A digital PCR kit for simultaneously detecting bovine tuberous skin disease virus, schmallenberg virus and peste des petits ruminants virus

Abstract

The application belongs to the technical field of virus detection, and discloses a digital PCR kit for simultaneously detecting bovine dermatophilosis virus, Schmallenberg virus and peste des petits ruminants virus, which comprises primers and probes shown as SEQ ID NO. 1-SEQ ID NO. 18. After single system tests on bovine dermatophilosis virus-ORF101 gene, bovine dermatophilosis virus-ORF126 gene, Schmallenberg virus, peste des petits ruminants virus-N gene and peste des petits ruminants virus-M gene, a six-repeated digital PCR system containing an internal standard is established. After tests on indexes such as linearity, specificity, minimum detection limit, repeatability, single and multiple digital PCR systems, the developed digital PCR multiple detection kit can fully meet the development requirements, has strong specificity, high sensitivity, and the minimum detection limit can reach below 10 Copies / μL.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and in particular relates to a digital PCR kit for simultaneously detecting bovine nodular dermatitis virus, Schmalenberg virus, and peste des petits ruminants virus. Background Technology

[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 global livestock farming in recent years, with expanding prevalence, complex transmission routes, and significant bottlenecks in detection technology. 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 the ORF126 gene is related to viral membrane protein synthesis and is a key target for distinguishing wild-type strains from gene-deleted vaccine strains (such as the Neethling strain). Schmallenberg Virus (SBV), belonging to the Bunyaviridae family, is transmitted through vectors such as Culicoides midges. Infecting ruminants such as cattle and sheep causes abortion, fetal malformations, and newborn mortality, and its spread expands with the northward shift of insect distribution areas. Pediatric peste des petits ruminants virus (PPRV) belongs to the Measlesvirus genus and primarily infects goats and sheep. Its N gene encodes the nucleocapsid protein, essential for viral replication, while the M gene encodes the matrix protein; both are diagnostic markers. PPRV has a mortality rate as high as 90%.

[0003] Traditional detection methods (such as virus isolation, ELISA, or singleton qPCR) require independent procedures, which are not only time-consuming and costly, but also lack sensitivity for low-virus-load samples (such as latent infections, animals in the recovery period, or environmental samples). For example, the detection limit of qPCR is typically 100 copies / reaction, resulting in a false negative rate of over 30% for early infections or asymptomatic carriers. Furthermore, multiplex qPCR is limited by fluorescence channels (usually ≤4 channels), making simultaneous detection of multiple pathogens difficult, and differences in amplification efficiency can easily lead to result deviations. More seriously, viral genetic heterogeneity (such as LSDV-ORF126 gene deletion mutations and PPRV-N gene point mutations) can cause primer-probe binding site failures, further reducing detection reliability. Against this backdrop, developing 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 control.

[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 is the first to integrate five gene targets of LSDV, SBV, and PPRV into a single detection system. Its significance lies in: ① Addressing the challenge of co-circulation: In areas with high incidence of disease, the same animal population may be exposed to multiple pathogens. Multiplex detection can significantly shorten the diagnostic time and avoid repeated sample consumption; ② Effectively overcoming the sensitivity problem 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) is used to achieve visual differentiation. ③ Enhanced result reliability: Dual-target probes (ORF101 and ORF126) are designed for LSDV, enabling simultaneous identification of wild-type and vaccine strains; combined detection of the N / M genes for PPRV avoids false negatives caused by single-target mutations; the SBV-N gene probe design considers viral evolutionary branches, reducing the risk of missed detections due to segmental recombination. ④ Improved detection efficiency and cost-effectiveness: Traditional methods require at least three independent experiments to detect three viruses, while this kit, through optimized four-color fluorescence channels and primer / probe combination design, achieves "six detections in one tube," including internal standards, increasing throughput by over 80%, making it particularly suitable for cross-border ports, large-scale epidemic screening, or vaccine efficacy evaluation. Furthermore, dPCR does not rely on a standard curve and can directly calculate viral copy number, providing data precision that qPCR cannot achieve for dynamic monitoring of viral load (such as evaluating the efficacy of antiviral treatment). In summary, the development of this invention not only solves the core deficiencies 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] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0006] (1) Traditional detection methods (such as virus isolation, ELISA or singleton qPCR) need to be performed independently, which is not only time-consuming and costly, but also lacks sensitivity for low-load samples (such as latent infections, animals in the recovery period or environmental samples). Taking qPCR as an example, its detection limit is usually 100 copies / reaction, and the false negative rate for early infection or latent carrier animals can reach more than 30%.

[0007] (2) Multiplex qPCR is limited by fluorescence channels (usually ≤4 channels), making it difficult to achieve simultaneous detection of multiple pathogens, and differences in amplification efficiency can easily lead to result deviations. More seriously, viral genetic heterogeneity (such as deletion mutations in the LSDV-ORF126 gene and point mutations in the PPRV-N gene) may cause primer-probe binding site failure, further reducing detection reliability. Against this backdrop, developing 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 disease control. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a digital PCR kit for the simultaneous detection of bovine nodular dermatitis virus, Schmalenberg virus, and peste des petits ruminants virus.

[0009] The present invention is achieved by providing a digital PCR kit for the simultaneous detection of bovine nodular dermatitis virus, Schmalenberg virus, and peste des petits ruminants virus, wherein the kit includes primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0010] A method for detecting bovine nodular dermatitis virus, Schmalenberg 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 primers as 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 amplification products are identified by probes as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15;

[0012] 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; and 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 set IPC internal standards including primers and probes as shown in SEQ ID NO.16~SEQ ID NO.18. For example, the 5' end of the probe shown in SEQ ID NO.18 is labeled with ROX fluorescent label and the 3' end is labeled with MGB fluorescent label; perform digital PCR fluorescence detection and collect the signal of the fluorescence channel;

[0014] (3) Results Analysis:

[0015] In this invention, the concentrations are 1.0 × 10⁻⁶. 3 A mixture of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard recombinant plasmids (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 graph, the darkest black dot represents a negative droplet, and the other colored droplets are the positive droplets for the corresponding channels. The negative control should have no positive droplets, while the positive control should show positive droplets with corresponding bands in all channels.

[0017] FAM channel band 1 represents Schmalenberg virus positive droplets, FAM channel band 2 represents bovine nodular dermatitis virus-ORF101 gene positive droplets, HEX channel band represents small ruminant virus-N gene positive droplets, ROX channel band 1 represents internal standard positive droplets, ROX channel band 2 represents bovine nodular dermatitis virus-ORF126 gene positive droplets, and CY5 channel band represents small ruminant virus-M gene positive droplets.

[0018] For the same sample: a positive FAM channel band 1 indicates Schmalenberg virus infection; a positive FAM channel band 2 and a positive ROX channel band 2 simultaneously indicate the presence of bovine nodular dermatitis virus in the sample, and that the virus originated from natural infection rather than vaccine immunization; a positive FAM channel band 2 and a negative ROX channel band 2 indicate the presence of bovine nodular dermatitis virus in the sample, and that the virus originated from vaccine immunization rather than natural infection; a positive HEX channel band and a positive CY5 channel band indicate peste des petits ruminants virus infection.

[0019] Based on the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solution to be protected by this invention from the following aspects:

[0020] First, after testing single-layer systems of bovine nodular dermatitis virus-ORF101 gene, bovine nodular dermatitis virus-ORF126 gene, Schmalenberg virus, peste des petits ruminants virus-N gene, and peste des petits ruminants virus-M gene, this invention established a six-layer digital PCR system including an internal standard. After testing indicators such as linearity, specificity, limit of detection, repeatability, and comparison of single-layer and multiplex digital PCR systems, the currently developed digital PCR multiplex detection kits fully meet the development requirements, exhibiting high specificity, high sensitivity, and a limit of detection of less than 10 copies / μL.

[0021] Secondly, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

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

[0023] This invention develops a six-target digital PCR kit that can simultaneously detect bovine nodular dermatitis virus (LSDV)-ORF101 and ORF126 genes, Schmalenberg virus (SBV), peste des petits ruminants virus (PPRV)-N and M genes, and an internal standard. Its commercial value lies in its high alignment with the urgent needs of the global animal disease detection market. According to data from the World Organisation for Animal Health (OIE), LSDV and PPRV alone cause billions of dollars in economic losses to the livestock industry annually, and the increased risk of cross-border disease transmission has heightened countries' reliance on efficient detection technologies. Traditional detection methods (such as qPCR or ELISA) require stepwise detection of different pathogens, are time-consuming (2-3 days per test), costly (approximately $50-$100 per sample), and struggle to differentiate between multiple infections. This kit completes the detection of six targets in a single reaction, reducing the detection time to less than 4 hours, lowering the cost to $30 per sample, and increasing efficiency by more than 5 times. It is particularly suitable for import / export quarantine, large-scale screening in large-scale farms, and high-incidence areas.

[0024] From a market perspective, this kit, with its high sensitivity (detection limit of 10 copies / μL), strong specificity (plasmid-verified no cross-reactivity), and standardized procedures, is poised to rapidly capture market share. Conservatively estimated, if it covers 10% of the global demand for bovine and ovine disease testing (approximately 200 million head per year), annual revenue could reach $600 million. Furthermore, the technology platform can be extended to other animal diseases (such as foot-and-mouth disease and African swine fever), forming a product matrix and further extending its commercial lifecycle.

[0025] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally:

[0026] The current technological gap in the field of digital PCR detection for animal diseases lies in the insufficient multiplex detection capabilities. According to statistics from the patent database (Derwent Innovation), as of 2023, among the publicly disclosed digital PCR-related patents for animal diseases worldwide, 91% are single or dual detection, and only 8% involve triple or higher detection, with targets mostly limited to a single pathogen (such as detecting only PPRV or LSDV). For example, Chinese patent CN112111572A achieves dual-gene detection of PPRV, but does not integrate other pathogens; although US patent US20210017562A1 proposes quadruple detection, the target is foot-and-mouth disease virus subtype typing, which does not overlap with the cross-pathogen detection of this invention. This kit is the first system to achieve six-fold detection of "three pathogens - five genes - plus internal standard". Its innovation is reflected in two aspects: In terms of technology, by optimizing primer and probe design (optimizing primer sequence and annealing temperature and introducing quenching group modification), the problems of channel interference and background signal in the six-fold system are solved; In terms of application, it is the first to integrate the detection of three major cross-border pathogens, LSDV, SBV and PPRV, covering the key conserved genes of the pathogens (such as LSDV-ORF126, which is the capsid protein encoding gene, and PPRV-N gene, which is the core target of nucleoprotein), significantly improving the clinical guidance value of the detection results.

[0027] From an industry standard perspective, existing international reagent kits (such as Thermo Fisher's VetMAX) TM The previous series only supported the qPCR platform and required multiple tube reactions to achieve the same detection volume. This invention breaks through the bottleneck of low multiplication count in digital PCR in the field of animal diseases, and provides a new technical option for the OIE "Handbook of Diagnostic Tests for Terrestrial Animals".

[0028] (3) Whether the technical solution of the present invention solves the technical problem that people have long wanted to solve but have never been able to solve successfully:

[0029] The core challenge of multiplex detection for animal diseases lies in achieving specific amplification of multiple targets without interference in highly complex samples (such as mixed-infected tissues). Traditional qPCR, limited by fluorescence channels (usually ≤4 channels) and primer dimer issues, struggles to achieve quadruple detection. Digital PCR, through optimization of probe concentration, surpasses the number of detection targets of traditional qPCR and offers absolute quantification. This breakthrough addresses four long-standing pain points in veterinary diagnostics: first, rapid identification of pathogens in the early stages of an outbreak to avoid misdiagnosis (e.g., similar clinical symptoms of LSDV and PPRV); second, accurate monitoring of viral mutations (e.g., ORF101 gene mutations may lead to vaccine escape), providing data support for vaccine development; third, absolute quantification without the need for standard curves and Ct values ​​required by traditional qPCR, solving the problem of limited accuracy due to lack of standards or cross-laboratory comparisons; and fourth, strong anti-interference capabilities, adapting to direct detection of complex samples, addressing the core pain point of difficult complex sample processing.

[0030] (4) Does the technical solution of the present invention overcome technical bias?

[0031] In the research and development of digital PCR technology for multiplex detection of animal diseases, two major technical biases have long existed in the industry: First, it is believed that multiplex detection systems (especially five-fold and above) will inevitably suffer from deteriorated linearity, decreased sensitivity, or loss of repeatability due to competition or cross-interference between primers and probes. Therefore, high-multiplex detection remains only at the theoretical verification level and is difficult to commercialize. Second, it is believed that there is a systematic bias in the quantitative results of single-fold and multiplex detection systems, making it impossible to guarantee data comparability, leading to the reliance on single-fold detection as the "gold standard" in clinical diagnosis. This 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, addressing the misconception that "high multiplicity leads to performance degradation," this kit achieves detection linearity, sensitivity, and repeatability for all targets (including internal standards) in a six-particle system that meet or even exceed single-particle detection standards. Experimental data show that, within the plasmid gradient dilution range, the linear regression coefficient (R²) for the six targets is [missing information]. 2 The values ​​are all greater than 0.99, proving that the multiplex system does not introduce nonlinear amplification interference. Meanwhile, the sensitivity of the six-fold detection reaches the single-copy level (lowest detection limit ≤ 10 copies / μL), and the repeatability is excellent (intra-batch CV < 1.5%). This result directly refutes the common perception that "multiplex detection inevitably sacrifices sensitivity."

[0033] Secondly, addressing industry concerns about the incomparability of single-plasm and multiplex quantification results, this kit demonstrates a high degree of consistency between the two quantification results through direct comparison experiments between single-plasm and six-plasm systems. In plasmid mixture samples (containing equal concentrations of 6 targets), the difference in quantitative concentration between single-plasm and six-plasm detection is less than 5% (CV < 2%), and there is no systematic trend in the direction of deviation (e.g., the six-plasm detection values ​​do not show an overall increase or decrease).

[0034] A deeper industry bias lies in the belief that plasmid-level validation cannot reflect the technical feasibility of real-world testing scenarios. However, the experimental design of this invention precisely demonstrates that achieving the performance limits of high-multiplexity detection within an idealized model (plasmid template) is a key prerequisite for overcoming technical bottlenecks. For example, plasmid mixing experiments can accurately quantify the cross-reactivity between primers and probes, while interference from inhibitors or complex matrices in clinical samples can be individually optimized through subsequent sample pretreatment modules (such as introducing anti-inhibition polymerases or magnetic bead purification). This modular development strategy breaks with the traditional thinking that "all technical difficulties must be solved simultaneously," providing a new paradigm for the phased industrialization of high-multiplexity digital PCR kits.

[0035] In summary, this technical solution demonstrates with rigorous experimental data that the six-fold digital PCR detection has achieved the core performance indicators of commercially available kits at the plasmid level, completely overcoming the industry's technical prejudice against the reliability of high-fold detection, and laying an irreplaceable theoretical and technical foundation for subsequent clinical sample validation and practical application. Attached Figure Description

[0036] Figure 1 This is a single-fluorescence PCR standard curve provided in this embodiment of the invention. Wherein, 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 The results are specific detection results of the single-system fluorescence PCR detection method provided in this embodiment of the invention.

[0038] Figure 3 This is a 1D diagram of positive droplets in each channel of the multiplex digital PCR reaction system provided in this embodiment of the invention.

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

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

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

[0042] Figure 5 This is a one-dimensional graph showing the linearity of positive droplets in each channel of the multiplex digital PCR reaction system provided in this embodiment of the invention. Wherein, A represents the FAM channel, B the HEX channel, C the ROX channel, and D 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 in this embodiment of the invention.

[0044] Figure 7 This is a one-dimensional graph showing the repeatability results of positive droplets in each channel of the multiplex digital PCR reaction system provided in this embodiment of the invention. Wherein, A represents the FAM channel, B represents the HEX channel, C represents the ROX channel, and D represents the CY5 channel.

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

[0046] Figure 9 This is a comparison chart of the results of multiplex digital PCR and individual digital PCR provided in this embodiment of the invention. The coefficients of variation (CV) of the results of single digital PCR and multiplex digital PCR are both less than 2%, indicating that the multiplex digital PCR and individual digital PCR have good consistency and that the multiplex digital PCR did not cause mutual interference. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0048] The purpose of this invention is to provide a digital PCR kit for the simultaneous detection of bovine nodular dermatitis virus, Schmalenberg virus, and peste des petits ruminants virus, wherein the kit includes primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0049] This invention also provides a method for detecting bovine nodular dermatitis virus, Schmalenberg 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 primers as 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 amplification products are identified by probes as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15;

[0051] 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; and 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 set IPC internal standards including primers and probes as shown in SEQ ID NO.16~SEQ ID NO.18. For example, the 5' end of the probe shown in SEQ ID NO.18 is labeled with ROX fluorescent label and the 3' end is labeled with MGB fluorescent label; perform digital PCR fluorescence detection and collect the signal of the fluorescence channel;

[0053] (3) Results Analysis:

[0054] In this invention, the concentrations are 1.0 × 10⁻⁶. 3A mixture of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard recombinant plasmids (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 graph, the darkest black dot represents a negative droplet, and the other colored droplets are the positive droplets for the corresponding channels. The negative control should have no positive droplets, while the positive control should show positive droplets with corresponding bands in all channels.

[0056] FAM channel band 1 represents Schmalenberg virus positive droplets, FAM channel band 2 represents bovine nodular dermatitis virus-ORF101 gene positive droplets, HEX channel band represents small ruminant virus-N gene positive droplets, ROX channel band 1 represents internal standard positive droplets, ROX channel band 2 represents bovine nodular dermatitis virus-ORF126 gene positive droplets, and CY5 channel band represents small ruminant virus-M gene positive droplets. Figure 3 );

[0057] For the same sample: a positive FAM channel band 1 indicates Schmalenberg virus infection; a positive FAM channel band 2 and a positive ROX channel band 2 simultaneously indicate the presence of bovine nodular dermatitis virus in the sample, and that the virus originated from natural infection rather than vaccine immunization; a positive FAM channel band 2 and a negative ROX channel band 2 indicate the presence of bovine nodular dermatitis virus in the sample, and that the virus originated from vaccine immunization rather than natural infection; a positive HEX channel band and a positive CY5 channel band indicate peste des petits ruminants virus infection.

[0058] In some specific implementation schemes, the amplification reaction system 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 a final volume of 20 μL.

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

[0060] Example 1: Design of primer and probe sequences

[0061] The complete genome sequence or N gene sequence of Schmalenberg virus (GenBank serial numbers: NC_043582.1, KT795133.1, LC309158.1, KC139376.1, KX384876.1, LC309162.1, LC309165.1, etc.) was obtained from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov); the complete genome sequence of bovine nodular dermatitis virus and... ORF101 and ORF126 gene sequences (GenBank serial 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 of peste des petits ruminants virus (PPR) and the N and M gene sequences (GenBank serial 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 is based on the conserved gene sequence of rRNA gene in sheep mitochondrial genome. After comparing the target gene sequence with SnapGene software, the relatively conserved nucleotide sequence of each pathogen is selected. According to the primer and TaqMan probe design principles and in combination with relevant national standards, relevant primer and probe sequences are designed. The kit detection targets and sequences are summarized in Table 1. The designed primers and probes are given to Sangon Biotech Co., Ltd. for synthesis.

[0062] Table 1. Primers and probes for the digital PCR kit used to detect bovine nodular dermatitis virus, Schmalenberg virus, and peste des petits ruminants virus.

[0063]

[0064]

[0065] Example 2: Construction of plasmid standards

[0066] The gene sequences of bovine nodular dermatitis virus, Schmalenberg virus, small ruminant 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 for later 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 standard curve for singleton fluorescence PCR reaction system

[0070] To verify whether the amplification efficiency and correlation coefficients of the primers and probes designed for each target met the requirements for establishing a digital PCR multiplex system, the positive plasmids of bovine nodular skin disease virus (LSDV) LSDV-ORF101 and LSDV-ORF126, the positive plasmid of Schmallenberg virus (SBV) SBV-N, and the positive plasmids of Peste des Petits Ruminants virus (PPRV) PPRV-N and PPRV-M were serially diluted 10-fold and then subjected to singlet fluorescent PCR. A concentration standard curve was plotted using the template concentration fluorescence Ct value. 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 fluorescent PCR reaction was performed according to the procedure shown in Table 3. The equipment used was a JLM QX600 from Jeremy Laboratories.

[0071] Table 2 Preparation of Single Fluorescence PCR Reaction System

[0072]

[0073] Table 3. Fluorescent PCR Reaction Parameter Settings

[0074]

[0075]

[0076] Concentration-Ct standard curves were prepared using LSDV-ORF101, SBV-N, PPRV-N, LSDV-ORF126, and PPRV-M positive plasmids, respectively. Figure 1 ).from Figure 1 As can be seen, in the singleton system, the standard curve PCR amplification efficiency E value, R value, and curve slope are all within the normal range, indicating good amplification efficiency, which can be used to establish a subsequent multiplex digital PCR system.

[0077] 2.2 Primer and probe specificity test

[0078] Eight other sheep disease-specific plasmids were selected for specificity testing, 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). Single-cell fluorescent PCR was performed using primers and probes from this kit for LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, and PPRV-M. Positive plasmids targeting LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, and PPRV-M 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. The fluorescent PCR reaction was carried out according to the procedure shown in Table 3. The equipment used was a JLMQX600 from Jeremy Laboratories.

[0079] Testing revealed that none of the target primers and probes in this kit exhibited nonspecific amplification compared to other sheep disease-positive plasmids, and each positive plasmid showed fluorescence intensity, indicating good specificity of the detection primers and probes, making them suitable for the subsequent construction of multiplex digital PCR systems. Results are shown below. Figure 2 Table 4 shows that the plasmid concentration is 1.0 × 10⁻⁶. 6 Copies / μL.

[0080] Table 4. Specificity testing of each target single-component system in this kit.

[0081]

[0082]

[0083] 2.3 Establishment of a digital PCR multiplex reaction system

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

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

[0086] Table 5. Preparation of digital PCR reaction system

[0087]

[0088] Table 6. Digital PCR Reaction Parameter Settings

[0089]

[0090] 2.4 Linearization and restriction enzyme digestion of target plasmids

[0091] The theoretical value of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and internal standard plasmid is 10. 6 Six positive plasmids with a ratio of 6 copies / μL were mixed together in equal volumes and linearized using two cutting enzymes, QuickCut Sac I and QuickCut Kpn I. The enzyme digestion system is shown in Table 7. The digestion was completed by incubating at 37℃ for 15 minutes.

[0092] Table 7. Plasmid linearization enzyme digestion system

[0093]

[0094] Using the six positive plasmids after enzyme digestion, tests were conducted according to the final concentrations of each target primer and probe obtained in Table 1 after optimization, the reaction system in Table 5, and the reaction procedure in Table 6. The 1D graph of the test results is shown below. Figure 3 2D image Figure 4 As shown in the 1D image, the bands between the targets in each channel are clearly layered and do not interfere with each other in detection. As shown in the 2D image, the targets are clearly distinguished and the bands are clearly divided. The 1D and 2D images show good results, and the experimental results prove that the multiplex digital PCR reaction system has been successfully established.

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

[0096] The theoretical concentration after enzyme digestion is 10.4 The plasmid mixture of Copies / μL was serially diluted 4-fold in 7 gradients for digital PCR reactions, with dd H2O set 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 limit of detection of this method. Sample concentration (Copies / μL) = (Result of set value (Copies / μL) × 20 (total reaction volume) ÷ 2 (sample loading amount).

[0097] Digital PCR results showed that the number of droplets was greater than 10,000, meeting the experimental analysis requirements, and the correlation coefficient of the standard curve (R) was [value missing]. 2 The linearity of the multiplex digital PCR method is greater than 0.98, which meets the performance requirements.

[0098] In the multiplex digital PCR system, the limits of detection (LODs) for 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. The LODs for all targets in the multiplex digital PCR system were below 10 copies / μL. (See attached figures.) Figure 5 , Figure 6 Table 8.

[0099] Table 8. Statistics of the lowest detection limit test results for 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, repeated eight times. The results were statistically analyzed, and the coefficient of variation was calculated to evaluate the reproducibility of the method.

[0103] Select 10 4 Copies / μL diluted 16-fold and mixed with plasmid as a positive template were used for intragroup replication experiments. The results showed that the number of droplets was greater than 10,000, meeting the experimental requirements. The intragroup coefficient of variation was between 0.85% and 1.18%, all less than 1.5%, indicating that the method has good reproducibility. The results are shown in [Figure number missing]. Figure 7 As shown in Table 9.

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

[0105]

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

[0107] Single- and multiple-system digital PCR reactions were performed for each target using five different positive mixed plasmid single copy number concentrations. The set concentrations corresponding to the same template concentration were compared, and the coefficient of variation of the set concentration results for single- and multiple-systems was calculated to compare the concordance between single- and multiple-system digital PCR.

[0108] The results showed that the number of droplets was greater than 10,000, meeting the experimental requirements. The coefficients of variation of copy number for both singlet and multiplex digital PCR were less than 2%, indicating that the singlet and multiplex digital PCR reaction systems showed little difference in the results for the same sample, thus meeting the experimental requirements. The experimental results are as follows: Figure 8 , Figure 9 As shown in Table 10.

[0109] Table 10. Statistical analysis of the comparison test results of single-particle and multiplex digital PCR systems of this kit.

[0110]

[0111] After testing single-particle systems of bovine nodular dermatitis virus-ORF101 gene, bovine nodular dermatitis virus-ORF126 gene, Schmalenberg virus, peste des petits ruminants virus-N gene, and peste des petits ruminants virus-M gene, a six-particle digital PCR system including an internal standard was established. After testing for linearity, specificity, limit of detection, repeatability, and comparison of single-particle and multiplex digital PCR systems, the developed digital PCR multiplex detection kit fully meets the development requirements.

[0112] I. Specific application areas or related products of the present invention.

[0113] Application areas:

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

[0115] Application scenario: 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 "fast customs clearance".

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

[0118] Application scenarios: Used for regular screening in farms, pathogen screening before introducing new species, and tracing the source of infection during outbreaks. For example, by detecting the ORF101 and ORF126 dual genes of LSDV, wild-type strains and vaccine strains can be distinguished, guiding precise immunization.

[0119] Technical advantages: High sensitivity (≤10 copies / μL) can detect latent infections at an early stage. 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 scenarios: As a quality control material or standard substance for third-party testing institutions, it is used to verify the accuracy of other testing methods (such as qPCR, ELISA).

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

[0123] 4. Vaccine development and efficacy evaluation

[0124] Application scenarios: Monitoring the effectiveness of virus inactivation during vaccine production (e.g., detecting residual LSDV nucleic acid), or evaluating the decrease in viral load 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, forming a complete technology ecosystem:

[0128] 1. Core Products

[0129] Six-fold digital PCR detection kit (premixed): contains liquid or lyophilized microsphere primers and probes, reaction buffer, and positive control. It is ready to use and suitable for mainstream digital PCR instruments (such as Bio-Rad QX200 and Thermo Fisher QuantStudio 3D).

[0130] Multi-pathogen mixed control: Contains simulated genomic fragments of LSDV, SBV, and PPRV, used for standardized validation of laboratory testing procedures, with concentration gradients covering the clinically common viral load range (1–10). 5 (copies / μL).

[0131] 2. Extended services

[0132] Animal disease testing and certification services: We provide testing data endorsement based on this kit to livestock enterprises, assisting them in obtaining OIE certification as "disease-free enterprises" and enhancing their export competitiveness.

[0133] Customized multi-target detection development: We provide “N+1” type detection solution development services by adding or replacing targets according to customer needs (such as expanding to foot-and-mouth disease virus, bluetongue virus, etc.).

[0134] The above description is merely 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 those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A digital PCR kit for simultaneous detection of bovine nodular dermatitis virus, Schmalenberg virus, and peste des petits ruminants virus, characterized in that, The kit includes: Primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18; In the probe, for example, FAM fluorescent label is set at the 5' end of SEQ ID NO.3 and SEQ ID NO.9, MGB fluorescent label is set at the 3' end of SEQ ID NO.3, and BHQ1 fluorescent label is set at the 3' end of SEQ ID NO.9; For example, ROX fluorescent markers are set at the 5' end of SEQ ID NO.6 and SEQ ID NO.18, MGB fluorescent markers are set at the 3' end of SEQ ID NO.6, and MGB fluorescent markers are set at the 3' end of SEQ ID NO.18; For example, the 5' end of SEQ ID NO.12 is marked with HEX fluorescent label and the 3' end is marked with BHQ1 fluorescent label; For example, SEQ ID NO.15 has a CY5 fluorescent label at the 5' end and a BHQ2 fluorescent label at the 3' end.

2. A detection method for non-disease diagnostic purposes using the kit as described in claim 1, characterized in that, The method includes the following steps: (1) Extract nucleic acid from the sample to be tested and amplify it using primers as 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; (2) The amplification products were detected 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, and collect fluorescence signals from channels FAM, HEX, ROX, and CY5.

3. The method as described in claim 2, characterized in that, The digital PCR amplification reaction system includes: 10 μL of digital PCR reaction premix; Primer Mix 1.1 μL; Probe Mix 0.64 μL; 2 μL of nucleic acid template; Add dd H2O to a final volume of 20 μL.

4. The method as described in claim 2, characterized in that, The amplification reaction procedure includes: Pre-denaturation at 95℃ for 10 minutes; Denaturation at 94 ℃ for 30 seconds, annealing and extending at 60 ℃ for 1 minute, cycled 40 times; Inactivate at 98 ℃ for 10 minutes; Cool at 20°C for 2 minutes; At the end of each cycle, fluorescence signals from four channels—FAM, HEX, ROX, and CY5—were collected.

5. The method as described in claim 2, characterized in that, The method includes a positive control, which comprises a concentration of 10... 3 A mixture of LSDV-ORF101, LSDV-ORF126, SBV-N, PPRV-N, PPRV-M, and IPC internal standard recombinant plasmids (copy / μL) was used as the negative control, with ultrapure water as the negative control.

6. The method as described in claim 2, characterized in that, In the digital PCR test results: The FAM channel is used to detect the Schmalenberg virus and bovine nodular dermatosis virus-ORF101 gene; The HEX channel is used to detect the peste des petits ruminants virus-N gene; The ROX channel is used to detect internal standards and the bovine nodular dermatitis virus-ORF126 gene; The CY5 channel is used to detect the peste des petits ruminants virus-M gene.

7. The method as described in claim 2, characterized in that, In the same sample: A positive FAM channel band 1 indicates the presence of Schmalenberg virus; Simultaneous positivity of both FAM channel band 2 and ROX channel band 2 indicates the presence of bovine nodular dermatitis virus; Simultaneous positivity of both the HEX and CY5 bands indicates the presence of peste des petits ruminants virus (PPR).

Images