Digital PCR kit for simultaneously detecting bovine nodular skin disease virus, schmallenberg virus and peste des petits ruminants virus

Abstract

The invention belongs to the technical field of virus detection, and discloses a digital PCR (Polymerase Chain Reaction) kit for simultaneously detecting bovine nodular skin disease virus, schmallenberg virus and peste des petits ruminants virus, and the kit comprises primers and probes as shown in SEQ ID NO.1-SEQ ID NO.18. The invention also discloses a kit for simultaneously detecting bovine nodular skin disease virus, schmallenberg virus and peste des petits ruminants virus. According to the invention, a sextuple digital PCR (Polymerase Chain Reaction) system containing an internal standard is established after a single system of a bovine nodular skin disease virus-ORF101 gene, a bovine nodular skin disease virus-ORF126 gene, a schmallenberg virus, a peste des petits ruminants virus-N gene and a peste des petits ruminants virus-M gene is tested; through tests of indexes such as linearity, specificity, minimum detection limit, repeatability, comparison of a single digital PCR system and a multiple digital PCR system, the currently developed digital PCR multiple detection kit completely meets development requirements and is high in specificity and sensitivity, and the minimum detection limit of the digital PCR multiple detection kit can reach 10 Copies / mu L or below.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a digital PCR kit for simultaneously detecting bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus. Background Art

[0002] Lumpy Skin Disease Virus (LSDV), Schmallenberg Virus (SBV), and Peste des Petits Ruminants Virus (PPRV) are three pathogens that have posed a serious threat to the global livestock industry in recent years. Their epidemics are expanding, their transmission pathways are complex, and their detection technology faces significant bottlenecks. LSDV, a member of the Poxviridae family, primarily infects cattle, causing skin nodules, high fever, and systemic symptoms. Its ORF101 gene encodes the viral capsid protein, while ORF126, involved in viral membrane protein synthesis, serves as a key target for distinguishing field strains from gene-deleted vaccine strains (such as the Neethling strain). Schmallenberg virus (SBV), a member of the Bunyaviridae family, is transmitted by vectors such as Culicoides. Infecting ruminants such as cattle and sheep, it causes abortion, fetal malformations, and neonatal mortality. Its spread has expanded as the insect's distribution range has shifted northward. Peste des petits ruminants virus (PPRV) belongs to the Morbillivirus genus and primarily infects goats and sheep. Its N gene encodes the nucleocapsid protein, essential for viral replication, while its M gene encodes the matrix protein, both of which are diagnostic markers. PPRV has a mortality rate of up to 90%.

[0003] Traditional detection methods (such as virus isolation, ELISA or single-plex qPCR) need to be carried out independently, which is not only time-consuming and costly, but also lacks sensitivity for low-load samples (such as latently infected, recovered animals or environmental samples). Taking qPCR as an example, its detection limit is usually 100 copies / reaction, and the missed detection rate for early-stage infected or latently infected animals can reach more than 30%; in addition, multiplex qPCR is difficult to achieve simultaneous detection of multiple pathogens due to fluorescence channel limitations (usually ≤4 channels), and differences in amplification efficiency can easily lead to biased results. What is more serious is that viral genetic heterogeneity (such as LSDV-ORF126 gene deletion mutations and PPRV-N gene point mutations) may cause the primer probe binding site to fail, further reducing the reliability of detection. In this context, the development of a highly sensitive and specific detection technology that can simultaneously cover the LSDV-ORF101 / ORF126, SBV and PPRV-N / M genes has become an urgent need for precise prevention and control of the disease.

[0004] Digital PCR (dPCR) technology achieves absolute quantification of nucleic acids at the single-molecule level by dividing the reaction system into tens of thousands of droplets. Its advantages are particularly prominent in multiplex detection and complex sample analysis. This invention integrates the five gene targets of LSDV, SBV and PPRV into a single detection system for the first time. Its significance lies in: 1. Meeting the challenge of co-epidemics: In areas with high incidence of epidemics, the same animal population may be exposed to multiple pathogens. Multiplex detection can greatly shorten the diagnosis time and avoid repeated consumption of samples; 2. Effectively overcome the sensitivity of traditional PCR to sample inhibitors. Through algorithm optimization (such as an absolute quantification model based on Poisson distribution), low-abundance pathogens can be accurately detected (detection limit ≤10 copies / μL), and four-color fluorescent labeling (FAM / HEX / ROX / CY5) can be used for visual distinction. ③ Enhance the reliability of results: Design ORF101 and ORF126 dual-target probes for LSDV, which can simultaneously identify wild strains and vaccine strains; the combined detection of the N / M genes of PPRV can avoid false negatives caused by single-target mutations; the design of the SBV-N gene probe takes into account the evolutionary branches of the virus, reducing the risk of missed detection due to segmental recombination; ④ Improve detection efficiency and economy: Traditional methods require at least three independent experiments to detect three viruses, while this kit achieves "one tube six tests" including internal standards through four-color fluorescence channel optimization and primer probe combination design, with a throughput increase of more than 80%, which is particularly suitable for cross-border ports, large-scale epidemic screening or vaccine effect evaluation. In addition, dPCR does not rely on a standard curve and can directly calculate the number of viral copies, providing data accuracy that qPCR cannot achieve for dynamic monitoring of viral load (such as evaluation of the efficacy of antiviral treatment). In summary, the development of the technology of the present invention not only solves the core defects of existing methods in terms of multiplicity, sensitivity and anti-interference, but also provides a technical paradigm for the comprehensive prevention and control of emerging and re-emerging animal diseases.

[0005] Through the above analysis, the problems and defects of the existing technology are as follows:

[0006] (1) Traditional detection methods (such as virus isolation, ELISA, or single-plex qPCR) must be performed independently, which is not only time-consuming and costly, but also lacks sensitivity for low-load samples (such as latently infected, recovered animals, or environmental samples). Taking qPCR as an example, its detection limit is usually 100 copies / reaction, and the missed detection rate for early-stage infected or latent carrier animals can reach over 30%.

[0007] (2) Multiplex qPCR is difficult to achieve simultaneous detection of multiple pathogens due to the limitation of fluorescence channels (usually ≤4 channels), and differences in amplification efficiency can easily lead to biased results. More seriously, viral gene heterogeneity (such as LSDV-ORF126 gene deletion mutation and PPRV-N gene point mutation) may cause primer probe binding site failure, further reducing detection reliability. In this context, the development of a highly sensitive and specific detection technology that can simultaneously cover LSDV-ORF101 / ORF126, SBV and PPRV-N / M genes has become an urgent need for precise disease prevention and control. Summary of the Invention

[0008] In response to the problems existing in the prior art, the present invention provides a digital PCR kit for simultaneously detecting bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus.

[0009] The present invention is achieved by providing a digital PCR kit for simultaneously detecting bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus, the kit comprising primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0010] A method for detecting bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus for non-disease diagnosis or treatment purposes, comprising the following steps:

[0011] (1) Using the extracted nucleic acid of the sample to be tested as a template, the sample to be tested is amplified using the primers shown in SEQ ID NO. 1-2, SEQ ID NO. 4-5, SEQ ID NO. 7-8, SEQ ID NO. 10-11 and SEQ ID NO. 13-14, and the amplified product is identified by the probes shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12 and SEQ ID NO. 15;

[0012] For example, the probe shown in SEQ ID NO. 3 has a FAM fluorescent label at its 5' end and an MGB fluorescent label at its 3' end; the probe shown in SEQ ID NO. 6 has a ROX fluorescent label at its 5' end and an MGB fluorescent label at its 3' end; the probe shown in SEQ ID NO. 9 has a FAM fluorescent label at its 5' end and a BHQ1 fluorescent label at its 3' end; the probe shown in SEQ ID NO. 12 has a HEX fluorescent label at its 5' end and a BHQ1 fluorescent label at its 3' end; the probe shown in SEQ ID NO. 15 has a CY5 fluorescent label at its 5' end and a BHQ2 fluorescent label at its 3' end;

[0013] (2) simultaneously setting an IPC internal standard including primers and probes as shown in SEQ ID NO. 16 to SEQ ID NO. 18, wherein the 5' end of the probe as shown in SEQ ID NO. 18 is set with a ROX fluorescent label, and the 3' end of the probe is set with an MGB fluorescent label; performing digital PCR fluorescence detection, and collecting the signal of the fluorescence channel;

[0014] (3) Result analysis:

[0015] In the present invention, the concentrations are 1.0×10 3 A mixture of recombinant plasmids containing LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard at 50 copies / μL was used as a positive control, and ultrapure water was used as a negative control.

[0016] Click "Analyze" on the biochip reader to analyze the experimental data. In the 2D or 1D plot, the darkest black dots represent negative droplets, and the remaining colored droplets represent positive droplets in the corresponding channels. The negative control should have no positive droplets, and the positive control should have positive droplets in all channels corresponding to the bands.

[0017] FAM channel band 1 represents Schmallenberg virus-positive droplets, FAM channel band 2 represents bovine lumpy skin disease virus-ORF101 gene-positive droplets, HEX channel band represents peste des petits ruminants virus-N gene-positive droplets, ROX channel band 1 represents internal standard-positive droplets, ROX channel band 2 represents bovine lumpy skin disease virus-ORF126 gene-positive droplets, and CY5 channel band represents peste des petits ruminants virus-M gene-positive droplets;

[0018] Judgment of the same sample: FAM channel band 1 is positive and is judged to be Schmallenberg virus infection; FAM channel band 2 is positive and ROX channel band 2 is positive at the same time, which is judged to contain bovine lumpy skin disease virus in the sample and the virus comes from natural infection rather than vaccine immunity; FAM channel band 2 is positive and ROX channel band 2 is not detected, then it is judged that the sample contains bovine lumpy skin disease virus and the virus comes from vaccine immunity rather than natural infection; HEX channel band and CY5 channel band are positive and are judged to be infected with peste des petits ruminants virus.

[0019] In combination with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

[0020] First, after testing the single-plex system of bovine lumpy skin disease virus-ORF101 gene, bovine lumpy skin disease virus-ORF126 gene, Schmallenberg virus, peste des petits ruminants virus-N gene, and peste des petits ruminants virus-M gene, the present invention established a six-plex digital PCR system including an internal standard. After testing indicators such as linearity, specificity, minimum detection limit, repeatability, and comparison of single-plex and multiplex digital PCR systems, the currently developed digital PCR multiplex detection kits fully meet development requirements, have strong specificity and high sensitivity, and the minimum detection limit can reach below 10 copies / μL.

[0021] Second, as auxiliary evidence for the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0022] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0023] The six-fold digital PCR kit developed by the present invention can simultaneously detect the bovine lumpy skin disease virus (LSDV)-ORF101 and ORF126 genes, Schmallenberg virus (SBV), peste des petits ruminants virus (PPRV)-N and M genes and internal standards. Its commercial value lies in its high degree of compliance with the urgent needs of the global animal disease detection market. According to data from the World Organization for Animal Health (OIE), the economic losses caused by LSDV and PPRV alone to the livestock industry exceed billions of dollars each year, and the risk of cross-border transmission of diseases has increased the dependence of various countries on efficient detection technologies. Traditional detection methods (such as qPCR or ELISA) require step-by-step detection of different pathogens, which is time-consuming (single detection requires 2-3 days), costly (the comprehensive cost of a single sample is about US$50-100), and difficult to distinguish multiple infections. This kit completes six-target detection through a single reaction, shortening the detection time to less than 4 hours, reducing the cost to US$30 per sample, and increasing the efficiency by more than 5 times. It is particularly suitable for import and export quarantine, large-scale farms and large-scale screening in high-incidence areas.

[0024] Market expectations suggest that this test kit, with its high sensitivity (detection limit of 10 copies / μL), strong specificity (plasmid-verified lack of cross-reactivity), and standardized workflow, is poised to rapidly capture the market. Conservative estimates suggest that if it covers 10% of global cattle and sheep disease testing needs (approximately 200 million head per year), annual revenue could reach $600 million. Furthermore, the technology platform can be expanded to cover other animal diseases (such as foot-and-mouth disease and African swine fever), forming a product pipeline and further extending the commercial lifecycle.

[0025] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0026] The current technological gaps in the field of digital PCR detection of animal diseases are concentrated on the lack of multiplex detection capabilities. According to the patent database (Derwent Innovation), as of 2023, 91% of the world's published digital PCR-related patents for animal diseases were single or double detection, and only 8% involved triple or more, and the targets were mostly limited to a single pathogen (such as only detecting PPRV or LSDV). For example, Chinese patent CN112111572A achieved dual-gene detection of PPRV, but did not integrate other pathogens; although U.S. patent US20210017562A1 proposed quadruple detection, the target was foot-and-mouth disease virus subtyping, which did not overlap with the cross-pathogen detection of the present invention. This kit is the first system to realize the "three pathogens - five genes - plus internal standard" six-fold detection. Its innovation is reflected in two aspects: in the technical dimension, by optimizing the primer probe design (optimizing the primer sequence and annealing temperature and introducing quenching group modification), the channel interference and background signal problems in the six-fold system are solved; in the application dimension, for the first time, the three major cross-border pathogens LSDV, SBV, and PPRV are integrated for detection, covering the key conserved genes of the pathogens (such as LSDV-ORF126 as the capsid protein encoding gene, and PPRV-N gene as the core target of the nucleoprotein), significantly improving the clinical guidance value of the test results.

[0027] From the perspective of industry standards, existing international test kits (such as Thermo Fisher's VetMAX TM Series) only supports qPCR platforms and requires multiple tube reactions to complete the same detection volume. This invention overcomes the bottleneck of digital PCR in the field of animal disease, which has long been limited by low multiplicity, and provides a new technical option for the OIE Manual of Diagnostic Tests for Terrestrial Animals.

[0028] (3) Whether the technical solution of the present invention solves the technical problems that people have been eager to solve but have not been able to solve successfully:

[0029] The core challenge of multiplex detection of animal diseases lies in how to achieve specific amplification of multiple targets without mutual interference in highly complex samples (such as mixed infected tissues). Traditional qPCR is difficult to break through quadruple detection due to the limitation of fluorescence channels (usually ≤4 channels) and primer dimer problems. Digital PCR optimizes the probe concentration to enable it to exceed the number of detection targets of traditional qPCR and has the advantage of absolute quantification. This breakthrough solves the four major pain points that have long existed in the field of veterinary diagnosis: first, rapid confirmation of pathogen types in the early stage of an outbreak to avoid misdiagnosis (such as the similar clinical symptoms of LSDV and PPRV); second, accurate monitoring of virus mutations (such as ORF101 gene mutations that may cause vaccine escape) to provide data support for vaccine development; third, there is no need for the standard curve and Ct value that traditional qPCR relies on for relative quantification, and absolute quantification is achieved, solving the problem of limited accuracy when lacking standards or cross-laboratory comparisons; fourth, strong anti-interference ability adapts to direct detection of complex samples, solving core pain points such as the difficulty in processing complex samples.

[0030] (4) Whether the technical solution of the present invention overcomes technical prejudice:

[0031] In the research and development of digital PCR technology for multiplex detection of animal diseases, the industry has long had two major technical biases: First, it is believed that multiplex detection systems (especially five-plex and above) will inevitably lead to degraded linear relationships, decreased sensitivity, or uncontrolled repeatability due to competition or cross-interference between primers and probes. Therefore, high-multiplex detection remains at the theoretical verification level and is difficult to commercialize; second, it is believed that there are systematic deviations in the quantitative results of single-plex and multiplex detection systems, and data comparability cannot be guaranteed, resulting in the reliance on single-plex detection as the "gold standard" in clinical diagnosis. This present invention, through systematic verification at the plasmid level, directly overturns the above technical biases with experimental data, providing scientific support for the practical application of high-multiplex digital PCR technology.

[0032] First, to address the prejudice that "high multiplex number leads to decreased performance", this kit achieves the detection linearity, sensitivity and repeatability of all targets (including internal standard) in the six-plex system, which reaches or even exceeds the single-plex detection standard. Experimental data show that: within the range of plasmid gradient dilution, the linear regression coefficient (R 2 ) were all greater than 0.99, demonstrating that the multiplex system introduced no nonlinear amplification interference. Furthermore, the sensitivity of the six-plex assay reached single-copy levels (minimum detection limit ≤10 copies / μL) with excellent reproducibility (intra-assay CV <1.5%). This result directly refutes the preconceived notion that multiplex testing necessarily sacrifices sensitivity.

[0033] Secondly, addressing industry concerns about the incomparability of singleplex and multiplex quantitative results, this kit demonstrated high consistency in quantitative results between singleplex and sextuple systems through direct comparison experiments. In mixed plasmid samples (containing equal concentrations of six targets), the difference in quantitative concentration between singleplex and sextuple assays was less than 5% (CV < 2%), with no systematic trend in the direction of deviation (e.g., the sextuple assay values did not appear to be generally higher or lower).

[0034] A deeper industry bias is that plasmid-level verification cannot reflect the technical feasibility of real-world detection scenarios. However, the experimental design of the present invention proves that achieving the performance limit of high-multiplicity detection in an idealized model (plasmid template) is a key prerequisite for breaking through technical bottlenecks. For example, cross-reactions between primer probes can be accurately quantified through plasmid mixing experiments, and interference from inhibitors or complex matrices in clinical samples can be optimized separately through subsequent sample pre-treatment modules (such as introducing anti-inhibition polymerases or magnetic bead purification). This modular development strategy breaks the traditional thinking that "all technical difficulties must be solved simultaneously" and provides a new paradigm for the phased industrialization of high-multiplicity digital PCR kits.

[0035] In summary, this technical solution, based on rigorous experimental data, demonstrates that hexaplex digital PCR detection has the core performance indicators of commercial test kits at the plasmid level, completely overcoming the industry's technical prejudice against the insufficient reliability of high-multiplex detection, and laying an irreplaceable theoretical and technical foundation for subsequent clinical sample verification and practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a single fluorescence PCR standard curve provided by an embodiment of the present invention. A represents the linearity of the LSDV-ORF101 gene target, B represents the linearity of the LSDV-ORF126 gene target, C represents the linearity of the SBV-N gene target, D represents the linearity of the PPRV-N gene target, and E represents the linearity of the PPRV-M gene target.

[0037] Figure 2 This is the specific detection result of the fluorescent PCR detection method using the single system provided in the embodiment of the present invention.

[0038] Figure 3 This is a 1-dimensional image of positive droplets in each channel of the multiplex digital PCR reaction system provided by an embodiment of the present invention.

[0039] Among them, A is the positive droplet of FAM channel band 1: SBV-N gene target and band 2: LSDV-ORF101 gene target; B is the HEX channel band: PPRV-N gene target; C is the positive droplet of ROX channel band 1: internal standard and band 2: LSDV-ORF126 gene target; D is the CY5 channel band: positive droplet of PPRV-M gene target.

[0040] Figure 4 This is a 2D image of positive droplets in each channel of the multiplex digital PCR reaction system provided by an embodiment of the present invention.

[0041] Among them, A is a 2D image of the positive droplets of each gene target in the FAM and HEX channels, B is a 2D image of the positive droplets of each gene target in the FAM and ROX channels, C is a 2D image of the positive droplets of each gene target in the FAM and CY5 channels, D is a 2D image of the positive droplets of each gene target in the HEX and ROX channels, E is a 2D image of the positive droplets of each gene target in the HEX and CY5 channels, and F is a 2D image of the positive droplets of each gene target in the ROX and CY5 channels.

[0042] Figure 5 This is a 1-dimensional graph of the linear results of positive droplets in each channel of the multiplex digital PCR reaction system provided by an embodiment of the present invention. Among them, A is the FAM channel, B is the HEX channel, C is the ROX channel, and D is the CY5 channel.

[0043] Figure 6 This is a statistical graph of the linear results of each target in the multiplex digital PCR reaction system provided by an embodiment of the present invention.

[0044] Figure 7 This is a 1-dimensional graph of the reproducibility results of positive droplets in each channel of the multiplex digital PCR reaction system provided by an embodiment of the present invention. Among them, A is the FAM channel, B is the HEX channel, C is the ROX channel, and D is the CY5 channel.

[0045] Figure 8 This is a 1-D comparison of the results of multiplex digital PCR and individual single digital PCR assays provided in an embodiment of the present invention. A compares the results of multiplex digital PCR and single digital PCR assays for the LSDV-ORF101 gene target, B compares the results of multiplex digital PCR and single digital PCR assays for the SBV-N gene target, C compares the results of multiplex digital PCR and single digital PCR assays for the PPRV-N gene target, D compares the results of multiplex digital PCR and single digital PCR assays for the LSDV-ORF126 gene target, and E compares the results of multiplex digital PCR and single digital PCR assays for the PPRV-M gene target.

[0046] Figure 9 This figure compares the results of multiplex digital PCR and individual single digital PCR assays provided by an embodiment of the present invention. The coefficient of variation (CV) values for both the single and multiplex digital PCR assays are less than 2%, demonstrating good agreement between the multiplex digital PCR assay and the individual single digital PCR assays, with no cross-talk between the multiplex digital PCR assays. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0048] The present invention aims to provide a digital PCR kit for simultaneously detecting bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus, wherein the kit comprises primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0049] The present invention also provides a method for detecting bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus for non-disease diagnosis or treatment purposes, comprising the following steps:

[0050] (1) Using the extracted nucleic acid of the sample to be tested as a template, the sample to be tested is amplified using the primers shown in SEQ ID NO. 1-2, SEQ ID NO. 4-5, SEQ ID NO. 7-8, SEQ ID NO. 10-11 and SEQ ID NO. 13-14, and the amplified product is identified by the probes shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12 and SEQ ID NO. 15;

[0051] For example, the probe shown in SEQ ID NO. 3 has a FAM fluorescent label at its 5' end and an MGB fluorescent label at its 3' end; the probe shown in SEQ ID NO. 6 has a ROX fluorescent label at its 5' end and an MGB fluorescent label at its 3' end; the probe shown in SEQ ID NO. 9 has a FAM fluorescent label at its 5' end and a BHQ1 fluorescent label at its 3' end; the probe shown in SEQ ID NO. 12 has a HEX fluorescent label at its 5' end and a BHQ1 fluorescent label at its 3' end; the probe shown in SEQ ID NO. 15 has a CY5 fluorescent label at its 5' end and a BHQ2 fluorescent label at its 3' end;

[0052] (2) simultaneously setting an IPC internal standard including primers and probes as shown in SEQ ID NO. 16 to SEQ ID NO. 18, wherein the 5' end of the probe as shown in SEQ ID NO. 18 is set with a ROX fluorescent label, and the 3' end of the probe is set with an MGB fluorescent label; performing digital PCR fluorescence detection, and collecting the signal of the fluorescence channel;

[0053] (3) Result analysis:

[0054] In the present invention, the concentrations are 1.0×10 3A mixture of recombinant plasmids containing LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard at 50 copies / μL was used as a positive control, and ultrapure water was used as a negative control.

[0055] Click "Analyze" on the biochip reader to analyze the experimental data. In the 2D or 1D plot, the darkest black dots represent negative droplets, and the remaining colored droplets represent positive droplets in the corresponding channels. The negative control should have no positive droplets, and the positive control should have positive droplets in all channels corresponding to the bands.

[0056] FAM channel band 1 represents Schmallenberg virus-positive droplets, FAM channel band 2 represents bovine lumpy skin disease virus-ORF101 gene-positive droplets, HEX channel band represents peste des petits ruminants virus-N gene-positive droplets, ROX channel band 1 represents internal standard-positive droplets, ROX channel band 2 represents bovine lumpy skin disease virus-ORF126 gene-positive droplets, CY5 channel band represents peste des petits ruminants virus-M gene-positive droplets ( Figure 3 );

[0057] Judgment of the same sample: FAM channel band 1 is positive and is judged to be Schmallenberg virus infection; FAM channel band 2 is positive and ROX channel band 2 is positive at the same time, which is judged to contain bovine lumpy skin disease virus in the sample and the virus comes from natural infection rather than vaccine immunity; FAM channel band 2 is positive and ROX channel band 2 is not detected, then it is judged that the sample contains bovine lumpy skin disease virus and the virus comes from vaccine immunity rather than natural infection; HEX channel band and CY5 channel band are positive and are judged to be infected with peste des petits ruminants virus.

[0058] In some specific embodiments, the reaction system of the amplification reaction includes: 10 μL of digital PCR reaction premix, 1.1 μL of primer mix, 0.64 μL of probe mix, 2 μL of nucleic acid template, and dd H2O to 20 μL.

[0059] In some specific embodiments, the amplification reaction is as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing and extension at 60°C for 1 min, 40 cycles; inactivation at 98°C for 10 min; cooling at 20°C for 2 min; and collecting fluorescence signals from four channels, namely, FAM, HEX, ROX, and CY5, at the end of each cycle.

[0060] Example 1 Design of primer and probe sequences

[0061] The complete genome sequence or N gene sequence of Schmallenberg virus (GenBank numbers: NC_043582.1, KT795133.1, LC309158.1, KC139376.1, KX384876.1, LC309162.1, LC309165.1, etc.) was retrieved from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov); the complete genome sequence of bovine lumpy skin disease virus and The ORF101 gene and ORF126 gene sequences (GenBank accession numbers: AF409137.1, MN995838.1, MN642592.1, KY829023.3, PP053747.1, OR134849.1, OK318001.1, KX683219.1, MW631933.1, OR393178.1, MN995838.1, MT643825.1, MW656253.1, OR23241 3.1, etc.); the complete genome sequence and N and M gene sequences of PPR virus (GenBank numbers: KX421387.1, OK274196.1, KX938427.1, KT895431.1, KT895431.1, KX033350.1, FJ905304.1, MN657232.1, OL310700.1, NC_006383.2, OK274205.1, KY628761.1, MT10 9379.1, OR286474.1, FJ905304.1, etc.); the IPC internal standard was based on the conserved gene sequence of the rRNA gene in the sheep mitochondrial genome. After aligning the target gene sequences using SnapGene software, relatively conserved nucleotide sequences of each pathogen were selected. According to the primer and TaqMan probe design principles and in combination with relevant national standards, the relevant primer and probe sequences were designed. The detection targets and sequences of the kit are summarized in Table 1. The designed primers and probes were synthesized by Sangon Biotech Co., Ltd.

[0062] Table 1 Primers and probes for digital PCR kits for bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus

[0063]

[0064]

[0065] Example 2 Construction of plasmid standards

[0066] The gene sequences of bovine lumpy skin disease virus, Schmallenberg virus, peste des petits ruminants virus, and internal standard were synthesized by Sangon Biotech Co., Ltd. and named LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard, respectively. The plasmid DNA standards were then frozen at -80°C until use.

[0067] DNA copy number = (M × 6.02 × 10 23 ×10 -9 ) / (n×660)

[0068] M represents the plasmid DNA concentration, where n represents the recombinant plasmid length = T vector length + target fragment length.

[0069] 2.1 Establishment of the standard curve of single-plex fluorescence PCR reaction system

[0070] To verify whether the amplification efficiency and correlation coefficient of the primer probes designed for each target met the requirements for establishing a multiplex digital PCR system, the LSDV-ORF101 and LSDV-ORF126 positive plasmids of bovine lumpy skin disease virus (LSDV), the SBV-N positive plasmid of Schmallenberg virus (SBV), and the PPRV-N and PPRV-M positive plasmids of Peste des Petits Ruminants virus (PPRV) were diluted 10-fold and then subjected to single-plex fluorescence PCR. The concentration standard curve was prepared using the fluorescence Ct value of the template concentration. The configuration of each reaction system is shown in Table 2. The final concentration of each target primer was 200 nmol / L, and the final concentration of the probe was 100 nmol / L. The fluorescence PCR reaction was performed according to the procedure shown in Table 3. The equipment used was a JLM QX600 from JLM.

[0071] Table 2 Preparation of single-plex fluorescence PCR reaction system

[0072]

[0073] Table 3 Fluorescence PCR reaction parameter settings

[0074]

[0075]

[0076] The concentration-Ct value standard curve was made using LSDV-ORF101, SBV-N, PPRV-N, LSDV-ORF126, and PPRV-M positive plasmids respectively ( Figure 1 ).from Figure 1 It can be seen that in the single-plex system, the standard curve PCR amplification efficiency E value, R and curve slope are all within the normal range, indicating that the amplification efficiency is good and can be used for the subsequent establishment of a multiplex digital PCR system.

[0077] 2.2 Primer probe specificity test

[0078] Eight other sheep disease-positive plasmids, including Bluetongue virus (BTV), Foot-and-mouth disease virus (FMDV), Sheep pox virus (SPV), Border disease virus (BDV), Brucella spp (Bru), Toxoplasma gondii (Tox), Ovine Enzootic Abortion (OEA), and Caprine arthritis-encephalitis virus (CAEV), were selected for specificity testing. Single fluorescence PCR was performed using the primers and probes of this kit, LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, and PPRV-M. At the same time, the positive plasmids of the targets of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, and PPRV-M of this kit were used as positive controls. H2O was used as a negative control. The system configuration is shown in Table 2. The final concentration of each target primer was 200 nmol / L, and the final concentration of the probe was 100 nmol / L. Fluorescence PCR reactions were performed according to the procedure shown in Table 3. The equipment used was JLMQX600 (Jelemi).

[0079] After testing, it was found that the target primers and probes of this kit did not show non-specific amplification with other sheep disease positive plasmids, and each positive showed fluorescence intensity, indicating that the detection primers and probes have good specificity and can be used for the construction of subsequent multiplex digital PCR systems. Figure 2 , Table 4. The plasmid concentrations in Table 4 are all 1.0×10 6 Copies / μL.

[0080] Table 4 Specificity test of each target single-plex system of this kit

[0081]

[0082]

[0083] 2.3 Establishment of digital PCR multiplex reaction system

[0084] The primer and probe stock solutions were diluted and mixed thoroughly to prepare primer mixtures and probe mixtures. The final concentrations of each target primer are shown in Table 1. The configuration of each reaction system is shown in Table 5. The digital PCR reaction was performed according to the procedure shown in Table 6. The equipment used was JLM Digital Matrix-5000 digital PCR from JLM.

[0085] Sample concentration (Copies / μL) = fixed value result (Copies / μL) × 20 (total reaction system) ÷ 2 (sample volume).

[0086] Table 5 Digital PCR reaction system preparation

[0087]

[0088] Table 6 Digital PCR reaction parameter settings

[0089]

[0090] 2.4 Linearization of target plasmid

[0091] The theoretical value of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and internal standard plasmids is 10 6 Six positive plasmids with the same volume of copies / μL were mixed together and linearized using QuickCut Sac I and QuickCut Kpn I. The enzyme digestion system is shown in Table 7. The digestion was completed by incubation at 37°C for 15 minutes.

[0092] Table 7 Plasmid linearization enzyme digestion system

[0093]

[0094] The six positive plasmids after enzyme digestion were tested according to the final concentrations of target primers and probes in Table 1, the reaction system in Table 5, and the reaction procedures in Table 6 obtained after the optimization system. The 1D diagram of the test results is shown in FIG. Figure 3 , 2D graph as Figure 4 As shown, from the 1D graph, the bands between the targets of each channel are clearly layered and will not interfere with each other's detection. From the 2D graph, the targets are clearly distinguished and the bands are clearly divided. The 1D and 2D graphs have good effects. The experimental results prove that the multiplex digital PCR reaction system was successfully established.

[0095] 2.5 Establishment of the digital PCR multiplex reaction system standard curve and determination of the minimum detection limit

[0096] The theoretical concentration after enzyme digestion is 104 Digital PCR reactions were performed using seven 4-fold dilutions of the plasmid mixture (copies / μL). ddH2O was used as a negative control. The reaction system is shown in Table 5. Multiplex digital PCR reactions were performed according to the reaction conditions in Table 6 to evaluate the linearity and minimum detection limit of the method. Sample concentration (copies / μL) = calculated value (copies / μL) × 20 (total reaction volume) / 2 (sample load).

[0097] The digital PCR results showed that the number of droplets was greater than 10,000, which met the experimental analysis requirements. The correlation coefficient of the standard curve (R 2 ) were all greater than 0.98, and the linearity established by the multiplex digital PCR method met the performance requirements.

[0098] In the multiplex digital PCR system, the minimum detection limits of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, and PPRV-M were 1.02 Copies / μL, 6.79 Copies / μL, 4.08 Copies / μL, 1.02 Copies / μL, and 2.04 Copies / μL, respectively. In the multiplex digital PCR system, the minimum detection limit of each target can reach below 10 Copies / μL. The results are shown in Figure 5 、 Figure 6 , Table 8.

[0099] Table 8 Statistics of the minimum detection limit test results of the multiplex digital PCR system of this kit

[0100]

[0101] 2.6 Repeatability test of digital PCR multiplex reaction system

[0102] Multiplex digital PCR reactions were performed using single copy number concentrations of five positive mixed plasmids. The test was repeated eight times, and the test results were statistically analyzed to calculate the coefficient of variation and evaluate the repeatability of the method.

[0103] Select 10 4 Copies / μL were diluted 16 times and mixed with plasmid as a positive template. Repeated experiments were performed within the group. The results showed that the number of droplets was greater than 10,000, which met the experimental analysis requirements. The coefficient of variation within the group was between 0.85% and 1.18%, which was less than 1.5%, indicating that the method has good repeatability. Figure 7 , as shown in Table 9.

[0104] Table 9 Statistics of repeatability test results for each target in the multiplex digital PCR system of this kit

[0105]

[0106] 2.7 Comparison of singleplex and multiplex digital PCR reaction systems

[0107] Single-copy concentrations of five positive mixed plasmids were used to perform single-plex and multiplex digital PCR reactions for each target. The fixed concentrations corresponding to the same template concentration were compared, the coefficient of variation of the fixed results of the single-plex and multiplex systems was calculated, and the consistency of single-plex digital PCR and multiplex digital PCR was compared.

[0108] The results showed that the number of droplets was greater than 10,000, which met the experimental analysis requirements. The coefficient of variation of the copy number of single and multiplex fixed concentrations was less than 2%, indicating that the fixed concentration results of the single and multiplex digital PCR reaction systems for the same sample were not much different, which met the experimental requirements. The experimental results are as follows Figure 8 、 Figure 9 , as shown in Table 10.

[0109] Table 10 Comparative test results of single-plex and multiplex digital PCR systems of this kit

[0110]

[0111] After testing the single-plex system of bovine lumpy skin disease virus-ORF101 gene, bovine lumpy skin disease virus-ORF126 gene, Schmallenberg virus, peste des petits ruminants virus-N gene, and peste des petits ruminants virus-M gene, a six-plex digital PCR system including internal standards was established. After testing indicators such as linearity, specificity, minimum detection limit, repeatability, and comparison of single-plex and multiplex digital PCR systems, the digital PCR multiplex detection kits currently developed fully meet development requirements.

[0112] 1. Specific application fields or related products of the present invention.

[0113] Application areas:

[0114] 1. Import and export quarantine and cross-border animal trade

[0115] Application scenarios: Pathogen screening of live animals (cattle, sheep, etc.) and their products (semen, embryos, meat products) at customs and border ports to prevent the cross-border spread of OIE notifiable diseases such as LSDV, SBV, and PPRV.

[0116] Technical advantages: A single test can simultaneously confirm the infection status of multiple pathogens, avoiding customs clearance delays caused by step-by-step testing (traditional methods require 3-5 independent tests), and meeting the technical requirements of the International Animal Health Code for "rapid customs clearance".

[0117] 2. Disease monitoring and purification in large-scale farms

[0118] Application scenarios: Used for regular screening of farms, pathogen screening before introduction of new breeds, and tracing infection sources during outbreaks. For example, by detecting the ORF101 and ORF126 genes of LSDV, it is possible to distinguish between wild strains and vaccine strains, guiding targeted immunization.

[0119] Technical advantages: High sensitivity (≤10 copies / μL) can detect latent infection early. Combined with the absolute quantitative capability of digital PCR, it can dynamically monitor changes in viral load and evaluate the effectiveness of prevention and control measures.

[0120] 3. Reference standards for disease diagnostic laboratories

[0121] Application scenario: As a quality control product or standard substance for third-party testing agencies to verify the accuracy of other detection methods (such as qPCR, ELISA).

[0122] Technical advantages: The six-fold detection system includes an internal standard, which can correct deviations in sample extraction efficiency, provide standardized quantitative results, and address the industry pain point of poor comparability of test data between laboratories.

[0123] 4. Vaccine development and efficacy evaluation

[0124] Application scenarios: Monitoring the virus inactivation effect during vaccine production (such as detecting residual LSDV nucleic acid), or evaluating the level of viral load reduction after immunization of animals.

[0125] Technical advantages: Multiple detection can simultaneously monitor vaccine target genes (such as PPRV-N) and potential contaminating pathogens (such as SBV), improving the efficiency of vaccine safety assessment.

[0126] Products related to this invention:

[0127] Based on the technical solution of this invention, the following core products and supporting services can be derived to form a complete technology ecosystem:

[0128] 1. Core Products

[0129] Six-plex Digital PCR Detection Kit (Premix): Contains liquid or lyophilized microsphere primer probes, reaction buffer, and positive control. It is ready for use right out of the box and is compatible with mainstream digital PCR instruments (such as the Bio-Rad QX200 and Thermo Fisher QuantStudio 3D).

[0130] Multi-pathogen mixed quality control product: contains simulated genome fragments of LSDV, SBV, and PPRV, used for standardized verification of laboratory testing procedures, with a concentration gradient covering the common clinical load range (1 to 10 5 copies / μL).

[0131] 2. Extended services

[0132] Animal disease testing and certification services: Provide breeding companies with test data endorsement based on this kit to assist them in obtaining OIE-certified "disease-free enterprise" qualifications and enhance their export competitiveness.

[0133] Customized multiplex detection development: We provide "N+1" detection solution development services by adding or replacing targets based on customer needs (such as expanding to foot-and-mouth disease virus, bluetongue virus, etc.).

[0134] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A digital PCR kit for simultaneous detection of bovine lumpy skin disease virus, Schmallenberg virus, and peste des petits ruminants virus, characterized in that: The kit comprises: Primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18; In the probes, for example, the 5' end of SEQ ID NO.3 and SEQ ID NO.9 is fluorescently labeled with FAM, the 3' end of SEQ ID NO.3 is fluorescently labeled with MGB, and the 3' end of SEQ ID NO.9 is fluorescently labeled with BHQ1; For example, the 5' end of SEQ ID NO.6 and SEQ ID NO.18 is set with ROX fluorescent label, the 3' end of SEQ ID NO.6 is set with MGB fluorescent label, and the 3' end of SEQ ID NO.18 is set with MGB fluorescent label; For example, the 5' end of SEQ ID NO. 12 is labeled with HEX fluorescent marker, and the 3' end is labeled with BHQ1 fluorescent marker; For example, the 5' end of SEQ ID NO. 15 is labeled with CY5 fluorescent marker, and the 3' end is labeled with BHQ2 fluorescent marker.

2. A detection method of the kit according to claim 1, characterized in that: The method comprises the following steps: (1) extracting nucleic acid from the sample to be tested, and amplifying the nucleic acid using primers shown in SEQ ID NOs. 1-2, SEQ ID NOs. 4-5, SEQ ID NOs. 7-8, SEQ ID NOs. 10-11, and SEQ ID NOs. 13-14; (2) Detecting the amplified product using probes such as SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, and SEQ ID NO. 15; (3) Perform digital PCR fluorescence signal acquisition, collecting the fluorescence signals of channels FAM, HEX, ROX, and CY5.

3. The method according to claim 2, wherein The digital PCR amplification reaction system comprises: 10 μL of digital PCR reaction master mix; Primer Mix 1.1 μL; Probe Mix 0.64 μL; 2 μL nucleic acid template; Add dd H2O to 20 μL.

4. The method according to claim 2, wherein The amplification reaction procedure includes: Pre-denaturation at 95°C for 10 minutes; Denaturation at 94°C for 30 seconds, annealing and extension at 60°C for 1 minute, 40 cycles; Inactivate at 98°C for 10 minutes; Cool at 20°C for 2 minutes; At the end of each cycle, fluorescence signals of four channels, FAM, HEX, ROX, and CY5, were collected.

5. The method according to claim 2, wherein The positive control included a concentration of 10 3 The recombinant plasmid mixture of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard was 100 copies / μL, and the negative control was ultrapure water.

6. The method according to claim 2, wherein In the digital PCR test results: The FAM channel is used to detect Schmallenberg virus and bovine lumpy skin disease virus-ORF101 genes; The HEX channel is used to detect the peste des petits ruminants virus-N gene; The ROX channel was used to detect the internal standard and the bovine lumpy skin disease virus-ORF126 gene; The CY5 channel is used to detect the peste des petits ruminants virus-M gene.

7. The method according to claim 2, wherein In the same sample: The positive FAM channel band 1 was determined to be the presence of Schmallenberg virus; The simultaneous positive results of FAM channel band 2 and ROX channel band 2 indicate the presence of bovine lumpy skin disease virus; The simultaneous positive results of HEX channel bands and CY5 channel bands indicate the presence of PPR virus.

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