Universal and differential detection of human circovirus by PCR detection primer group, kit and application

Abstract

This invention discloses a universal and typing PCR detection primer set, kit, and its applications for human porcine circovirus (HCirV). The typing primer set sequences are shown in SEQ ID NO:2-9, and the universal primer set sequences are shown in SEQ ID NO:10-11. The PCR detection primer set and kit provided by this invention can specifically detect human porcine circovirus (HCirV), as well as HCirV-A, HCirV-B, HCirV-C, and HCirV-D, without cross-reactivity with other DNA pathogens, exhibiting excellent specificity. The primer set and kit provided by this invention enable low-cost detection and typing of human circovirus samples in a short time, and have broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of viral biological detection technology, specifically to a universal and typing PCR detection primer set, kit, and application for detecting human porcine circovirus. Background Technology

[0002] Circoviruses belong to the genus Circovirus in the family Circoviridae. They are non-enveloped, single-stranded DNA viruses with a diameter of approximately 15-20 nm, and are considered one of the smallest known autonomously replicating viruses. Circoviruses were initially widely reported in animals such as pigs and poultry, and are closely associated with a range of diseases, including porcine circovirus-associated disease (PCVAD). In recent years, the newly discovered human circovirus (HCirV) has gradually attracted significant attention from the international academic and clinical communities. In 2022, HCirV was first detected and reported in plasma samples from injecting drug users in Yunnan Province, my country. In the same year, HCirV was also detected in liver tissue from a patient with acute hepatitis in France, and in situ hybridization confirmed its replication in hepatocytes. In 2024, HCirV was re-isolated from a sample of an immunocompromised patient in Switzerland, and the virus remained detectable for up to 21 months after the patient's recovery, suggesting that HCirV may exhibit characteristics of chronic or latent infection. Epidemiological surveys in Hong Kong, my country, show that the detection rate of HCIrV among hepatitis patients is approximately 2.9%, further supporting its association with the onset of hepatitis. PCR technology, as a mature molecular detection method, has low equipment requirements and is easy to operate, making it suitable for use in grassroots laboratories or large-scale screening. However, there is a critical gap in the detection of HCIrV: there is currently no detection kit that can simultaneously achieve universal screening and specific genotyping of AD subtypes, resulting in problems such as insufficient specificity, complex operation, and high cost. Therefore, there is an urgent need for a detection method that combines universal detection and AD subtype genotyping capabilities, is easy to operate, highly sensitive, and inexpensive to meet the needs of clinical diagnosis and public health surveillance. Summary of the Invention

[0003] The purpose of this invention is to overcome the problems of existing HCirV detection methods, such as reliance on expensive equipment and lack of genotyping capabilities, and to achieve universal detection and genotyping of HCirV-A, HCirV-B, HCirV-C, and HCirV-D on a conventional PCR platform, while considering sensitivity, specificity, repeatability, and applicability. To achieve the above objectives, this invention adopts the following technical solution: A first aspect of the present invention provides a specific primer pair for the human circovirus HCirV-D subtype, the primer pair binding to a specific variant region of the HCirV-D subtype genome for amplification; the amino acid sequence of the HCirV-D subtype is shown in SEQ ID NO:1.

[0004] In a specific embodiment of the present invention, the primer parameters are optimized to be 18-27 bp in length, 45-55% GC content, 55-60℃ Tm value, and ≤2℃ difference in Tm value between upstream and downstream primers; hairpin structures within primers and complementarity between primers (especially at the 3' end) are avoided to prevent dimer formation; all 3' end bases are avoided to be T to reduce the risk of mismatch; specificity verification: BLAST alignment is used to ensure no homology with other pathogens.

[0005] Furthermore, the first primer also includes a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO:8.

[0006] Furthermore, the second primer also includes a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO:9.

[0007] In this invention, the term "human circovirus" refers to a class of circoviruses capable of infecting humans or originating from human samples. Their genomes are typically single-stranded circular DNA, with a genome length of approximately 1.7 kb to 2.2 kb, and usually contain open reading frames (ORFs) encoding replication-related proteins and capsid proteins. The human circoviruses include known subtypes, strains, variants, and their derivatives, as well as the newly discovered human circovirus D subtype and its sequence-homogeneous variants, recombinants, mutants, natural isolates, or artificially modified strains. The term "upstream primer" refers to an oligonucleotide sequence that can bind complementary to the target nucleic acid sequence and provide a DNA strand extension initiation site during nucleic acid amplification, with its binding site located upstream of the 5' end of the target amplification region. The term "downstream primer" refers to an oligonucleotide sequence that can bind to the complementary strand of the target nucleic acid sequence and form an amplification termination boundary during nucleic acid amplification, with its binding site located downstream of the 3' end of the target amplification region. The downstream primer is typically used in conjunction with the upstream primer to achieve specific amplification of the target nucleic acid region.

[0008] A second aspect of the invention provides a set of primer pairs for detecting different subtypes of human circovirus, the set including the primer pairs described in the first aspect of the invention.

[0009] Furthermore, the set also includes specific primer pairs for human circovirus HCirV-A, HCirV-B, and HCirV-C subtypes.

[0010] Furthermore, the specific primer pair for the HCirV-A subtype consists of the sequences shown in SEQ ID NO:2-3.

[0011] Furthermore, the nucleotide sequences of the specific primer pairs for the HCirV-B subtype are shown in SEQ ID NO:4-5.

[0012] Furthermore, the specific primer pair for the HCirV-C subtype consists of the sequences shown in SEQ ID NO:6-7.

[0013] Furthermore, the set also includes universal primer pairs against human circovirus, the universal primer pairs consisting of the sequences shown in SEQ ID NO:10-11.

[0014] In this invention, the term "universal primer pair" refers to a pair of primers capable of simultaneously recognizing, binding to, and amplifying conserved nucleic acid regions in multiple different viral subtypes, genotypes, strains, or variants. The universal primer pair is designed to target conserved sequence regions common to different human circovirus subtypes, thereby enabling simultaneous detection, pre-screening, or broad-spectrum amplification of multiple human circovirus subtypes. The universal primer pair may contain one or more degenerate bases to improve compatibility and coverage with different variant sequences.

[0015] A third aspect of the present invention provides a reagent combination for detecting human porcine circovirus, characterized in that the reagent combination comprises the primer pairs described in the first aspect of the present invention or the primer pair set described in the second aspect of the present invention.

[0016] Furthermore, the reagent combination also includes reagents for nucleic acid amplification.

[0017] Furthermore, the reagents used for nucleic acid amplification are selected from DNA polymerase, dNTPs, buffer solutions, and reagents containing Mg. 2+ Any one of the following in the solution.

[0018] In a specific embodiment of the present invention, the reagent for nucleic acid amplification comprises Taq DNA polymerase (5 U / μL), a mixture of dNTPs (10 mM each), PCR buffer (200 mM Tris-HCl pH 8.3, 1000 mM KCl) and 25 mM MgCl2.

[0019] Furthermore, the primer pairs and / or primer pair sets exist in a physically mixed or physically separated form with the reagents used for nucleic acid amplification.

[0020] A fourth aspect of the present invention provides a nucleic acid chip, wherein the primer pairs described in the first aspect or the primer pair set described in the second aspect of the present invention are fixed on the chip.

[0021] In this invention, the term "nucleic acid chip" refers to a detection platform that uses a solid-phase carrier to immobilize specific nucleic acid probes and utilizes the principle of nucleic acid hybridization to achieve high-throughput detection, analysis, or typing of target nucleic acids. The nucleic acid chip includes, but is not limited to, DNA chips, primer chips, microarray chips, biochips, liquid-phase chips, and microfluidic chips. The nucleic acid chip can be used for the simultaneous detection, identification, and typing analysis of different human circovirus subtypes in this invention. The solid-phase carrier includes, but is not limited to, glass slides, silicon wafers, polymer materials, nanomaterials, magnetic beads, or microspheres.

[0022] A fifth aspect of the present invention provides a kit for detecting human porcine circovirus, the kit comprising any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the reagent combination described in the third aspect, and the nucleic acid chip described in the fourth aspect.

[0023] Furthermore, the kit also includes one or more of a positive control and a negative control.

[0024] In this invention, the term "positive control" refers to a control sample containing a known target nucleic acid sequence and capable of producing the expected amplification result or detection signal, used to verify whether the detection system, amplification system, or reagent performance is normal. The positive control includes, but is not limited to, positive plasmids, artificially synthesized nucleic acids, positive samples, standards, or recombinant vectors.

[0025] Furthermore, the positive controls include recombinant plasmids containing the complete HCirV-A genome, recombinant plasmids containing the complete HCirV-B genome, recombinant plasmids containing the complete HCirV-C genome, and recombinant plasmids containing the complete HCirV-D genome.

[0026] The term "negative control" refers to a control sample that does not contain the target nucleic acid sequence and should theoretically not produce a specific amplification signal, used to exclude contamination, nonspecific amplification, or false positive results. Negative controls include, but are not limited to, template-free controls (NTCs), negative samples, or nucleic acid systems that do not contain the target sequence.

[0027] Furthermore, the negative control is nuclease-free water.

[0028] Furthermore, the kit also includes instructions for interpreting test results, which are determined based on the amplification curve or the size of the electrophoretic bands.

[0029] A sixth aspect of the present invention provides a method for detecting human porcine circovirus, the method comprising the following steps: (1) A method for detecting human porcine circovirus, the method comprising the following steps: performing PCR amplification of nucleic acid in a sample to be tested using any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the reagent combination described in the third aspect, the nucleic acid chip described in the fourth aspect, and the reagent kit described in the fifth aspect; detecting the amplification product, and determining the presence and subtype of human porcine circovirus based on the detection results; (2) A method for screening drugs for treating diseases caused by human porcine circovirus infection, the method comprising the following steps: a) The test drug came into contact with a cell model infected with human porcine circovirus; b) Use any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the reagent combination described in the third aspect, the nucleic acid chip described in the fourth aspect, or the kit described in the fifth aspect to detect the change in human circovirus genome copy number in cell models before and after drug administration; c) Screening for test drugs with significant inhibitory effects as candidate drugs for the treatment of diseases caused by human circovirus infection.

[0030] In this invention, the test samples include, but are not limited to, biological samples derived from humans, animals, or the environment, including blood, plasma, serum, whole blood, tissue, saliva, nasopharyngeal swabs, anal swabs, feces, urine, bronchoalveolar lavage fluid, cell culture medium, tissue homogenate, pathological sections, environmental monitoring samples, and their derivative products. The test samples can be used for virus detection in this invention after nucleic acid extraction, lysis, purification, enrichment, reverse transcription, or pre-amplification.

[0031] Furthermore, the samples to be tested include blood, nasopharyngeal swabs, oropharyngeal swabs, sputum, and liver tissue.

[0032] In specific embodiments of this invention, DNA can be extracted from samples using either a silica gel membrane centrifuge column method or a magnetic bead method. For the silica gel membrane centrifuge column method, follow the kit instructions: take 200 μL of sample, add lysis buffer and proteinase K, incubate at 56°C for 30 min, add ethanol and mix well, centrifuge, wash twice with washing buffer, and finally elute nucleic acids with 50 μL of elution buffer. For the magnetic bead method, take 200 μL of sample, add magnetic beads and binding buffer, incubate at room temperature for 5 min, allow the magnetic beads to adhere using a magnetic field, wash three times with washing buffer, and elute nucleic acids with elution buffer. The extracted nucleic acid is measured using a spectrophotometer to determine the A260 / A280 ratio, which should be greater than 1.8 to ensure acceptable purity.

[0033] In this invention, the term "PCR amplification" refers to the process of exponentially amplifying a target nucleic acid sequence in vitro through cyclic reactions such as denaturation, annealing, and extension in the presence of primers, polymerase, nucleotides, and a reaction buffer system. The PCR amplification includes, but is not limited to, conventional PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), multiplex PCR, reverse transcription PCR (RT-PCR), nested PCR, isothermal amplification, and their derivative forms.

[0034] Furthermore, the method also includes a PCR reaction system, specifically comprising: 12.5 μL of PCR premix, 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 2 μL of template DNA, and nuclease-free water to a final volume of 25 μL.

[0035] Furthermore, the PCR reaction conditions set in the method are as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min. These reaction conditions were optimized through annealing temperature gradient experiments (55-60℃), with 58℃ being the optimal annealing temperature, which effectively balances amplification efficiency and specificity.

[0036] Furthermore, the method also includes electrophoresis detection of the PCR product, and determining whether the sample contains HCirV or a specific genotype based on the size of the amplified band: Take 5 μL of PCR product and mix it with 1 μL of loading buffer, add it to the loading well of a 1.0% agarose gel (containing 0.01% nucleic acid dye), use 1× TBE buffer as the electrophoresis buffer, electrophores at 120V for 30 min, and observe the results under UV light.

[0037] Further, in step (3), if the universal primer pair amplifies a band of 725-742 bp, the sample is determined to contain human circovirus; if the specific primer pair for HCirV-A subtype amplifies a band of 869 bp, the sample is determined to contain HCirV-A; if the specific primer pair for HCirV-B subtype amplifies a band of 1739 bp, the sample is determined to contain HCirV-B; if the specific primer pair for HCirV-C subtype amplifies a band of 310 bp, the sample is determined to contain HCirV-C; if the specific primer pair for HCirV-D subtype amplifies a band of 937 bp, the sample is determined to contain HCirV-D.

[0038] A seventh aspect of the present invention provides an apparatus for detecting human porcine circovirus, the apparatus comprising any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the reagent combination described in the third aspect, the nucleic acid chip described in the fourth aspect, and the reagent kit described in the fifth aspect.

[0039] Furthermore, the device also includes a hardware module for performing nucleic acid amplification and detection.

[0040] In this invention, the hardware module can automatically determine the subtype of the virus to be tested based on preset thresholds, amplification curves, melting curves, fluorescence intensity changes, machine learning models, or a viral subtype database. The device can connect to cloud databases, hospital information systems, disease monitoring systems, or laboratory information management systems to achieve virus detection result sharing, epidemiological monitoring, and remote analysis.

[0041] The eighth aspect of the present invention provides any of the following applications: (1) The application of the primer pairs described in the first aspect and the primer pair sets described in the second aspect of the present invention in the preparation of reagents and kits for non-diagnostic purposes for the detection of human porcine circovirus; (2) The application of any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the nucleic acid chip described in the third aspect, the reagent combination described in the fourth aspect, the reagent kit described in the fifth aspect, the method described in the sixth aspect, and the device described in the seventh aspect in the detection of human circovirus for non-diagnostic purposes; (3) Any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the nucleic acid chip described in the third aspect, the reagent combination described in the fourth aspect, the reagent kit described in the fifth aspect, the method described in the sixth aspect, and the device described in the seventh aspect may be used in the preparation of products for diagnosing human circovirus infection; (4) Any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the nucleic acid chip described in the third aspect, the reagent combination described in the fourth aspect, the reagent kit described in the fifth aspect, the method described in the sixth aspect, and the device described in the seventh aspect may be used in the preparation of products for diagnosing diseases caused by human porcine circovirus infection; (5) The use of any one of the primer pairs described in the first aspect of the present invention, the primer pair set described in the second aspect, the nucleic acid chip described in the third aspect, the reagent combination described in the fourth aspect, the reagent kit described in the fifth aspect, the method described in the sixth aspect, and the device described in the seventh aspect in screening drugs for treating diseases caused by human circovirus infection.

[0042] Furthermore, the diseases caused by the human circovirus infection include, but are not limited to, respiratory diseases, gastrointestinal diseases, febrile diseases, immune-related diseases, virus-related inflammatory diseases, liver diseases, nervous system-related diseases, and infectious diseases of unknown cause. Even further, the diseases include diarrhea, gastroenteritis, respiratory infections, pneumonia, fever, abnormal immune function, liver damage, encephalitis-like symptoms, and related diseases accompanied by viremia.

[0043] In this invention, the application in the preparation of reagents and kits for non-diagnostic purposes for the detection of human porcine circovirus refers to the use of the primer sets and primer pairs provided by this invention for industrial or scientific research purposes in the preparation of chemical reagents for virus-related evolutionary studies, and does not involve specific medical procedures.

[0044] The application of non-diagnostic detection of human circovirus refers to detection that is detached from specific animal subjects and focuses on the physicochemical detection of the virus itself, such as screening environmental samples in epidemiological surveys or determining viral load in basic scientific research.

[0045] The application in the preparation of products for diagnosing diseases caused by human circovirus infection refers to the use of the product to determine whether an animal is infected with human circovirus; the application in the preparation of products for diagnosing diseases caused by human circovirus infection refers to detecting the presence of the virus while also associating it with the specific pathological state caused by the virus, for guiding medication or assessing the course of the disease; the application in screening drugs for treating diseases caused by human circovirus infection refers to using any one of the primer pairs described in the first aspect, the primer pair set described in the second aspect, the nucleic acid chip described in the third aspect, the reagent combination described in the fourth aspect, the reagent kit described in the fifth aspect, the method described in the sixth aspect, and the device described in the seventh aspect as tools to evaluate the efficacy of candidate compounds.

[0046] The advantages and beneficial effects of this invention are as follows: This invention utilizes PCR technology to establish a universal and typing method for detecting human porcine circovirus. This method is suitable for rapid screening and typing of human porcine circovirus, and is simple to operate, accurate, highly sensitive, and highly specific. It can be widely applied in clinical diagnosis, epidemiological surveys, and public health monitoring, and is particularly suitable for large-scale screening in primary healthcare institutions and resource-limited areas. Attached Figure Description

[0047] Figure 1 The evolutionary relationship between the newly discovered HCirV subtype and previously reported subtypes is shown in Figure A, which displays the pairwise alignment values ​​of the HCirV whole genome sequence; Figure B shows the evolutionary relationship of the HCirV whole genome. Figure 2This is a sliding window analysis diagram showing the genome-wide consistency of different HCirV subtypes, with a window size of 200 bp and a step size of 1 bp. Figure 3 This diagram illustrates the coverage areas of universal primers and HCirV AD-specific primers; where primer ad represents HCirV AD-specific primers, and the bar and line graphs above represent the Shannon index obtained statistically based on all available HCirV sequences. Figure 4 Figure A shows gel electrophoresis images of universal primers and subtype-specific primers for specific detection. Figure A shows the results of universal primer detection of HCirV AD type positive control, negative control, and other viral DNA; lane 1 is the HCirV-A positive control plasmid; lane 2 is the HCirV-B positive control plasmid; lane 3 is the HCirV-C positive control plasmid; lane 4 is the HCirV-D positive control plasmid; lane 5 is a saliva DNA sample from a healthy volunteer; lane 6 is a blood DNA sample from a healthy volunteer; lane 7 is a urine DNA sample from a healthy volunteer; lane 8 is a fecal DNA sample from a healthy volunteer; lane 9 is the HDV-1 virus plasmid; lane 10 is the HBV-D virus plasmid; and lane 11 is the HEV-3 virus plasmid. Figure B shows the specificity verification of the subtype-specific primers, using primers a, b, c, and d to amplify and detect plasmids carrying the complete genome of human circovirus subtypes A, B, C, and D, respectively.

[0048] Figure 5 The detection sensitivity of universal primers and subtype-specific primers is shown (10-fold serial dilution of plasmid template). The templates for each sub-lane from left to right are: 1×10 9 Up to 1×10 1 Copy / μL plasmid template. (AD) Amplification of human circovirus AD subtype plasmid template using universal primers; (EH) Amplification of human circovirus AD subtype plasmid template using subtype-specific primers. Detailed Implementation

[0049] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0050] Example 1: Primer Design and Synthesis The newly discovered HCirV subtypes share 78.91%-81.21% genome-wide identity with all previously reported sequences. Furthermore, they occupy different branches on the phylogenetic tree compared to other sequences. Referring to the ICTV standard of classifying circoviruses as separate species based on less than 80% genome-wide identity, we have divided HCirV into four subtypes based on their reporting time, with the evolutionary relationships as follows: Figure 1 Based on HCirV-A (GenBank: ON226770), HCirV-B (GenBank: ON677309), HCirV-C (GenBank: OZ282268), and HCirV-D (a newly discovered subtype) reported in NCBI, sequence alignment was performed using Geneious software to determine conserved regions. The alignment results are as follows: Figure 2 As shown.

[0051] Universal primers: targeting highly conserved genomic regions shared by all subtypes ( Figure 3 The "universal primer" covers the region to ensure that all HCirV subtypes can be amplified.

[0052] Genotyping primers (HCirV AD): Designed specifically for the variant regions of subtypes A, B, C, and D, each primer pair binds specifically to the genomic sequence of its corresponding subtype. Figure 3 For the regions corresponding to primers a, b, c, and d, Geneious was used to design universal and genotyping PCR primer sets, following these principles: primer length 18-27 bp; GC content 45-55%; Tm value 55-60℃; avoid hairpin structures within primers and dimers between primers, and avoid the last base at the 3' end being T; specificity verification: ensure no homology with other pathogens by BLAST; the designed primer sequences are shown in Table 1, synthesized by Suzhou Genewise Biotechnology Co., Ltd., purified by PAGE, and diluted to 10 μM for later use.

[0053] Table 1 Primer sequences and parameters for human porcine circovirus PCR detection

[0054] Example 2: Kit Composition and Preparation The kit of the present invention includes: PCR premix: 12.5 μL of PCR premix containing Taq DNA polymerase (5 U / μL), a mixture of dNTPs (10 mM each), PCR buffer (200 mM Tris-HCl pH 8.3, 1000 mM KCl) and 25 mM MgCl2; The upstream primer (10 μM) 0.5 μL and the downstream primer (10 μM) 0.5 μL (HCirV-AF / R, HCirV-BF / R, HCirV-CF / R, HCirV-DF / R, HCirV-ADF / R) described in Example 1 were both at a concentration of 10 μM. Positive control: 2 μL template DNA, containing a recombinant plasmid with the complete HCirV-A to D genome (concentration 1×10⁻⁶). 9 Method for preparing the positive control (copy / μL):

Claims

1. A specific primer pair for the human porcine circovirus HCirV-D subtype, characterized in that, The primer pair binds to the specific variant region of the HCirV-D subtype genome for its amplification; the nucleotide sequence of the HCirV-D subtype is shown in SEQ ID NO:

1.

2. The primer pair according to claim 1, characterized in that, The primer pair includes an upstream primer as shown in SEQ ID NO:8 and a downstream primer as shown in SEQ ID NO:

9. Preferably, the first primer further includes a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO:8; Preferably, the second primer further includes a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO:

9.

3. A primer pair set for detecting different subtypes of human porcine circovirus, characterized in that, The set includes the primer pairs as described in claims 1-2; Preferably, the set further includes specific primer pairs for human circovirus HCirV-A, HCirV-B, and HCirV-C subtypes; Preferably, the specific primer pair for the HCirV-A subtype consists of the sequences shown in SEQ ID NO:2-3; Preferably, the specific primer pair for the HCirV-B subtype consists of the sequences shown in SEQ ID NO:4-5; Preferably, the specific primer pair for the HCirV-C subtype consists of the sequences shown in SEQ ID NO:6-7; Preferably, the set further includes universal primer pairs against human circovirus, the universal primer pairs consisting of the sequences shown in SEQ ID NO:10-11.

4. A reagent combination for detecting human porcine circovirus, characterized in that, The reagent combination comprises the primer pairs as described in claims 1-2 or the primer pair set as described in claim 3; Preferably, the reagent combination further includes reagents for nucleic acid amplification; Preferably, the reagent for nucleic acid amplification is selected from DNA polymerase, dNTPs, buffer, and reagents containing Mg. 2+ Any one of the following in the solution; Preferably, the primer pairs and / or primer pair sets exist in a physically mixed or physically separated form with the reagents used for nucleic acid amplification.

5. A nucleic acid chip, wherein the primer pairs of claims 1-2 or the primer pair set of claim 3 are immobilized on the chip.

6. A kit for detecting human porcine circovirus, characterized in that, The kit comprises any one of the primer pairs of claims 1-2, the primer pair set of claim 3, the reagent combination of claims 4-5, and the nucleic acid chip of claim 5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes one or more of a positive control and a negative control; Preferably, the positive control includes a recombinant plasmid containing the complete HCirV-A genome, a recombinant plasmid containing the complete HCirV-B genome, a recombinant plasmid containing the complete HCirV-C genome, and a recombinant plasmid containing the complete HCirV-D genome; Preferably, the negative control is nuclease-free water.

8. A method for detecting human porcine circovirus, characterized in that, The method includes the following steps: Any of the following methods: (1) A method for detecting human porcine circovirus, the method comprising the following steps: performing PCR amplification of nucleic acid in a sample to be tested using any one of the primer pairs of claims 1-2, the primer pair set of claim 3, the reagent combination of claims 4-5, the nucleic acid chip of claim 5, and the kit of claims 6-7; detecting the amplification product, and determining the presence and subtype of human porcine circovirus based on the detection results; (2) A method for screening drugs for treating diseases caused by human porcine circovirus infection, the method comprising the following steps: a) The test drug came into contact with a cell model infected with human porcine circovirus; b) Use any one of the primer pairs of claims 1-2, the primer pair set of claim 3, the reagent combination of claims 4-5, the nucleic acid chip of claim 5, or the kit of claims 6-7 to detect the change in human circovirus genome copy number in cell models before and after drug administration; c) Screening for test drugs with significant inhibitory effects as candidate drugs for the treatment of diseases caused by human porcine circovirus infection; Preferably, the sample to be tested includes blood, nasopharyngeal swabs, oropharyngeal swabs, sputum, and liver tissue.

9. A device for detecting human porcine circovirus, characterized in that, The device comprises any one of the primer pairs of claims 1-2, the primer pair set of claims 3, the reagent combination of claims 4, the nucleic acid chip of claims 5, and the kit of claims 6-7; Preferably, the device further includes a hardware module for performing nucleic acid amplification and detection.

10. Any of the following applications: (1) The use of the primer pairs of claims 1-2 and the primer set of claim 3 in the preparation of reagents and kits for the detection of human porcine circovirus for non-diagnostic purposes; (2) The application of any one of the primer pairs of claims 1-2, the primer pair set of claims 3, the reagent combination of claims 4-5, the nucleic acid chip of claims 5, the kit of claims 6-7, the method of claims 8, and the device of claims 9 in the detection of human porcine circovirus for non-diagnostic purposes; (3) The primer pair of claim 1-2, the primer pair set of claim 3, the reagent combination of claim 4-5, the nucleic acid chip of claim 5, the kit of claim 6-7, the method of claim 8, and the device of claim 9 are used in the preparation of products for diagnosing human porcine circovirus infection; (4) The primer pair of claim 1-2, the primer pair set of claim 3, the reagent combination of claim 4-5, the nucleic acid chip of claim 5, the kit of claim 6-7, the method of claim 8, and the device of claim 9 are used in the preparation of products for diagnosing diseases caused by human porcine circovirus infection; (5) The use of any one of the primer pairs of claims 1-2, the primer pair set of claims 3, the reagent combination of claims 4-5, the nucleic acid chip of claims 5, the kit of claims 6-7, the method of claims 8, and the device of claims 9 in screening drugs for treating diseases caused by human circovirus infection.

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