all six viruses was >99.5%. The detection of each virus is unaffected by interference from the other five viruses or by human genomic DNA. For example, when the BK virus concentration is... When the concentration of human genomic DNA was as high as 100 ng / μL, the detection results of the six viruses were negative (Ct>40); when the concentration of human genomic DNA was as high as 100 ng / μL, the detection results of the six viruses were less than 5% compared with the detection results without background DNA, proving that the present invention has extremely high specificity.

[0037] (4) The multi-index non-invasive detection kit for graft injury will bring significant social benefits when it is promoted and applied: (1) By detecting viral infection early, graft injury and failure can be reduced, and the lifespan of transplanted organs can be extended. It is expected that the 5-year survival rate of grafts can be increased by 8-10%; (2) Unnecessary tissue biopsies can be reduced, and the risk of complications for patients can be reduced; (3) Immunosuppression regimens can be optimized to balance rejection and infection risks and improve the quality of life of patients.

[0038] (5) This multi-indicator non-invasive detection kit for graft injury not only achieves high sensitivity and high specificity for simultaneous detection of six key viruses, but also provides a scientific basis for post-organ transplant management through an innovative risk assessment model. Experimental data fully demonstrate that this invention is significantly superior to existing technologies in terms of sensitivity, specificity, detection efficiency, and clinical application value, making an important contribution to improving the long-term survival rate and quality of life of transplant recipients. Attached Figure Description

[0039] Figure 1 This is a performance verification graph of the BK virus standard curve of the present invention; Figure 2 This is a performance verification graph of the JC virus standard curve for this invention; Figure 3 This is a performance verification graph of the B19 virus standard curve of the present invention; Figure 4 This is a performance verification graph of the CMV virus standard curve of the present invention; Figure 5 This is a graph showing the performance verification of the adenovirus standard curve of the present invention; Figure 6 This is a standard curve performance verification graph for the TTV virus of this invention. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Please see Figure 1-6 The present invention provides seven technical solutions: a multi-index combined non-invasive detection kit for graft injury, specifically including the following embodiments: Example 1: Design and screening of virus-specific primers and probes: 1. Sequence Acquisition and Alignment Analysis: The whole genome sequences of BK virus (BKV), JC virus (JCV), human parvovirus B19 (B19V), cytomegalovirus (CMV), adenovirus (AdV), and transviral circovirus (TTV) were downloaded from the NCBI GenBank database, and viral sequence databases were constructed accordingly. Multiple sequence alignment was performed using ClustalW software to screen conserved regions of each virus as potential detection targets: the VP1 gene region was selected for BK virus, the VP1 gene region for JC virus, the NS1 gene region for B19 virus, the UL83 gene region for CMV virus, the Hexon gene region for adenovirus, and the non-coding region for TTV virus.

[0042] 2. Primer and Probe Design: Multiple primer and probe pairs were designed using Primer software for each conserved region of the virus. Primer design parameters were: length 18-25 nt, Tm value 58-62℃, GC content 40-60%. Probe design parameters were: length 19-24 nt, Tm value 5-10℃ higher than the primers, 5' end labeled with a fluorescent group, and 3' end labeled with a quencher group. Primers and probes homologous to the human genome and other common pathogens were excluded by BLAST alignment. The secondary structures of primers and probes were analyzed using OligoAnalyzer software to exclude sequences that formed dimers or hairpin structures.

[0043] 3. Primer and probe screening, synthesis of candidate primers and probes, experimental verification, and sensitivity testing (10¹-) Primer combinations with high amplification efficiency were screened using the (copies / mL) method; primer combinations with high specificity were screened using a specificity test (cross-reaction with other 5 viruses and human genomic DNA); primer combinations that can work synergistically in the same reaction system were screened using a multiplex qPCR compatibility test; the final selected primer and probe sequences are shown in Table 2.

[0044] Table 2 Primer and Probe Screening Table

[0045] Example 2: Optimization of multiplex qPCR reaction system: 1. Optimization of reaction system components: Preparation of 10 × qPCR Buffer: Tris-HCl (pH 8.3) 100 mM, KCl 500 mM, 80 mM; Prepare 6 × virus-specific primer mixture: Mix the six virus primers screened in Example 1 in equal amounts to make the final concentration of each primer 1.2-2.4 μM; Prepare 6 × virus-specific probe mixture: Mix the six virus probes screened in Example 1 in equal amounts to make the final concentration of each probe 0.6-1.2 μM.

[0046] Optimize the concentrations of each component in the reaction system: Primer concentration: tested in the range of 0.1-0.5 μM, and determined the optimal final concentration to be 0.3 μM; Probe concentration: tested in the range of 0.05-0.3 μM, and determined the optimal final concentration to be 0.15 μM. Concentration: Test within the range of 2-5 mM, and determine the optimal final concentration as 3 mM; Taq enzyme concentration: Test within the range of 0.5-2.0 U / reaction, and determine the optimal dosage as 1.0 U.

[0047] 2. Optimization of reaction conditions: Annealing / extending temperature: tested in the range of 55-65℃, and determined the optimal temperature to be 60℃; Number of loops: Test 40-50 loops and determine the optimal number of loops to be 45; Pre-denaturation time: Test for 2-10 minutes, with 5 minutes being the optimal time.

[0048] 3. The final determined multiplex qPCR reaction system is shown in Table 3.

[0049] Table 3 qPCR reaction system

[0050] 4. The reaction procedure is shown in Table 4. Table 4 Reaction Procedure

[0051] Example 3: Improvement of viral DNA extraction method: 1. Sample collection and plasma separation: Collect 5-8 mL of peripheral blood using a professional cell-free DNA sample preservation tube and ensure that it is delivered to the laboratory within 24 hours for plasma separation. Centrifuge the blood sample at 1600×g for 10 minutes, carefully aspirate the supernatant plasma into a new centrifuge tube, centrifuge the plasma again at 4℃ at 16000×g for 10 minutes, and transfer the supernatant to a new centrifuge tube.

[0052] 2. Viral DNA extraction: Take 200 μL of plasma sample, add 400 μL of lysis buffer (containing proteinase K 1 mg / mL), incubate at 56℃ for 30 min, mixing every 10 min during incubation. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), vortex to mix, centrifuge at 12,000×g for 10 min, collect the supernatant, add 0.8 volumes of binding buffer and 50 μL of magnetic beads, mix at room temperature for 10 min, place on a magnetic rack for separation, discard the supernatant, wash twice with 70% ethanol, air dry for 5 min, add 50 μL of elution buffer, incubate at 56℃ for 3 min, place on a magnetic rack for separation, and collect the supernatant as template DNA.

[0053] 3. DNA quality testing: DNA concentration was measured using a Qubit 4.0 fluorescence quantitative PCR instrument, and DNA purity was measured using a spectrophotometer (A260 / A280 ratio should be between 1.8 and 2.0) to ensure that the extracted DNA was free from significant degradation and could be used for subsequent qPCR detection.

[0054] Example 4: Preparation of Standards 1. Plasmid construction: Specific fragments were amplified by PCR from each virus-positive sample, cloned into the pUC57 vector, and the recombinant plasmid was transformed into E. coli DH5α. Positive clones were screened, plasmid DNA was extracted, and the concentration was determined using Nanodrop.

[0055] 2. Copy number calculation: The copy number is calculated based on the plasmid molecular weight using the following formula: Copy number / mL = (plasmid concentration × 6.02 × 10²³) / (plasmid length × 660 × ... Dilute the plasmid to Copies / mL were used as the stock solution.

[0056] 3. Standard Dilution: Perform 10-fold serial dilutions using TE buffer to prepare 10¹- Standards in copies / mL should be aliquoted and stored at -80°C, avoiding repeated freeze-thaw cycles.

[0057] Example 5: Establishment of Standard Curve: 1. Standard curve detection: Take the standard prepared in Example 4 and perform qPCR detection according to the reaction system and procedure of Example 2. Each concentration is repeated 3 times and the Ct value is recorded.

[0058] 2. Data Analysis: (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) Plot a standard curve with (copy number) on the x-axis and Ct value on the y-axis, calculate the linear regression equation and correlation coefficient, and calculate the amplification efficiency: E = (10^(-1 / slope)1) × 100%.

[0059] 3. Results: Six viruses were detected in 10¹- It exhibited good linearity within the range of copies / mL, with R² > 0.995, and an amplification efficiency of 95-105%, meeting the requirements for qPCR detection. The limit of detection was 10¹ copies / mL, and the limit of quantification was [missing information]. copies / mL.

[0060] Example 6: Sensitivity and Specificity Tests: 1. Sensitivity test: for 10¹- The kit was tested using a series of diluted standards at 10¹ copies / mL, with each concentration repeated five times. The results showed that the detection limit for all six viruses using this kit was 10¹ copies / mL. - Within the range of copies / mL, the CV value for repeated detections is <5%.

[0061] 2. Specificity test: Cross-reactivity is tested using a single viral standard: The single viral standard ( When the detection system (copies / mL) was mixed with the detection systems of five other viruses, the results showed that the detection of each virus was not affected by the other viruses.

[0062] Non-specific amplification was tested using human genomic DNA (100 ng / μL): The results showed that the detection of all six viruses was not affected by human genomic DNA.

[0063] Specificity was tested using common clinical pathogens (such as HSV, EBV, HBV, etc.): The results showed that this kit had no cross-reactivity with non-target viruses.

[0064] Example 7: Clinical sample testing for organ transplantation: One plasma sample from a clinical kidney transplant patient was selected and subjected to multiplex qPCR detection according to the method of this invention, with the clinical test results used as a control. The test results are shown in the table below. As can be seen from the results, the method of this invention can accurately detect the viral load in plasma samples from heart transplant, kidney transplant, and lung transplant patients. The pathogen detection results are shown in Table 5.

[0065] Table 5 Pathogen detection results

[0066] Figure 1 The standard curve for BK virus qPCR detection is shown, with the x-axis representing... (Virus concentration) (copies / mL), with the ordinate representing the Ct value. The curve is shown in 10¹- The method exhibits excellent linearity within the range of copies / mL, with a linear regression equation of y = -2.3436x + 41.626 and a correlation coefficient R² = 0.9967, indicating high accuracy and reliability. The slope of the curve is -2.3436, corresponding to an amplification efficiency of 98.7%, which meets the requirements for qPCR detection (90-110%). The limit of detection reaches 10¹ copies / mL, and the limit of quantification is [missing information]. The number of copies / mL meets the testing needs of clinical samples with low viral load.

[0067] Figure 2The standard curve for JC virus qPCR detection is shown, with the x-axis representing... (Virus concentration) (copies / mL), with the ordinate representing the Ct value. The curve is shown in 10¹- The method exhibits excellent linearity within the range of copies / mL, with a linear regression equation of y = -2.1898x + 38.478 and a correlation coefficient R² = 0.9992, indicating high accuracy and reliability. The slope of the curve is -2.1898, corresponding to an amplification efficiency of 103.5%, which meets the requirements for qPCR detection (90-110%). The limit of detection is 10¹ copies / mL, and the limit of quantification is [missing information]. The number of copies / mL meets the testing needs of clinical samples with low viral load.

[0068] Figure 3 The standard curve for human parvovirus B19 (qPCR detection) is shown, with the horizontal axis representing the curve. (Virus concentration) (copies / mL), with the ordinate representing the Ct value. The curve is shown in 10¹- The method exhibits excellent linearity within the range of copies / mL, with a linear regression equation of y = -1.9921x + 38.537 and a correlation coefficient R² = 0.9994, indicating high accuracy and reliability. The slope of the curve is -1.9921, corresponding to an amplification efficiency of 109.4%, which meets the requirements for qPCR detection. The limit of detection reaches 10¹ copies / mL, and the limit of quantification is [missing information]. The number of copies / mL meets clinical testing needs.

[0069] Figure 4 The standard curve for cytomegalovirus (CMV) qPCR detection is shown, with the x-axis representing the curve. (Virus concentration) (copies / mL), with the ordinate representing the Ct value. The curve is shown in 10¹- The method exhibits excellent linearity within the range of copies / mL, with a linear regression equation of y = -2.4321x + 40.495 and a correlation coefficient R² = 0.9994, indicating high accuracy and reliability. The slope of the curve is -2.4321, corresponding to an amplification efficiency of 96.1%, which meets the requirements for qPCR detection. The limit of detection reaches 10¹ copies / mL, and the limit of quantification is [missing information]. The number of copies / mL meets the testing needs of clinical samples with low viral load.

[0070] Figure 5 The standard curve for adenovirus (AdV) qPCR detection is shown, with the x-axis representing the curve. (Virus concentration) (copies / mL), with the ordinate representing the Ct value. The curve is shown in 10¹- The method exhibits excellent linearity within the range of copies / mL, with a linear regression equation of y = -2.3152x + 39.863 and a correlation coefficient R² = 0.9991, indicating high accuracy and reliability. The slope of the curve is -2.3152, corresponding to an amplification efficiency of 99.2%, which meets the requirements for qPCR detection. The limit of detection reaches 10¹ copies / mL, and the limit of quantification is [missing information]. The number of copies / mL meets clinical testing needs.

[0071] Figure 6 The standard curve for TTV (transverse circovirus) qPCR detection is shown, with the x-axis representing the curve. (Virus concentration) (copies / mL), with the ordinate representing the Ct value. The curve is shown in 10¹- The method exhibits excellent linearity within the range of copies / mL, with a linear regression equation of y = -3.3747x + 40.59 and a correlation coefficient R² = 0.9989, indicating high accuracy and reliability. The slope of the curve is -3.3747, corresponding to an amplification efficiency of 84.6%, which meets the requirements for qPCR detection. The limit of detection reaches 10¹ copies / mL, and the limit of quantification is [missing information]. The number of copies / mL meets clinical testing needs.

[0072] Furthermore, any content not described in detail in this specification is existing technology known to those skilled in the art.

[0073] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation, characterized in that: By employing optimized multiplex real-time quantitative polymerase chain reaction (qPCR) technology, we can achieve highly sensitive and specific quantitative detection of six types of DNA viruses closely related to post-transplant complications. These DNA viruses are BK virus, JC virus, human parvovirus B19, CMV virus, adenovirus, and circovirus. The quantitative detection of these six types of DNA viruses closely related to post-transplant complications includes: designing and screening six virus-specific primers and probes, optimizing the multiplex qPCR reaction system, improving the viral DNA extraction method, and quantifying viral load.

2. The multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation according to claim 1, characterized in that: The design and screening of the six virus-specific primers and probes are as follows: S1. Using a set of primers for detecting pathogens in organ transplantation, the genomes of BK virus, JC virus, B19 virus, CMV virus, adenovirus and TTV virus were compared and analyzed based on the NCBI database and a self-built virus sequence database, and conserved regions of each virus were screened as detection targets. S2. Design specific primers and probes for conserved regions of DNA viruses. Design multiple pairs of primers and probes, and screen out the best primer-probe combination through computer simulation and experimental verification. Each pathogen contains 1-3 pairs of specific primers, and the amplicon length is 70-100bp. S3. This primer and probe combination can be synthesized into one tube, enabling multiplex PCR detection within a single tube.

3. The multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation according to claim 2, characterized in that: In step S2, multiple pairs of primers and probes are designed and hydrolyzed using TaqMan. The probes are labeled with a fluorescent group at their 5' end and a quenching group at their 3' end. The annealing temperature of the probes must be higher than that of the primers to preferentially bind to the template.

4. The multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation according to claim 1, characterized in that: The optimization of the multiplex qPCR reaction system is as follows: T1. First, collect 8-10 mL of peripheral blood using a cell-free DNA sample preservation tube and ensure that it is delivered to the laboratory for processing within 3 days. If an EDTA anticoagulant tube is used for collection, plasma separation must be completed within 8 hours. T2. The plasma separation process adopts a two-step centrifugation method: first, centrifuge at 1600×g for 10 minutes, and aspirate the upper plasma layer to a new centrifuge tube, avoiding aspirating the middle layer of white blood cells; then, centrifuge at 12000×g at 4℃ for 10 minutes, and transfer the supernatant to a new centrifuge tube. The separated plasma can be stored at -20℃ for later use. T3. Viral DNA extraction was performed using a modified magnetic bead method. 200 μL of plasma sample was taken and 400 μL of lysis buffer was added. The sample was then incubated at 56°C for 30 minutes for complete lysis. T4. Then add an equal volume of phenol-chloroform-isoamyl alcohol, centrifuge at 12,000×g for 10 minutes, take the supernatant, add 0.8 times the volume of binding buffer and 50 μL of magnetic beads, mix at room temperature for 10 minutes, then place on a magnetic rack for separation, discard the supernatant, wash twice with 70% ethanol, air dry for 5 minutes, add 50 μL of elution buffer, incubate at 56℃ for 3 minutes, and collect the supernatant as template DNA. T5. The concentration of the extracted DNA was detected by a Qubit 4.0 fluorescence quantitative instrument, and the purity was determined by a spectrophotometer. For T6, in terms of standard curve preparation, each virus-specific fragment was cloned into the pUC57 vector, and the copy number was calculated after concentration determination. - Starting at a concentration of copies / μL, the solution was serially diluted 10-fold to prepare 10¹- Standards in copies / μL; T7. Plot a logarithmic standard curve of Ct value versus copy number using qPCR amplification to verify the linear range, ensure the accuracy and sensitivity of the detection system, and provide a reliable basis for the accurate quantification of BK virus, JC virus, CMV virus, B19 virus, adenovirus, and circovirus in subsequent clinical samples.

5. The multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation according to claim 4, characterized in that: The lysis buffer in step T3 contains 1 mg / mL proteinase K.

6. The multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation according to claim 4, characterized in that: In step T5, the A260 / A280 ratio in the purity is determined by a spectrophotometer to be 1.8-2.

7. The multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation according to claim 4, characterized in that: In step T7, the linear range is verified to be R²≥0.99, and the amplification efficiency is 90%-110%.

8. A multi-index combined non-invasive detection kit for graft injury, characterized in that: This method, employing any one of claims 1-7, is a multi-virus combined non-invasive detection method for assessing graft injury risk after solid organ transplantation. It is used for continuous monitoring of viral infections after clinical lung, heart, kidney, and liver transplantation, enabling timely detection of changes in the patient's physical condition after organ transplantation and assisting doctors in providing targeted treatment.

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