Primer and kit for multiple nucleic acid detection of organ transplantation infection virus and application of primer and kit

Abstract

The invention relates to the technical field of biology, in particular to primers and a kit for multiple nucleic acid detection of organ transplantation infection viruses and application of the primers and the kit. Only one-step amplification is adopted, thirteen organ transplantation infection viruses can be detected in the same reaction system at the same time, the specificity is good, and the sensitivity is high. The kit provided by the invention further comprises a reaction internal reference which can be used for monitoring the whole detection process of nucleic acid extraction, PCR amplification, capillary electrophoresis and the like, and an anti-pollution system can effectively avoid aerosol pollution.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to primers, a kit and applications thereof for multiple nucleic acid detection of organ transplant infection viruses. Background Art

[0002] Healthy people have normal immune function and can effectively resist infection by viruses and other microorganisms. However, in immunosuppressed or immunodeficient patients, such as stem cell / bone marrow transplant recipients who have undergone myeloablative pretreatment, solid organ transplant patients who use immunosuppressants for a long time, etc., the risk of viral infection or reactivation of latent viral infection in the body is significantly increased. These infections are not only the primary cause of death from non-primary diseases in patients, but also greatly increase the incidence of other side effects such as graft-versus-host disease and graft failure. In addition, the symptoms caused by various viral infection sources often overlap with the clinical manifestations of other side effects of transplant recipients, and the consequences and treatment options caused by different viruses are different. Therefore, timely and accurate diagnosis of pathogens is particularly critical. Molecular diagnostic methods based on polymerase chain reaction and sequencing technology have been widely used in clinical practice, but problems such as poor timeliness, high detection costs, and complex result analysis still plague clinical staff.

[0003] With the continuous advancement of medical technology, the introduction of new immunosuppressants and the optimization of postoperative care, the survival rate of various types of transplant patients has been significantly improved. However, viral infection is still one of the common complications after transplantation, and it is often the main cause of transplantation failure. BK virus, JC virus, cytomegalovirus and Epstein-Barr virus can remain dormant in the body for a long time after the initial infection. Due to the general low immune function of transplant patients, these viruses can be transformed from a latent state to an activated state, resulting in poor clinical prognosis. Therefore, postoperative monitoring of these viruses is crucial to ensure the success of the transplant. Common infectious viruses and their hazards include:

[0004] Herpes simplex virus (HSV): HSV includes type 1 and type 2. Type 1 mainly causes oral herpes (such as blisters around the lips and mouth), and can also cause eye infections, encephalitis, etc. Type 2 mainly causes genital herpes, and symptoms include blisters and ulcers in the genital area.

[0005] Varicella-zoster virus (VZV): causes chickenpox (primary infection) and shingles (recurrent infection), etc., sometimes accompanied by neuralgia.

[0006] Cytomegalovirus (CMV): Humans are the only host of CMV, and CMV can infect almost all organs. The active CMV infection rate within 6 months after organ transplantation is 50%-70%, of which 10%-30% develop into CMV disease. CMV infection can cause fever, organ dysfunction, pneumonia, hepatitis, etc. Severe infection may cause organ rejection and affect the long-term function of the transplanted organ.

[0007] Epstein-Barr virus (EBV): When the body is immunodeficient or the immune system is in a suppressed state, it is very susceptible to EB virus infection, causing serious lymphoproliferative diseases, plasma cell proliferation, polymorphic lymphoproliferative disorder, malignant non-Hodgkin's lymphoma, multiple myeloma, etc., which may eventually lead to a decline in the function of the transplanted organ.

[0008] Human herpesvirus (HHV): HHV includes human herpesvirus types 6, 7, and 8. Types 6 and 7 mainly cause inflammation of the nervous system and internal organs and are associated with roseola and other skin lesions. Type 8 is mainly associated with Kaposi's sarcoma and is particularly common in AIDS patients.

[0009] JC polyomavirus: More than 80% of adults are positive for JC virus antibodies, but most people do not have obvious symptoms after being infected with the JC virus. The immune system of healthy adults can keep the JC virus dormant. However, for patients with immune deficiency and long-term use of immunosuppressive drugs, it can cause progressive multifocal leukoencephalopathy, leading to extensive damage to the white matter of the brain, resulting in cognitive impairment, vision loss, poor motor coordination, hemiplegia and other symptoms.

[0010] BK polyomavirus: BK virus primary infection occurs in childhood. About 0.5% to 20% of infected people will experience BKV reactivation, leading to viral replication, which generally does not cause renal damage. However, in people receiving immunosuppressive therapy, especially those who have undergone kidney and bladder transplantation, the rate of BKV reactivation will increase significantly and may lead to BKV-associated nephropathy (BKVAN). BKV can be detected in the urine of 10% to 60% of patients after kidney transplantation.

[0011] Human parvovirus B19: It is widely present in the population and is mainly transmitted through respiratory secretions. The B19 virus can infect and destroy red blood cell-producing cells in the bone marrow, leading to acute or chronic anemia; it can also cause joint and muscle pain, accompanied by a rash.

[0012] Adenovirus: In immunosuppressed patients, adenovirus infection may cause systemic inflammatory response syndrome (SIRS), which manifests as high fever, general malaise, etc. It may also lead to severe viral pneumonia, adenovirus gastroenteritis, etc.

[0013] Transplant-transmitted virus (TTV): A small circular single-stranded DNA virus that is transmitted through transplantation, blood transfusion, or other blood contact and may be associated with certain liver diseases (e.g., hepatitis, cirrhosis, etc.).

[0014] Therefore, regular viral monitoring is crucial in the above-mentioned transplant patients. Effective detection methods can help doctors detect viral infections as early as possible, adjust treatment plans in a timely manner, avoid worsening of the disease, and improve the success rate of transplantation. At the same time, since different viral infections may require different antiviral drugs and treatment strategies, accurate detection can also guide personalized treatment. Regular data can reflect the dynamic changes of patients' viral infections and help develop individualized management plans. In addition, postoperative monitoring can also help identify and isolate patients at risk of infection, reduce the spread of the virus in hospitals or communities, and protect other immunosuppressed patients.

[0015] However, existing detection methods have problems such as insufficient sensitivity and cumbersome operation, and the targets that can be detected simultaneously are limited. As the number of targets increases, the difficulty of primer design also increases. So far, there has been no report on the simultaneous detection of the above ten viruses. Summary of the invention

[0016] In view of this, the present invention provides primers, kits and applications thereof for multiple nucleic acid detection of organ transplant infection viruses. The primer combination simultaneously and rapidly detects thirteen viruses, including herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus type 6, human herpes virus type 7, human herpes virus type 8, JC polyomavirus, BK polyomavirus, human parvovirus B19, adenovirus and TTV virus, and has the advantages of good specificity and high sensitivity. The present invention provides more accurate digital analysis in combination with an automated system, which helps to more quickly and clearly identify different viruses in multiple infections.

[0017] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0018] The primer combination comprises at least one pair of the following primers:

[0019] Primer pair 1 for detecting JC polyomavirus: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 1 to 2, or sequences having at least 80% homology to SEQ ID NOs: 1 to 2;

[0020] Primer pair 2 for detecting transplant-transmitted viruses: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 3-4, or sequences having at least 80% homology to SEQ ID NOs: 3-4;

[0021] Primer pair 3 for detecting adenovirus: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 5 to 6, or sequences having at least 80% homology to SEQ ID NOs: 5 to 6;

[0022] Primer pair 4 for detecting BK polyomavirus: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 7 to 8, or sequences having at least 80% homology to SEQ ID NOs: 7 to 8;

[0023] Primer pair 5 for detecting cytomegalovirus: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 9 to 10, or sequences having at least 80% homology to SEQ ID NOs: 9 to 10;

[0024] Primer pair 6 for detecting human herpesvirus type 6: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 11 to 12, or sequences having at least 80% homology to SEQ ID NOs: 11 to 12;

[0025] Primer pair 7 for human parvovirus B19: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 13-14, or sequences having at least 80% homology to SEQ ID NOs: 13-14;

[0026] Primer pair 8 for detecting Epstein-Barr virus: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 15 to 16, or sequences having at least 80% homology to SEQ ID NOs: 15 to 16;

[0027] Primer pair 9 for detecting herpes simplex virus type 1: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 17 to 18, or sequences having at least 80% homology to SEQ ID NOs: 17 to 18;

[0028] Primer pair 10 for detecting herpes simplex virus type 2: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 19 to 20, or sequences having at least 80% homology to SEQ ID NOs: 19 to 20;

[0029] Primer pair 11 for detecting varicella zoster virus: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 21-22, or sequences having at least 80% homology with SEQ ID NOs: 21-22;

[0030] Primer pair 12 for detecting human herpesvirus type 8: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 23-24, or sequences having at least 80% homology to SEQ ID NOs: 23-24;

[0031] Primer pair 13 for detecting human herpesvirus type 7: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 25-26, or sequences having at least 80% homology with SEQ ID NOs: 25-26.

[0032] In the primer combination of the present invention, the 5' end of the upstream primer of the primer pair is modified with a fluorescent group. The fluorescent group is FAM, HEX, ROX, VIC, CY3 or CY5. In a specific embodiment of the present invention, the 5' end of the upstream primer is modified with FAM.

[0033] The present invention also provides the use of the primer combination in preparing a kit for detecting multiple nucleic acid detection of organ transplant infection viruses.

[0034] The present invention also provides a kit for multiple nucleic acid detection of organ transplant infection viruses, which comprises the primer combination.

[0035] In the present invention, the kit also includes a primer pair for detecting human internal reference genes; the primer pair for detecting human internal reference genes has a sequence as shown in SEQ ID NOs: 27-28, or a sequence having at least 80% homology with SEQ ID NOs: 27-28.

[0036] In the present invention, the kit further comprises a primer pair for detecting an internal reference gene of the reaction; the primer pair for detecting an internal reference gene of the reaction has a sequence as shown in SEQ ID NOs: 29 to 30, or a sequence having at least 80% homology with SEQ ID NOs: 29 to 30.

[0037] In the present invention, the kit further comprises at least one of a PCR reaction reagent, a nucleic acid extraction reagent, and a nucleic acid purification reagent; the PCR reaction reagent comprises a buffer, MgCl 2 , KCL, dimethyl sulfoxide, dNTP and enzyme. The present invention has no special restrictions on the specific types and sources of nucleic acid extraction reagents and nucleic acid purification reagents, and any reagent commonly used in the art can be used, and commercial reagents can be purchased or prepared by oneself.

[0038] The present invention also provides a method for detecting organ transplant infection viruses for non-diagnostic purposes, comprising: using the primer combination or the kit to perform PCR amplification on the sample, using capillary gel electrophoresis to separate and determine the amplification products, and obtaining detection results of characteristic targets.

[0039] The criteria for determination include: if the peak height of the characteristic target is ≥500RFU, the characteristic target is determined to be positive.

[0040] The present invention combines multiplex PCR technology with capillary electrophoresis fragment analysis, and can detect multiple viruses efficiently and sensitively in a single test. The virus detection results are intuitively displayed through electrophoresis patterns, which simplifies the result interpretation process and facilitates clinical doctors to make judgments.

[0041] The primer combination of the present invention is used for in vitro qualitative detection of 13 common organ transplant infection viruses in human serum or plasma, urine and cerebrospinal fluid samples. The viruses include: herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus type 6, human herpes virus type 7, human herpes virus type 8, JC polyoma virus, BK polyoma virus, human parvovirus B19, adenovirus and TTV virus. The present invention adopts a new generation of fragment analysis technology (Advanced Fragment Analysis, AFA), takes the highly conserved sequences of 13 common organ transplant infection virus nucleic acids as the target region, designs 13 groups of specific primers, and performs multiple PCR amplification in one amplification tube; uses capillary electrophoresis to separate amplification products of different lengths to obtain the detection results of pathogens; monitors the sample quality by simultaneously detecting human DNA in the sample; the reagent of the present invention carries a reaction internal reference, which can be used to monitor the entire detection process such as nucleic acid extraction, PCR amplification and capillary electrophoresis, and the anti-pollution system can effectively avoid aerosol contamination.

[0042] Compared with traditional multiplex PCR, the present invention has the following advantages:

[0043] 1) Easy to operate: The operation steps of multiplex PCR are simplified, and multiple viruses can be quickly detected simultaneously through only one amplification in fewer body fluid specimens.

[0044] 2) High sensitivity: PCR capillary electrophoresis fragment analysis combines the high amplification efficiency of PCR technology and the high resolution of capillary electrophoresis, which can more accurately distinguish the gene fragments of different viruses and reduce false positive and false negative results.

[0045] 3) Semi-quantitative analysis capability: Capillary electrophoresis fragment analysis can perform semi-quantitative evaluation by analyzing the height and area of ​​the electrophoretic peak, which is of great significance for evaluating changes in viral load and helping doctors better monitor the disease and adjust treatment plans.

[0046] 4) High-throughput detection: Capillary electrophoresis technology has the characteristics of high degree of automation and can process multiple samples simultaneously. It is suitable for large-scale screening and detection, especially when monitoring multiple viral infections after organ transplantation, which improves the detection efficiency.

[0047] 5) The results are more intuitive: The capillary electrophoresis pattern can intuitively display the size and number of different viral fragments, which facilitates the interpretation of the results and subsequent analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Shows the peak type reference diagram of the positive control for organ transplantation;

[0049] Figure 2 Reference graph of negative control peak type for organ transplantation

[0050] Figure 3 Shows the positive detection rate of various viruses;

[0051] Figure 4 The positive sample is shown as one;

[0052] Figure 5 The second positive sample. DETAILED DESCRIPTION

[0053] The present invention provides primers, kits and applications for multiple nucleic acid detection of organ transplant infection viruses. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0054] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0055] The present invention will be further described below in conjunction with embodiments:

[0056] Example 1 Detection primer probe of the present invention

[0057] The primer and probe sequences are shown in Table 1.

[0058] Table 1 Primer probe sequences

[0059]

[0060]

[0061] Example 2 Detection method

[0062] 1. Sample nucleic acid extraction

[0063] Extract nucleic acids according to the instructions of nucleic acid extraction or purification reagents. Each sample involved in the extraction needs to be added with 2 μL of organ transplant reaction internal reference for co-extraction.

[0064] 2. PCR amplification

[0065] Add reagents and samples to the 96-well sample plate / eight-well tube strip / PCR tube according to the following ratios.

[0066] Table 3

[0067]

[0068]

[0069] The organ transplant premix contains all amplification primers, PCR reaction reagents and water, specifically including: 7 μL of total primers, 5 μL of PCR reaction reagents, and 2 μL of water; the concentration of total primers in the premix is ​​60 μM.

[0070] PCR reaction reagents are reaction reagents other than enzymes, including buffer, MgCl 2 , KCl, dimethyl sulfoxide, dNTP.

[0071] After mixing, perform thermal cycling reaction at the following temperatures:

[0072] Table 4

[0073]

[0074] 3. Capillary electrophoresis separation of samples

[0075] 1) Preparation of electrophoresis samples

[0076] Table 5

[0077]

[0078] 2) Separate samples by capillary electrophoresis on the 3500 Gene Analyzer: Select the “Fragment” electrophoresis method (see the 3500 Gene Analyzer manual for details).

[0079] 3) Positive judgment value

[0080] If the peak height of the characteristic target is ≥500RFU, the target is judged to be positive.

[0081] 4) Interpretation of test results

[0082] (1) In the peak pattern diagram using the organ transplantation positive control as a template, 14 characteristic peaks must appear, and the peak heights of the 14 characteristic peaks must all be ≥500 RFU, and the absolute difference between the actual fragment length and the reference fragment length must be ≤1.5 nt ( Figure 1 ), the corresponding amplified fragment sizes of each detection target are shown in Table 6.

[0083] (2) The characteristic peak of human DNA internal reference (huDNA) must appear in the peak pattern graph using the organ transplantation negative control as the template, and the peak height must be ≥500RFU, and the absolute difference between the actual fragment length and the reference fragment length must be ≤1.5nt ( Figure 2); the target peak for pathogen detection by this kit should not appear.

[0084] (3) If the huDNA characteristic peak appears in the sample to be tested and the peak height is ≥500RFU, it means that the sampling is successful, the sample nucleic acid is not significantly degraded, and the nucleic acid extraction is successful. Otherwise, it means that the sampling failed, the nucleic acid was severely degraded, or the nucleic acid extraction failed.

[0085] (4) IC is used as a built-in reaction internal reference to monitor the entire operation process. If this peak appears in the electrophoresis results and the peak height is ≥1000RFU, it means that the PCR process and capillary electrophoresis are normal; otherwise, it means that the PCR or capillary electrophoresis has failed. However, if other characteristic peaks are obvious, the reaction internal reference peak may be lower or even disappear due to competitive amplification.

[0086] (5) When the above four points are met at the same time, the pathogen detection results are analyzed.

[0087] Table 6 Gene loci and detection fragment size information (size deviation within ±1.5nt)

[0088] Test content Target Fragment size (nt) ±1.5nt Herpes simplex virus type 1 HSV-1 260.71 Herpes simplex virus type 2 HSV-2 267.26 Varicella-zoster virus V Z 303.29 Epstein-Barr virus EBV 210.74 Cytomegalovirus CMV 159.90 Human herpesvirus 6 HHV-6 173.06 Human herpesvirus-7 HHV-7 333.41 Human herpesvirus-8 HHV-8 327.09 JC polyomavirus JbXj 103.63 BK polyomavirus BkV 142.8 Human parvovirus B19 HPV B19 196.73 Adenovirus ADV / ADV-2 131.35 / 150.00 Human genomic DNA HuDNA 185.75 Transplant-transmitted virus TTV 123.13 Internal reference IC 232.08

[0089] Note: The 3500 platform here uses SIZE-500Plus as the internal standard, 50cm capillary, and POP7 gel for the fragment size.

[0090] Example 3 Detection rate test

[0091] The primer pair of Example 1 was used to detect 148 samples according to the method of Example 2. The sample types included plasma, serum and urine. Among them, 4 samples were negative and 144 samples were positive. The positive detection rate was shown in Figure 3 Among the 144 positive samples, 76 (52.78%) were infected with at least two viruses, and the results were easy to distinguish. The results of two random positive samples are detailed in the attached Figure 4 and Figure 5 .

[0092] Example 4 Sensitivity Test

[0093] After synthesizing an artificially synthesized plasmid containing all detection targets and accurately quantifying the plasmid using digital PCR, each target is detected using the primers of the present invention according to its own gradient. The minimum detection limit of each virus is shown in the following table:

[0094] Table 7

[0095]

[0096] The results showed that the primer pair of the present invention can detect the above viruses simultaneously with high sensitivity.

[0097] Example 5 Specificity Test

[0098] The 13 viruses and other pathogens described in the present invention were detected using the primers in Example 1 and the method in Example 2. The specific results are shown in Table 8.

[0099] Table 8

[0100]

[0101]

[0102] Note: “-” indicates negative, “+” indicates positive, and “ / ” indicates that the sample or result does not exist.

[0103] The results showed that the present invention only tested positive for the samples of the 13 organ transplantation infection virus plasmids, i.e., the positive control, and the other tubes (other viruses, bacteria, and fungi) were all negative. This shows that the detection kit of the present invention has high specificity and can accurately and specifically detect the 13 viruses of the present invention without non-specific amplification.

[0104] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer combination, characterized in that: Includes at least one pair of primers as follows: Primer pair 1: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 1 to 2, or sequences having at least 80% homology to SEQ ID NOs: 1 to 2; Primer pair 2: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 3-4, or sequences having at least 80% homology to SEQ ID NOs: 3-4; Primer pair 3: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 5-6, or sequences having at least 80% homology to SEQ ID NOs: 5-6; Primer pair 4: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 7 to 8, or sequences having at least 80% homology to SEQ ID NOs: 7 to 8; Primer pair 5: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 9 to 10, or sequences having at least 80% homology to SEQ ID NOs: 9 to 10; Primer pair 6: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 11-12, or sequences having at least 80% homology to SEQ ID NOs: 11-12; Primer pair 7: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 13-14, or sequences having at least 80% homology to SEQ ID NOs: 13-14; Primer pair 8: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 15-16, or sequences having at least 80% homology to SEQ ID NOs: 15-16; Primer pair 9: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 17-18, or sequences having at least 80% homology to SEQ ID NOs: 17-18; Primer pair 10: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 19 to 20, or sequences having at least 80% homology to SEQ ID NOs: 19 to 20; Primer pair 11: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 21-22, or sequences having at least 80% homology to SEQ ID NOs: 21-22; Primer pair 12: the upstream and downstream primers have the sequences shown in SEQ ID NOs: 23-24, or sequences having at least 80% homology to SEQ ID NOs: 23-24; Primer pair 13: The upstream and downstream primers have the sequences shown in SEQ ID NOs: 25-26, or sequences having at least 80% homology to SEQ ID NOs: 25-26.

2. The primer combination according to claim 1, characterized in that The 5' end of the upstream primer is modified with a fluorescent group.

3. The primer combination according to claim 2, characterized in that: The fluorescent group includes FAM.

4. Use of the primer combination according to any one of claims 1 to 3 in the preparation of a kit for multiple nucleic acid detection of organ transplant infection viruses.

5. A kit, characterized in that: It comprises the primer combination according to any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that Also included are primer pairs for human internal reference genes; the primer pairs for detecting human internal reference genes have sequences as shown in SEQ ID NOs: 27-28, or sequences with at least 80% homology to SEQ ID NOs: 27-28.

7. The kit according to claim 5, characterized in that Also included are primer pairs for reaction internal references; the primer pairs for detecting reaction internal reference genes have sequences as shown in SEQ ID NOs: 29-30, or sequences with at least 80% homology to SEQ ID NOs: 29-30.

8. The kit according to any one of claims 5 to 7, characterized in that It also includes at least one of a PCR reaction reagent, a nucleic acid extraction reagent, and a nucleic acid purification reagent; the PCR reaction reagent includes a buffer, MgCl2, KCl, dimethyl sulfoxide, dNTP, and an enzyme.

9. A method for detecting organ transplant virus infection for non-diagnostic purposes, characterized in that: The sample is amplified by PCR using the primer combination described in any one of claims 1 to 3 or the kit described in any one of claims 5 to 8, and the amplified product is separated and determined to obtain the detection result of the characteristic target.

10. The method according to claim 9, characterized in that The separation method is capillary gel electrophoresis; The criteria for determination include: if the peak height of the characteristic target is ≥500RFU, the characteristic target is determined to be positive.

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