A digital PCR kit for simultaneous detection of African horse sickness virus, West Nile virus, and equine influenza H3N8

Abstract

This invention belongs to the field of virus detection technology and discloses a digital PCR kit for the simultaneous detection of African horse sickness virus (ESV), West Nile virus (WNVI), and equine influenza H3N8. The kit includes primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.21. After testing single-particle systems of the ESV-VP7 gene, ESV-NS2 gene, WNVI-NS5 gene, WNVI-NS2a gene, equine influenza H3N8-HA gene, and equine influenza H3N8-NA gene, a seven-particle digital PCR system including an internal standard was established. After testing 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, exhibiting high specificity and sensitivity, with limits of detection all below 10 copies / μL.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus detection, and particularly relates to a digital PCR kit for simultaneously detecting African horse sickness virus, West Nile virus and equine influenza H3N8. BACKGROUND

[0002] African Horse Sickness Virus (AHSV), West Nile Virus (WNV) and Equine Influenza Virus H3N8 (H3N8) are core pathogens that threaten the health of global equine animals. African Horse Sickness Virus belongs to the Reoviridae family and is transmitted by Culicoides. Its high mortality rate (up to 90%) and rapid transmission characteristics cause devastating damage to the racing and livestock industries. The genome of AHSV contains 10 segments, of which the VP7 gene encodes the major capsid protein and is the core marker for virus typing, and the NS2 gene is related to virus particle assembly and host cell immune escape, and its variation may affect the pathogenicity of the strain. West Nile Virus is a mosquito-borne flavivirus that can infect horses, humans and birds. Its NS5 gene encodes an RNA-dependent RNA polymerase, which is a key element of viral replication, and the NS2a gene is involved in the inhibition of the host interferon signaling pathway and is closely related to viral pathogenicity. Due to the easy mutation of the genome of WNV, the conserved regions of NS5 and NS2a can be used as detection targets, covering epidemic strains of different geographical strains, and revealing the mechanism of viral escape from host immunity. Equine influenza H3N8 subtype virus is the main pathogen of respiratory diseases in equine animals. The HA (hemagglutinin) and NA (neuraminidase) genes are responsible for virus adsorption to host cells and release of progeny viruses, respectively, and are key targets for subtype typing and vaccine design. Joint detection of HA and NA genes can simultaneously monitor viral antigen drift and vaccine matching, providing double protection for epidemic early warning.

[0003] Currently, the detection techniques for these three viruses mainly rely on single-pathogen fluorescent quantitative PCR (qPCR), serological methods or virus isolation, which have significant defects. First, the traditional detection of African horse sickness virus (AHSV) relies on virus isolation or ELISA, which is time-consuming (3-5 days) and has low sensitivity, especially in latent period or low viral load samples (such as Culex or environmental samples), with a high risk of missed detection. Although some kits use multiplex PCR to detect the VP7 gene, they lack simultaneous analysis of the NS2 gene, which cannot comprehensively evaluate the virus replication capacity. The detection of West Nile virus (WNV) often uses IgM antibody capture ELISA, but the antibody window period (5-7 days after infection) limits its early diagnostic value. The detection of equine influenza H3N8 subtype also faces challenges. Although the RT-LAMP technique can complete HA gene amplification within 75 minutes, it cannot distinguish the co-variation of HA and NA genes, and has insufficient detection ability for mixed infections (such as H3N8 and WNV). In addition, existing methods such as hemagglutination inhibition test (HI) rely on virus culture, which is complex to operate and has high bio-safety risk. More importantly, there is a lack of multi-pathogen joint detection technology, and breeding farms need to purchase kits separately, resulting in a doubling of sample consumption, high cost, and difficulty in revealing the synergistic pathogenic mechanism of pathogens (such as immune suppression after H3N8 infection, which may exacerbate the neuroinvasiveness of WNV).

[0004] Digital PCR (dPCR) provides an innovative solution for multi-pathogen joint detection due to its absolute quantification, ultra-high sensitivity, and strong resistance to inhibitors. First, dPCR can simultaneously detect AHSV-VP7, NS2, WNV-NS5, and NS2a, H3N8-HA and NA through microdroplet partitioning technology, breaking through the bottleneck of cross interference of multiple primers in traditional methods. The significance of multi-target joint detection is: ①Improved comprehensive diagnostic efficiency: a single reaction can cover the key virulence and typing markers of the three viruses, suitable for entry and exit quarantine, racehorse screening, and epidemic area monitoring; ②Analysis of mixed infection and co-evolution mechanism: reveal the impact of H3N8 and WNV co-infection on the nervous system of horses, or the seasonal transmission correlation of AHSV and mosquito-borne viruses, providing a basis for precise medication and prevention and control strategies; ③Vaccine development and wild virus monitoring: through HA / NA gene variation tracking, the effectiveness of vaccines can be evaluated in real time, and the detection of NS2 and NS2a provides data support for antiviral drug target screening. In addition, the modular design of the kit allows flexible adjustment of target combinations according to regional epidemic strains (such as African horse sickness type 9 or WNV North American strain), combining the advantages of standardization and customization, and providing key technical tools for global horse disease prevention and control network construction.

[0005] Through the above analysis, the problems and defects of the prior art are:

[0006] (1) Current detection techniques for these three viruses rely on single-pathogen fluorescent quantitative PCR (qPCR), serological methods or virus isolation, which have significant limitations.

[0007] (2) Traditional detection of African horse sickness virus relies on virus isolation or ELISA, which is time-consuming (3-5 days) and has low sensitivity, especially in latent or low viral load samples (such as Culex or environmental samples), with a high risk of missed detection. Although some kits use multiplex PCR to detect the VP7 gene, they lack simultaneous analysis of the NS2 gene, which cannot comprehensively evaluate viral replication capacity. IgM antibody capture ELISA is often used for West Nile virus detection, but the antibody window period (5-7 days after infection) limits its early diagnostic value.

[0008] (3) The detection of equine influenza H3N8 subtype also faces challenges. RT-LAMP technology can complete HA gene amplification within 75 minutes, but it cannot distinguish between HA and NA gene co-variation, and has insufficient detection capability for mixed infections (such as H3N8 and WNV).

[0009] (4) Existing methods such as hemagglutination inhibition test (HI) rely on virus culture, which is complex to operate and has high bio-safety risk. More importantly, there is a lack of multi-pathogen joint detection technology, and farms need to purchase kits separately, resulting in a doubling of sample consumption, high costs, and difficulty in revealing the pathogenic mechanism of pathogen synergy (such as immune suppression after H3N8 infection, which may exacerbate the neuroinvasiveness of WNV). SUMMARY

[0010] To solve the problems of the prior art, the present application provides a digital PCR kit for simultaneously detecting African horse sickness virus, West Nile virus and equine influenza H3N8.

[0011] The present application is realized in that a digital PCR kit for simultaneously detecting African horse sickness virus, West Nile virus and equine influenza H3N8 subtype, the kit comprises primers and probes as shown in SEQ ID NO. 1-SEQ ID NO. 21.

[0012] A method for detecting African horse sickness virus, West Nile virus and equine influenza H3N8 subtype for non-disease diagnosis or treatment purposes, comprising the following steps:

[0013] (1) using extracted nucleic acid of the sample to be tested as a template, 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, SEQ ID NO. 13-14, SEQ ID NO. 16-17 to amplify the sample to be tested, and using probes as shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18 to identify the amplification product;

[0014] the 5' end of the probe as shown in SEQ ID NO. 3 is provided with FAM fluorescent label, and the 3' end is provided with MGB fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 6 is provided with ROX fluorescent label, and the 3' end is provided with BHQ2 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 9 is provided with FAM fluorescent label, and the 3' end is provided with BHQ1 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 12 is provided with CY5 fluorescent label, and the 3' end is provided with BHQ2 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 15 is provided with HEX fluorescent label, and the 3' end is provided with BHQ1 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 18 is provided with CY5 fluorescent label, and the 3' end is provided with BHQ2 fluorescent label;

[0015] (2) the IPC internal standard is set at the same time, including primers and probes as shown in SEQ ID NO. 19-SEQ ID NO. 21, the 5' end of the probe as shown in SEQ ID NO. 21 is provided with ROX fluorescent label, and the 3' end is provided with MGB fluorescent label; fluorescence quantitative detection is carried out, and the signals of the fluorescence channels are collected;

[0016] (3) result analysis:

[0017] In the present application, the mixture of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, H3N8-NA, and IPC internal standard recombinant plasmids with a concentration of 103 copies / μL respectively is used as a positive control, and the negative control is ultrapure water.

[0018] Click "analysis" on the biochip reader to analyze the experimental data. In the 2-dimensional graph or 1-dimensional graph, the darkest black dot represents the negative droplet, and the remaining color droplets are positive droplets corresponding to the channels. The negative control should have no positive droplets, and all channels of the positive control should have positive droplets corresponding to the bands;

[0019] FAM channel band 1 represents West Nile virus-NS5 gene positive microdroplets, FAM channel band 2 represents African horse sickness virus-VP7 gene positive microdroplets, HEX channel band represents equine influenza H3N8-HA gene positive microdroplets, ROX channel band 1 represents internal standard positive microdroplets, ROX channel band 2 represents African horse sickness virus-NS2 gene positive microdroplets, CY5 channel band 1 represents equine influenza H3N8-NA gene positive microdroplets, CY5 channel band 2 represents West Nile virus-NS2a gene positive microdroplets;

[0020] The same sample is determined: FAM channel band 1 and CY5 channel band 2 are positively determined as West Nile virus infection, HEX channel band and CY5 channel band 1 are positively determined as equine influenza H3N8 subtype infection, and FAM channel band 2 and ROX channel band 2 are determined as African horse sickness virus infection.

[0021] In combination with the technical solutions and the technical problems solved above, the advantages and positive effects of the technical solutions to be protected by the present application are analyzed from the following aspects:

[0022] First, in view of the technical problems existing in the prior art and the difficulty in solving the problems, the technical solutions to be protected by the present application and the results and data in the research and development process are combined in detail and deeply to analyze how the technical solutions solve the technical problems and bring some creative technical effects after solving the problems. The specific description is as follows:

[0023] Technical problems existing in the prior art and difficulty in solving:

[0024] 1. Limitation of fluorescence channel and insufficient multiplex detection capability: The traditional digital PCR technology is limited by the number of hardware fluorescence channels (usually 4 channels), and the existing technology can only realize fourfold detection through the "one channel one target" mode, which cannot break the linear relationship between the number of targets and channels. For example, the "four-channel four-target" horse disease detection scheme disclosed in patent CN114395594A requires additional detection times or equipment upgrading (such as six-channel instruments) to realize more target detection, resulting in a sharp increase in cost and an increase in operation complexity. For the multi-gene synchronous detection demand of equine animal diseases (such as AHSV, WNV, EIV), the existing technology is difficult to cover 7 targets (including internal standard) in a single reaction, which seriously restricts the detection efficiency.

[0025] 2. Imbalance of multiplex amplification efficiency at a single annealing temperature: In a multiplex PCR system, if the annealing temperature (Tm value) of different primers differs by more than 5℃, the amplification efficiency of part of the targets will be significantly reduced (such as high Tm value primers prefer to bind, and low Tm value targets are delayed in amplification).

[0026] 3. Compatibility defect of internal standard and sample matrix: The existing internal standard mostly uses exogenous additives (such as lambda DNA, artificially synthesized plasmid), and there is a significant difference in extraction efficiency between the exogenous additives and equine DNA (recovery rate deviation > 30%). Especially when dealing with high inhibitor samples (such as equine whole blood, spleen tissue), the exogenous internal standard may be degraded or inhibited in advance, resulting in calibration failure. In addition, the exogenous internal standard may introduce cross contamination risk, which does not meet the OIE requirement of "zero contamination" for epidemic disease detection.

[0027] 4. Technical problem of performance degradation in high number detection: With the increase of the number of detection targets, the sensitivity, linearity and repeatability of digital PCR will decrease significantly in the existing technology. For example, the sensitivity of a five-fold or more detection system is usually reduced by more than 10 times (LoD from ≤5 copies / μL to ≥50 copies / μL) compared with single target detection, and the repeatability (CV) is more than 8%. Therefore, the existing technology limits the multiplexing capability of digital PCR to 3-4 targets (such as the three-fold equine influenza detection of patent EP3567089A1), which cannot meet the clinical demand for simultaneous detection of multiple pathogens and multiple genes.

[0028] Innovative breakthrough of the technical solution of the present application:

[0029] 1. Four-channel seven-target probe concentration gradient coding technology: When detecting two targets in the same fluorescence channel (such as FAM), the target can be distinguished by accurately controlling the probe concentration gradient (target A probe concentration: 250nM, target B probe concentration: 125nM) and using the difference in the end-point fluorescence signal intensity of digital PCR. For example, AHSV-VP7 (high concentration probe) and WNV-NS5 (low concentration probe) share the FAM channel, and accurate differentiation (resolution > 95%, verified by plasmid) is achieved by setting the signal amplitude threshold (high signal for VP7 and low signal for NS5).

[0030] 2. Tm uniformization by primer and MGB probe modification: In order to achieve Tm uniformization, the length and sequence of the primer are adjusted, and degenerate bases are introduced to increase the coverage of the primer. For some low Tm value probes (such as AHSV-VP7 original Tm value of 53.5℃), MGB (minor groove binder) modification is introduced at the 3' end of the probe to increase the actual Tm value to more than 65℃, ensuring stable hybridization of all probes at a uniform annealing temperature (60℃) and avoiding non-specific signals.

[0031] 3. Selecting equine mitochondrial rRNA gene as internal standard (ROX channel detection), which has the following advantages: Each equine cell contains hundreds of mitochondria, and the copy number of rRNA gene is much higher than that of nuclear genome (> 10^4 copies / cell), which can sensitively reflect the extraction efficiency of trace samples (such as nasal swabs).

[0032] 4. Limit breakthrough of high multiplex detection performance: under seven-fold system, the linear range of all detection targets covers 1-10^5 copies / μL (R 2 >0.99), the minimum detection limit is ≤10 copies / μL (as low as 2.82 copies / μL for African horse sickness virus AHSV-VP7 gene), which is in the same order of magnitude as the single detection sensitivity (≤5 copies / μL); the CV value of the logarithmic concentration of batch repeatability is less than 2%, which is significantly better than the traditional multiplex detection (CV>5%); after specific test, there is no mutual amplification between targets, and the specificity is good.

[0033] After testing the single system of African horse sickness virus-VP7 gene, African horse sickness virus-NS2 gene, West Nile virus-NS5 gene, West Nile virus-NS2a gene, equine influenza H3N8-HA gene, and equine influenza H3N8-NA gene, the present application establishes a seven-fold digital PCR system containing an internal standard. After testing the indicators such as linearity, specificity, minimum detection limit, repeatability, single and multiplex digital PCR system, the developed digital PCR multiplex detection kit fully meets the development requirements, has strong specificity, high sensitivity, and the minimum detection limit can reach 10 copies / μL or less.

[0034] Second, as the creative evidence of the claims of the present application, it is also reflected in the following important aspects:

[0035] (1) The expected income and commercial value of the technical solution of the present application after transformation are:

[0036] The seven-fold digital PCR kit of the present application has significant commercial transformation potential in the field of animal disease detection. The global equine animal disease detection market is expected to exceed 1.2 billion US dollars in 2025, and the cross-border transmission risk of African horse sickness (AHSV), West Nile virus (WNV) and equine influenza (EIV) has intensified the demand for efficient detection technology in various countries. Traditional single detection requires 7 independent experiments (2 genes / pathogen x 3 pathogens + internal standard), and the cost of a single sample is as high as 140 US dollars (based on 20 US dollars per single detection), and the time-consuming is more than 14 hours. While the present application can complete all target screening through four-channel seven-fold detection technology in a single reaction, the detection time is shortened to 4 hours, the cost is reduced to 28 US dollars per sample, the efficiency is improved by 3.5 times, and the cost is saved by 80%.

[0037] From the application scene, the present application can cover three core markets:

[0038] ① Import and export quarantine: According to OIE data, the global horse trade exceeded 500,000 heads in 2022, and the EU alone lost 230 million euros annually due to quarantine caused by diseases. This kit can meet the requirements of the International Animal Health Code for "one inspection and multiple judgments", shorten the customs clearance time by more than 50%, and conservatively estimate that it covers 10% of the quarantine needs (50,000 heads / year), with annual revenue of up to $14 million.

[0039] ② Disease purification in large-scale horse farms: The global horse racing and breeding industry has an annual output value of over $100 billion, but the disease-induced horse culling rate is as high as 15%. Early screening (sensitivity ≤10 copies / μL) by this kit can reduce the risk of disease outbreaks by 70%, and according to a 5% market penetration rate, annual revenue is approximately $50 million.

[0040] ③ Vaccine research and development quality control: The global horse vaccine market is approximately $800 million, and this kit is used for virus inactivation verification and potency evaluation in vaccine production, expected to occupy 15% of the quality control market share, with annual revenue of $120 million.

[0041] (2) The technical solution of the present application fills the technical gap in the industry at home and abroad:

[0042] The current technical gap in the global animal disease digital PCR detection field is concentrated in the lack of high multiplexing and simultaneous detection capability. According to Derwent patent database statistics, as of 2023, 91% of patents related to horse animal disease detection are single or double detection, only 9% involve three or more, and all are limited to a single pathogen (such as patent CN113622342A only detects AHSV double genes). International mainstream products (such as Thermo Fisher VetMAX TM Horse Influenza Kit) still rely on qPCR technology, with a maximum of 3 targets detected at a time. The seven-reagent digital PCR kit of the present application is the first commercial solution to achieve four-channel seven-target (including cross-pathogen) simultaneous detection, and its technical gap filling is reflected in:

[0043] ① Target dimension: For the first time, the multi-gene detection of arbovirus (AHSV, WNV) and respiratory virus (H3N8) is integrated, covering all core horse disease pathogens specified by OIE, and including an internal standard correction system. For example, simultaneous detection of AHSV-VP7 (capsid protein gene) and NS2 (non-structural protein gene) can distinguish between wild strains and vaccine strains, guiding precise immunization.

[0044] ② Technical dimension: Breakthrough four-channel hardware limitations, through probe concentration gradient coding (such as a 2:1 concentration ratio of two target probes in the FAM channel) and AI signal analysis algorithms (error rate <1.5%), achieve a target-channel ratio of 1.75:1, with an efficiency improvement of 75% compared to traditional four-reagent detection.

[0045] ③Standardization: There is no standard for multi-pathogen detection of Equidae by digital PCR in the world. The kit has been tested, and the linearity (R 2 >0.99), repeatability (CV<3%) and single-multiple consistency (deviation<3%) all meet the requirements of ISO 17025 certification, providing a technical reference for the revision of OIE guidelines.

[0046] (3) Does the technical solution of the application solve the technical problems that people have long desired to solve but have always failed to succeed:

[0047] There are two technical problems in the field of animal disease multiplex detection: single and multiple detection results are not comparable, and high number detection performance is deteriorated. The traditional view believes that primer competition, signal interference and droplet distribution deviation in the multiplex system will inevitably lead to quantitative results deviating from single detection (literature "Veterinary Research" points out that the deviation is >15%), and the sensitivity decreases by >10 times when the number of targets exceeds five. The present application solves these problems through two major technical innovations:

[0048] ① Single-multiple equivalent design: through MGB probe modification (improve the binding stability of low Tm value target) and primer Tm equalization (7 groups of primer Tm homogenization), the amplification efficiency of all targets is between 90-105% at the same annealing temperature (60℃). Experimental data shows that the logarithmic deviation of quantitative concentration of single and seven detection is <3% (such as WNV-NS2a gene single value: 209 copies / μL, seven value: 217.5 copies / μL), breaking the industry cognition of "multiple unreliable".

[0049] ②Maintain single level sensitivity (LoD≤10 copies / μL) and linearity (R 2 >0.99) in seven system. For example, the minimum detection limit of AHSV-VP7 gene is 2.82 copies / μL, which is several times more sensitive than the existing five detection (LoD≥50 copies / μL).

[0050] (4) Does the technical solution of the application overcome the technical bias:

[0051] There are two technical biases in the field of digital PCR: "high number must sacrifice performance" and "multiplex detection cannot replace single detection". The present application completely overturns these biases through experimental data and innovative design:

[0052] ① The prejudice that "high multiplex deteriorates sensitivity": the industry generally believes that the sensitivity of detection above five times is reduced by more than 10 times (Analytical Chemistry 2022). However, the kit still achieves single copy level detection (LoD≤10 copies / μL) under a seven-time system.

[0053] ② The prejudice that "multiplex detection is unreliable": the prior art believes that multiplex detection needs to rely on complex correction algorithms (such as the compensation model of patent US20220056321A), and the present application makes the seven-time detection results highly consistent with single-time (concentration logarithmic deviation<3%) through internal standard driven dynamic correction and probe concentration coding. For example, the single-time value of WNV-NS2a gene is 209 copies / μL, and the seven-time value is 217.5 copies / μL. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 is a single fluorescence PCR standard curve provided by an embodiment of the present application. Among them, A is the linear target of AHSV-VP7 gene, B is the linear target of AHSV-NS2 gene, C is the linear target of WNV-NS5 gene, D is the linear target of WNV-NS2a gene, E is the linear target of H3N8-HA gene, and F is the linear target of H3N8-NA gene.

[0055] Figure 2 is the specificity detection result of the fluorescence PCR detection method provided by the embodiment of the present application.

[0056] Figure 3 is a 1-dimensional graph of positive droplets of each channel of a multiplex digital PCR reaction system provided by an embodiment of the present application.

[0057] Among them, A is the positive droplet of FAM channel strip 1: WNV-NS5 gene target and strip 2: AHSV-VP7 gene target; B is the positive droplet of HEX channel strip: H3N8-HA gene target; C is the positive droplet of strip 1: internal standard and strip 2: AHSV-NS2 gene target; D is the positive droplet of CY5 channel strip 1: H3N8-NA gene target and strip 2: WNV-NS2a gene target.

[0058] Figure 4 is a 2-dimensional graph of positive droplets of each channel of a multiplex digital PCR reaction system provided by an embodiment of the present application.

[0059] Wherein, A is the 2D plot of each gene target positive microdroplet of FAM and HEX channels, B is the 2D plot of each gene target positive microdroplet of FAM and ROX channels, C is the 2D plot of each gene target positive microdroplet of FAM and CY5 channels, D is the 2D plot of each gene target positive microdroplet of HEX and ROX channels, E is the 2D plot of each gene target positive microdroplet of HEX and CY5 channels, and F is the 2D plot of each gene target positive microdroplet of ROX and CY5 channels.

[0060] Figure 5 The 1D plot of linear results of each channel positive microdroplet of the multiplex digital PCR reaction system provided by the embodiment of the application is shown in FIG. 4. Wherein, A is the FAM channel, B is the HEX channel, C is the ROX channel, and D is the CY5 channel.

[0061] Figure 6 The statistical chart of linear results of each target of the multiplex digital PCR reaction system provided by the embodiment of the application is shown in FIG. 5.

[0062] Figure 7 The 1D plot of repeatability results of each channel positive microdroplet of the multiplex digital PCR reaction system provided by the embodiment of the application is shown in FIG. 6. Wherein, A is the FAM channel, B is the HEX channel, C is the ROX channel, and D is the CY5 channel.

[0063] Figure 8 The 1D result comparison chart of the multiplex digital PCR and each single digital PCR provided by the embodiment of the application is shown in FIG. 7. A is the comparison of the value results of the AHSV-VP7 gene target multiplex digital PCR and single digital PCR, B is the comparison of the value results of the WNV-NS5 gene target multiplex digital PCR and single digital PCR, C is the comparison of the value results of the H3N8-HA gene target multiplex digital PCR and single digital PCR, D is the comparison of the value results of the AHSV-NS2 gene target multiplex digital PCR and single digital PCR, E is the comparison of the value results of the H3N8-NA gene target multiplex digital PCR and single digital PCR, and F is the comparison of the value results of the WNV-NS2a gene target multiplex digital PCR and single digital PCR.

[0064] Figure 9 The result comparison chart of the multiplex digital PCR and each single digital PCR provided by the embodiment of the application is shown in FIG. 8. The coefficient of variation CV value of the value results of the single digital PCR and the multiplex digital PCR is less than 3%, which indicates that the multiplex digital PCR and each single digital PCR are in good compliance, and the multiplex digital PCR does not produce mutual interference. DETAILED DESCRIPTION

[0065] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0066] The present application aims to provide a digital PCR kit for simultaneously detecting African horse sickness virus, West Nile virus and equine influenza H3N8 subtype, which comprises primers and probes as shown in SEQ ID NO. 1-21.

[0067] The present application also provides a method for detecting African horse sickness virus, West Nile virus and equine influenza H3N8 subtype for non-disease diagnosis or treatment purposes, which comprises the following steps:

[0068] (1) using extracted nucleic acid of the sample to be tested as a template, 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, SEQ ID NO. 13-14, SEQ ID NO. 16-17 to amplify the sample to be tested, and using probes as shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18 to identify the amplification product;

[0069] the 5' end of the probe as shown in SEQ ID NO. 3 is provided with FAM fluorescent label, and the 3' end is provided with MGB fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 6 is provided with ROX fluorescent label, and the 3' end is provided with BHQ2 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 9 is provided with FAM fluorescent label, and the 3' end is provided with BHQ1 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 12 is provided with CY5 fluorescent label, and the 3' end is provided with BHQ2 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 15 is provided with HEX fluorescent label, and the 3' end is provided with BHQ1 fluorescent label; the 5' end of the probe as shown in SEQ ID NO. 18 is provided with CY5 fluorescent label, and the 3' end is provided with BHQ2 fluorescent label;

[0070] (2) simultaneously setting IPC internal standard including primers and probes as shown in SEQ ID NO. 19-21, the 5' end of the probe as shown in SEQ ID NO. 21 is provided with ROX fluorescent label, and the 3' end is provided with MGB fluorescent label; performing fluorescence quantitative detection and collecting signals of the fluorescence channel;

[0071] (3) result analysis:

[0072] In the present application, the concentration of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, H3N8-NA, IPC internal standard recombinant plasmid mixture is 10 3 The mixture of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, H3N8-NA, IPC internal standard recombinant plasmid with a concentration of 10 copies / μL is used as a positive control, and ultrapure water is used as a negative control.

[0073] Click "analysis" 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 remaining color droplets are positive droplets corresponding to the channel. The negative control should have no positive droplets, and all channels of the positive control should have positive droplets corresponding to the bands;

[0074] FAM channel band 1 West Nile virus-NS5 gene positive droplet, FAM channel band 2 represents African horse sickness virus-VP7 gene positive droplet, HEX channel band represents horse influenza H3N8-HA gene positive droplet, ROX channel band 1 represents internal standard positive droplet, ROX channel band 2 represents African horse sickness virus-NS2 gene positive droplet, CY5 channel band 1 represents horse influenza H3N8-NA gene positive droplet, CY5 channel band 2 represents West Nile virus-NS2a gene positive droplet Figure 3

[0075] The same sample is determined: FAM channel band 1 and CY5 channel band 2 positive judgment as West Nile virus infection, HEX channel band and CY5 channel band 1 positive judgment as horse influenza H3N8 subtype infection, FAM channel band 2 and ROX channel band 2 judgment as African horse sickness virus infection.

[0076] In some specific embodiments, the reaction system of the amplification reaction includes: digital PCR reaction premix 10 μL, primer Mix 1.36 μL, probe Mix 0.61 μL, nucleic acid template 2 μL, and dd H2O to 20 μL.

[0077] In some specific embodiments, the reaction of the amplification reaction is: 95°C pre-denaturation for 10 min; 94°C denaturation for 30 s, 60°C annealing and extension for 1 min, 40 cycles; 98°C inactivation for 10 min; 20°C cooling for 2 min; collect the fluorescence signals of channels FAM, HEX, ROX, and CY5 at the end of each cycle.

[0078] Example 1 Design of primer and probe sequence

[0079] ​The VP7 gene, NS2 gene sequences of African horse sickness virus were retrieved from the NCBI nucleic acid database GenBank (GenBank No. KT030336.1, KT715607.1, KT030426.1, AM883171.1, FJ183371.1, KF859993.1, KT030456.1, U90337.1, KT030488.1, KT030378.1, KT030518.1, KP940202.1, AF545434.1, FJ183372.1, KF860043.1, KF860043.1, M69090.1, KT030458.1, etc.); the whole genome sequence, NS5 gene, NS2a gene sequences of West Nile virus (GenBank No. AY688948.1, ON813218.1, MN238670.1, OP345105.1, MN812761.1, MH939154.1, OR757501.1, MH939153.1, KY594040.1, AY688948.1, KM659876.1, MF984348.1, MW751837.1, MZ605382.4, OK129334.1, OL840879.1, OQ204315.1, OX442308.1, etc.); the whole genome sequence, HA gene, NA gene sequences of equine influenza H3N8 subtype (GenBank No. KP693700.1, EU794511.1, MK215821.1, FJ195395.3, EU794535.1, MK215822.1, MK215815.1, EU794567.1, KF806985.1, MK215820.1, KF309035.1, EU794497.1, MF067529.1, MK215817.1, EU794529.1, EU794569.1, KF806987.1, etc.). The IPC internal standard is based on the conserved gene sequence of the rRNA gene in the mitochondrial genome. After aligning each target gene sequence with SnapGene software, the relatively conserved nucleotide sequences of each pathogen were selected, and the primer and TaqMan probe sequences were designed according to the primer and TaqMan probe design principles and combined with the requirements of the relevant national standards. The target sequences detected by the kit are shown in Table 1, and the designed primers and probes were synthesized by Shengong Bioengineering Co., Ltd.

[0080] Table 1 Digital PCR kit for detecting African horse sickness virus, West Nile virus, and equine influenza H3N8 subtype

[0081]

[0082]

[0083] Example 2 Construction of plasmid standard

[0084] The gene sequences of African horse sickness virus, West Nile virus, Equine Influenza Virus H3N8, and internal standard were synthesized by Shenguo Bioengineering Co., Ltd., and were named AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, H3N8-NA, and IPC internal standard, respectively. Subsequently, the plasmid DNA standard was stored at -80°C for later use.

[0085] DNA copy number = (M x 6.02 x 10 23 x 10 -9 ) / (n x 660)

[0086] M represents the concentration of plasmid DNA, and n represents the length of the recombinant plasmid = the length of the T vector + the length of the target fragment.

[0087] 2.1 Establishment of standard curve for single fluorescent PCR reaction system

[0088] In order to verify whether the amplification efficiency and correlation coefficient of the primers and probes designed for each target meet the establishment of a multiplex system of digital PCR, the AHSV-VP7 and AHSV-NS2 positive plasmids of African Horse Sickness Virus (AHSV), the WNV-NS5 and WNV-NS2a positive plasmids of West Nile Virus (WNV), and the H3N8-HA and H3N8-NA positive plasmids of Equine Influenza Virus H3N8 (H3N8) were diluted by 10 times in gradient and then subjected to single fluorescent PCR. The template concentration fluorescence Ct value was used to make a concentration standard curve. Each reaction system was configured as shown in Table 2, and the fluorescent PCR reaction was performed according to the procedure shown in Table 3. The device used was JLM QX600 of Jeleme Co.

[0089] Table 2 Preparation of single fluorescent PCR reaction system

[0090]

[0091] Table 3 Parameters for fluorescent PCR reaction

[0092]

[0093] The positive plasmids of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, and H3N8-NA were used to make the standard curve of concentration-Ct value, respectively. Figure 1 Figure 1 It can be seen that in the single system, the values of PCR amplification efficiency E, R and curve slope are in the normal range, indicating that the amplification efficiency is good and can be used for the establishment of subsequent multiplex digital PCR system.

[0094] 2.2 Primer probe specificity test

[0095] The AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, and H3N8-NA horse disease related plasmids involved in the kit were selected for mutual specificity test within the kit. Single fluorescence PCR was performed using the primers and probes of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, and H3N8-NA of the kit, and the positive plasmids of HSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, and H3N8-NA target of the kit were used as positive controls, and dd H2O was used as negative control. The system configuration is shown in Table 2, the final concentration of each target primer is 200 nmol / L, the final concentration of the probe is 100 nmol / L, and the fluorescence PCR reaction was performed according to the procedure shown in Table 3, and the device used was JLM QX600 of Jeleme Company.

[0096] It was found through testing that there was no amplification between each target primer probe of the kit and the remaining target positive plasmids, and each had a strong fluorescence intensity, indicating that the detection primers and probes had good specificity and could be used for the construction of subsequent multiplex digital PCR system. The results are shown in Figure 2 , Table 4, and the concentration of the plasmid in Table 4 is 1.0 x 10 6 Copies / μL.

[0097] Table 4 Specificity test of each target single system of the kit

[0098]

[0099] 2.3 Establishment of multiplex digital PCR reaction system

[0100] The primer and probe stock solutions were diluted and mixed thoroughly to prepare primer mixture and probe mixture. The final concentration of each target primer is shown in Table 1, and each reaction system is configured as shown in Table 5. The digital PCR reaction was performed according to the procedure shown in Table 6, and the device used was JLM Digital Matrix-5000 digital PCR of Jeleme Company.​

[0101] Sample concentration (Copies / μL) = fixed value result (Copies / μL) x 20 (total reaction system) ÷ 2 (loading amount).

[0102] Table 5 digital PCR fixed value reaction system preparation

[0103]

[0104] Table 6 digital PCR reaction parameter settings

[0105]

[0106]

[0107] 2.4 Linearization enzyme digestion of target plasmid

[0108] The theoretical value of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA, H3N8-NA, and internal standard plasmid was 10 6 Copies / μL of the 7 positive plasmids were mixed together in the same volume, and linearization enzyme digestion was performed using QuickCut Sac I and QuickCut Kpn I cutting enzymes. The enzyme digestion system is shown in Table 7, and enzyme digestion can be completed at 37°C for 15 minutes.

[0109] Table 7 Plasmid linearization enzyme digestion system

[0110]

[0111] Using the 7 positive plasmids after enzyme digestion, the final concentration of each target primer probe according to Table 1 and the reaction system of Table 5 and the reaction program of Table 6 were tested, and the 1D graph of the test results is shown in Figure 3 , and the 2D graph is shown in Figure 4 From the 1D graph, the band layering between each channel target is obvious and will not interfere with the detection. From the 2D graph, each target is clearly distinguished, and the band partition is clear. The 1D graph and 2D graph have good effects, and the test results prove that the multiplex digital PCR reaction system is successfully established.

[0112] 2.5 Establishment of digital PCR multiplex reaction system standard curve and determination of minimum detection limit

[0113] The theoretical concentration of the 7 positive plasmids after enzyme digestion was selected as 10 4The 4-fold dilution of the plasmid mixture was performed in sequence, and 7 gradients were used for digital PCR reaction. The ddH2O was set as a negative control. The reaction system was shown in Table 5, and the multiplex digital PCR reaction was performed according to the reaction conditions in Table 6 to evaluate the linearity and the minimum detection limit of the method. The sample concentration (Copies / μL) = the fixed value result (Copies / μL) x 20 (total reaction system) ÷ 2 (loading amount).

[0114] The digital PCR results showed that the number of microdroplets was greater than 10000, meeting the experimental analysis requirements. The correlation coefficient (R 2 ) of the standard curve was greater than 0.98, and the linearity of the multiplex digital PCR method met the performance requirements. In the multiplex digital PCR system, the minimum detection limits of AHSV-VP7, AHSV-NS2, WNV-NS5, WNV-NS2a, H3N8-HA and H3N8-NA were 2.82 Copies / μL, 6.57 Copies / μL, 3.75 Copies / μL, 3.76 Copies / μL, 2.82 Copies / μL and 0.94 Copies / μL, respectively. The results were shown in Figure 5 、 Figure 6 and Table 8.

[0115] Table 8. The minimum detection limit test results of the multiplex digital PCR system of the kit were statistically analyzed.

[0116]

[0117] 2.6. Reproducibility test of the multiplex digital PCR reaction system

[0118] The single copy number concentration of the 7 positive mixed plasmids was used for the multiplex digital PCR reaction, which was repeated for 8 times, and the test results were statistically analyzed to calculate the coefficient of variation and evaluate the reproducibility of the method.

[0119] The 10 4 fold diluted 16 times mixed plasmids were selected as the positive template for the intra-group repeated experiment. The results showed that the number of microdroplets was greater than 10000, meeting the experimental analysis requirements. The intra-group coefficient of variation was between 0.551% and 1.795%, which was less than 2%, indicating that the method had good reproducibility. The results were shown in Figure 7 and Table 9.

[0120] Table 9. The reproducibility test results of each target of the multiplex digital PCR system of the kit were statistically analyzed.

[0121]

[0122] 2.7. Comparison of the single and multiplex digital PCR reaction systems

[0123] Using 6 positive mixed plasmid single copy number concentration respectively for each target single and multiple system digital PCR reaction, the same template concentration corresponding to the fixed concentration is compared, the variation coefficient of single system and multiple system fixed value result is calculated, and the conformity of single digital PCR and multiple digital PCR is compared.

[0124] The results show that the number of microdroplets is greater than 10000, meeting the experimental analysis requirements, the variation coefficient of single and multiple fixed concentration copy number is less than 3%, indicating that the single and multiple digital PCR reaction system has little difference in fixed value results of the same sample, meeting the experimental requirements, and the experimental results are shown in Table 10. Figure 8 、 Figure 9 , Table 10.

[0125] Table 10 Single and multiple digital PCR system comparison test results of the kit

[0126]

[0127] After testing the single system of African horse sickness virus-VP7 gene, African horse sickness virus-NS2 gene, West Nile virus-NS5 gene, West Nile virus-NS2a gene, horse influenza H3N8 subtype-HA gene, horse influenza H3N8 subtype-NA gene, a seven-reaction digital PCR reaction system containing an internal standard is established. After testing the linear, specificity, minimum detection limit, repeatability, single and multiple digital PCR system comparison indicators, the developed digital PCR multiple detection kit completely meets the development requirements.

[0128] I. The specific application field or related product of the application.

[0129] Application field:

[0130] 1. Cross-border horse trade and import and export quarantine

[0131] Core pain point: African horse sickness (AHSV) and West Nile virus (WNV) are listed as mandatory reporting diseases by the World Organization for Animal Health (OIE), and cross-border transportation needs to provide a disease-free certificate, but traditional detection needs multiple independent experiments (such as AHSV double gene detection + WNV double gene detection + H3N8 detection + internal standard), which takes a long time of 2-3 days.

[0132] Technical advantage: through single seven-reaction detection (completed within 4 hours), the genotype information of all pathogens (such as AHSV-VP7 and NS2 double gene detection) can be obtained simultaneously, meeting the requirements of OIE International Animal Health Code for "one test and multiple judgments", and shortening the customs clearance time by more than 50%.

[0133] Typical user: customs quarantine department, international equestrian event organizing committee, and horse import and export enterprise.

[0134] 2. Wildlife disease traceability and ecological research

[0135] Core pain points: The transmission mechanism of wild equine diseases (such as Przewalski's horse and zebra) is complex, and traditional methods cannot efficiently detect multiple pathogens.

[0136] Technical advantages: Seven detection combined with equine mitochondrial rRNA internal standard (cross-species conservation) can simultaneously analyze pathogen load and host genetic background, supporting disease transmission dynamics research.

[0137] Typical users: Wildlife conservation organizations and research institutions.

[0138] Products related to the present application:

[0139] Sevenplex digital PCR detection kit (pre-mixed): Contains liquid or lyophilized microspherical primers and probes, reaction buffer, positive control, ready-to-use after opening, suitable for mainstream digital PCR instruments (such as Bio-Rad QX200, Thermo Fisher QuantStudio 3D).

[0140] Multi-pathogen mixed quality control: Contains simulated genomic fragments of AHSV, WNV, H3N8 and gradient concentration standards (1-10 5 copies / μL) for laboratory detection process verification and standardization control.

[0141] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principles of the present application, should be covered within the protection scope of the present application.

Claims

1. A digital PCR kit for simultaneous detection of African horse sickness virus, West Nile virus, equine influenza H3N8, characterized in that, The kit comprises: Primers and probes with sequences as shown in SEQ ID NO. 1~SEQ ID NO. 21; wherein the 5' end of SEQ ID NO. 3 and SEQ ID NO. 9 is provided with FAM fluorescent label, the 3' end of SEQ ID NO. 3 is provided with MGB fluorescent label, and the 3' end of SEQ ID NO. 9 is provided with BHQ1 fluorescent label; the 5' end of SEQ ID NO. 6 and SEQ ID NO. 21 is provided with ROX fluorescent label, and the 3' end is provided with BHQ2 fluorescent label; the 5' end of SEQ ID NO. 12 and SEQ ID NO. 18 is provided with CY5 fluorescent label, the 3' end of SEQ ID NO. 12 is provided with BHQ2 fluorescent label, and the 3' end of SEQ ID NO. 18 is provided with BHQ2 fluorescent label; the 5' end of SEQ ID NO. 15 is provided with HEX fluorescent label, and the 3' end is provided with BHQ1 fluorescent label.

2. A method for detecting African horse sickness virus, West Nile virus, equine influenza H3N8 using the kit according to claim 1, characterized in that, The method comprises the following steps: (1) extracting nucleic acid of the sample to be tested, and performing digital PCR amplification reaction using primers with sequences as shown in SEQ ID NO. 1~2, SEQ ID NO. 4~5, SEQ ID NO. 7~8, SEQ ID NO. 10~11, SEQ ID NO. 13~14, SEQ ID NO. 16~17, SEQ ID NO. 19~SEQ ID NO. 20; (2) detecting the amplification product using probes with sequences as shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 21; (3) collecting digital PCR fluorescent signals, and collecting fluorescent signals of channels FAM, HEX, ROX, and CY5; The method is for non-diagnostic or therapeutic purposes.

3. The method of claim 2, wherein, The digital PCR amplification reaction system comprises: 10 μL of digital PCR reaction premix; 1.36 μL of primer Mix; 0.61 μL of probe Mix; 2 μL of nucleic acid template; dd H2O is supplemented to 20 μL.

4. The method of claim 2, wherein, The digital PCR amplification reaction program comprises: 95 ℃ pre-denaturation for 10 minutes; 94 ℃ denaturation for 30 seconds, 60 ℃ annealing and extension for 1 minute, and cycling for 40 times; 98 ℃ inactivation for 10 minutes; 20 ℃ cooling for 2 minutes; Collecting fluorescent signals of four channels FAM, HEX, ROX, and CY5 at the end of each cycle.

5. The method of claim 2, wherein, In the detection result, the FAM channel is used for detecting African horse sickness virus VP7 gene and West Nile virus NS5 gene, the HEX channel is used for detecting equine influenza H3N8-HA gene, the ROX channel is used for detecting internal standard and African horse sickness virus NS2 gene, and the CY5 channel is used for detecting equine influenza H3N8-NA gene and West Nile virus NS2a gene.

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