Metagenome sequencing-based rapid early warning method for pathogenic microorganisms of skin of imported animal

Abstract

The invention belongs to the technical field of port animal quarantine, and discloses an imported animal skin pathogenic microorganism rapid early warning method based on metagenome sequencing, and the method comprises the following steps: optimization pretreatment of an imported animal skin sample: taking the imported animal skin sample, carrying out sterile grinding to prepare a homogenate, and carrying out spray drying on the homogenate; carrying out nucleic acid enrichment by combining protease K + lysozyme composite enzymolysis with a magnetic bead method; carrying out metagenome sequencing and bioinformatics analysis; performing biological safety risk research, judgment and early warning: performing risk grading on pathogens and generating early warning information on the basis of an analysis result in the step S2 and in combination with an imported non-edible animal product monitoring technical table; efficient and accurate detection of target pathogens: aiming at the high-risk pathogens early-warned in the step S3, developing a standardized detection reagent by adopting at least one technology of CRISPR / Cas, recombinant RPA and digital PCR, and carrying out targeted verification detection on the sample. The sample pretreatment is adaptive to the characteristics of animal skin, so that the enrichment efficiency of pathogenic nucleic acid is improved; a multi-technology detection system is combined, and the requirements of'broad-spectrum early warning 'and'accurate verification' are considered; technical support is provided for port animal quarantine, and biological safety hazards are effectively prevented and controlled.

Description

TECHNICAL FIELD

[0001] The present application belongs to, but not limited to, the technical field of animal quarantine at ports, and particularly relates to a method for rapid early warning of pathogenic microorganisms in imported animal hides based on metagenomic sequencing. BACKGROUND

[0002] Imported animal hides (including raw hides, salted hides, etc. of livestock such as cattle, sheep, and pigs) are common commodities in international trade, but as animal-derived products, they carry a high risk of carrying pathogenic microorganisms (including viruses, bacteria, fungi, etc.) and are one of the important carriers of cross-border biological safety transmission. With the expansion of international trade, the pressure of quarantine of imported animal hides continues to increase, but the current quarantine technology system still has significant shortcomings, which are embodied in the following aspects: 1. Insufficient coverage of existing pathogen detection methods Current port detection of pathogenic microorganisms in imported animal hides mainly relies on single targeted detection methods (such as conventional PCR, ELISA, etc.), which can only detect known pathogens (such as foot-and-mouth disease virus, anthrax bacillus) listed in the "Imported Animal Quarantine Disease List". However, there are two types of pathogens that are not effectively covered in the actual transmission risk: Newly emerging pathogens: In recent years, new animal diseases (such as African swine fever variant strains, animal-derived variants of new coronaviruses) have emerged globally, and existing targeted detection reagents cannot match their new genetic sequences; Unknown pathogens: There are a large number of potential pathogenic microorganisms in nature that have not been identified, and traditional methods cannot identify them, forming a biological safety "blind area".

[0003] 2. Poor adaptability of sample pretreatment technology The matrix characteristics of animal hides (containing a large amount of collagen, fat, impurities, and usually very low pathogen load) are significantly different from conventional animal tissue samples, but the existing pretreatment methods mostly follow the "universal tissue grinding - nucleic acid extraction" process: Collagen, fat and other impurities can inhibit the efficiency of nucleic acid extraction, resulting in insufficient enrichment of pathogenic nucleic acids; Under low pathogen load, conventional extraction methods are prone to "false negatives", which cannot meet the detection needs of trace pathogens; The pretreatment process lacks optimization for hide matrix, and is tedious and time-consuming (2-3 hours for a single treatment), which is difficult to adapt to the efficiency requirements of "fast clearance" at ports.

[0004] 3. Lack of systematic analysis capability for pathogenic community Existing detection only targets a single or a few pathogens for "point-to-point" screening, and cannot analyze the community structure and diversity characteristics of pathogenic microorganisms in hides: Unable to identify the "dominant pathogen group" (e.g., when multiple pathogens are present in a batch of skin, it is difficult to determine the main source of risk); Unable to detect "synergistic risk between pathogens" (some pathogens have low risk when they exist alone, but may increase pathogenicity or transmissibility when they exist in combination); It is difficult to provide early warnings for "potentially risky pathogens" (such as microorganisms with low abundance but high pathogenicity). Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a rapid early warning method for pathogenic microorganisms on the skin of imported animals based on metagenomic sequencing.

[0006] This invention is implemented as follows: a rapid early warning method for pathogenic microorganisms in the skin of imported animals based on metagenomic sequencing, the method comprising: S1: Optimized pretreatment of imported animal hide samples: Imported animal hide samples were aseptically ground into a homogenate, and then nucleic acid enrichment was performed using a combination of proteinase K and lysozyme and magnetic beads to obtain pathogen nucleic acid templates. S2: Metagenomic sequencing and bioinformatics analysis: Construct a metagenomic library from the nucleic acid templates obtained in step S1 and perform high-throughput sequencing. Upload the raw sequencing data to the "Metagenomic Data Analysis Platform for Pathogens of Skin of Imported Animals" to complete biological category annotation, microbial diversity analysis and community structure analysis. S3: Biosafety Risk Assessment and Early Warning: Based on the analysis results of step S2, and combined with the monitoring technical table for imported non-edible animal products, pathogens are classified according to risk and early warning information is generated; S4: Highly efficient and accurate detection of target pathogens: For high-risk pathogens identified in step S3, standardized detection reagents are developed using at least one of the following technologies: CRISPR / Cas, recombinant RPA, and digital PCR, to perform targeted verification detection on samples.

[0007] Furthermore, in S1, the sampling amount of the imported animal hide sample is 2-5g, the final concentration of proteinase K is 15-25mg / mL, the final concentration of lysozyme is 40-60mg / mL, and the enzymatic hydrolysis conditions are incubation at 35-39℃ for 25-35min.

[0008] Furthermore, the length of the inserted fragment in the metagenomic library in S2 is 200-600 bp, and the amount of raw data from high-throughput sequencing is not less than 3 Gb.

[0009] Furthermore, the "Metagenomic Data Analysis Platform for Pathogens of Imported Animal Skin" mentioned in S2 includes databases that cover at least bacterial, viral, and fungal databases.

[0010] Furthermore, the risk classification in S3 includes: Level I (High Risk): Corresponds to major animal disease pathogens listed in the "List of Quarantine Diseases for Imported Animals"; Level II (Medium Risk): Corresponding zoonotic pathogens; Level III (Low Risk): Corresponds to newly emerging or unknown pathogens.

[0011] Furthermore, the high-risk pathogens in S4 include at least one of foot-and-mouth disease virus, anthrax bacillus, Brucella, and Q fever Rickettsia, and the target of the standardized detection reagent is the specific functional gene of the pathogen.

[0012] Furthermore, the specific functional gene of the foot-and-mouth disease virus is the VP1 gene, and the specific functional gene of the anthrax bacillus is the pagA gene.

[0013] Furthermore, the CRISPR / Cas detection reagent in S4 contains guide RNA targeting pathogen-specific genes, the recombinant RPA detection reagent contains primer pairs targeting pathogen-specific genes, and the digital PCR detection reagent contains primers and probes targeting pathogen-specific genes.

[0014] The application of the rapid early warning method for pathogenic microorganisms in imported animal hides based on metagenomic sequencing in the port quarantine of imported animal hides.

[0015] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: This invention constructs a complete rapid pathogen early warning technology chain around the complex sample system of imported animal hides. Its significant technical effect is first reflected in the adaptability of the pretreatment stage. Animal hides undergo multiple treatments such as pickling, drying, storage, and transportation, resulting in low numbers of pathogenic microorganisms, hard cell walls, and often embedded states, making it difficult for traditional lysis methods to release intact nucleic acids. This invention uses a combination of proteinase K and lysozyme enzymatic hydrolysis with magnetic bead enrichment, specifically optimized for the high collagen and high lipid structure of hides. This significantly improves the extractability of pathogen nucleic acids, enabling downstream detection to be based on real pathogen signals rather than background noise, thus solving the core difficulty of "difficulty in enriching nucleic acids" in hide samples from the source of the technology.

[0016] At the metagenomic sequencing level, this invention is the first to introduce a comprehensive identification system of "known pathogens + emerging or unknown pathogens" into the port leather quarantine process. By constructing an analysis platform with full sequence indexes for bacteria, viruses, and fungi, this method can simultaneously identify multiple types of pathogens in a single sequencing run and provide taxonomic approximations or potential risk indications for unknown microbial sequences. This eliminates the reliance on "pre-assumptions" in pathogen discovery and overcomes the structural limitation of traditional targeted detection methods that cannot identify unknown pathogens.

[0017] This invention further integrates metagenomic screening results with CRISPR, RPA, or digital PCR targeted rapid detection into a multi-technology synergistic system, enabling both "broad-spectrum early warning" and "precise verification" needs to be compatible within the same process. Metagenomic sequencing is responsible for comprehensive scanning and early warning, while targeted technology is responsible for confirmation within a short time, thus meeting the timeliness requirements of the "discovery-confirmation-disposal" three-stage process in port supervision.

[0018] This method ultimately forms a standardized and replicable cross-border quarantine technology model, providing strong support for the early identification of risks in imported animal hides, enabling port quarantine to shift from passive screening to proactive perception, and effectively reducing biosecurity risks caused by the importation of high-risk pathogens.

[0019] The technical solution of this invention has clear engineering potential and scalable deployment value. Firstly, the trade volume of imported hides is enormous, but a comprehensive pathogen screening technology is currently lacking. Therefore, the overall process of metagenomic detection combined with targeted confirmation has significant market adaptability. Its translational products may include: pretreatment kits, hide pathogen metagenomic analysis platforms, CRISPR or RPA rapid pathogen detection kits, etc., which can be widely used by port regulatory agencies, animal-derived processing enterprises, biosafety laboratories, and international trading companies. Secondly, the "non-hypothesized pathogen model" constructed by this invention upgrades regulatory capabilities from passive response to proactive detection, reducing economic losses and public health risks from the importation of major animal epidemics, while also reducing the testing costs associated with traditional multiple, itemized tests. Overall, this technical system has significant social, industrial, and regulatory benefits, and can form a specialized product line to support the modernization of cross-border quarantine.

[0020] Animal hide samples have always been one of the most challenging sample types in the quarantine system. Their complex matrix, highly processed characteristics, and extremely low microbial content make it difficult to reliably obtain effective pathogen signals using traditional culture methods, serological methods, and single-target PCR, especially for identifying unknown pathogens. The industry has long faced structural challenges such as "difficulty in nucleic acid extraction," "background noise far exceeding the effective pathogen signal," "inability to identify unknown pathogens," and "long detection cycles leading to ineffective early warnings." This invention, for the first time, integrates three technologies—specific compound enzymatic digestion and enrichment, metagenomic full-spectrum scanning, and rapid targeted confirmation—in a streamlined process, making comprehensive identification of hide pathogens technically feasible and meeting practical regulatory needs in terms of time scale. This system not only achieves panoramic pathogen identification under high-throughput data conditions but also resolves the technical contradiction of rapid confirmation for high-risk pathogens, filling a technological gap in port animal hide pathogen monitoring. It represents a key technological milestone that the industry has long awaited but failed to achieve. Attached Figure Description

[0021] Figure 1 This is a flowchart of a rapid early warning method for pathogenic microorganisms in the skin of imported animals based on metagenomic sequencing, provided in an embodiment of the present invention. Figure 2 This is a risk classification diagram provided in an embodiment of the present invention; Figure 3 This is a comparison chart of detection times provided in an embodiment of the present invention. Detailed Implementation

[0022] 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.

[0023] In the current regulatory system for imported animal products, the pathogen monitoring of animal hides has long relied on traditional culture and identification, serological reactions, or specific nucleic acid detection. While these methods have advantages in confirming certain single-target pathogens, they fall short in the context of complex microbial contamination. Hides often undergo multiple processes such as pickling, drying, and transportation, resulting in low microbial concentrations, poor culturability, and severe nucleic acid degradation. This leads to industrialization bottlenecks in the sensitivity, coverage, and timeliness of conventional detection technologies. Especially in high-throughput scenarios at ports of entry, existing processes cannot achieve rapid capture of unknown pathogens, co-infections, or trace amounts of exogenous nucleic acids, preventing regulatory authorities from taking timely risk mitigation measures. This invention addresses this limitation by using metagenomic sequencing as a foundation, combined with a specially constructed hide sample pretreatment process, to overcome the limitations of traditional detection systems.

[0024] This invention addresses the fundamental challenge of nucleic acid extractability in leather samples by employing a combination of enzymatic digestion and magnetic bead enrichment. Bacterial cells, viral particles, and spores adhering to the leather surface are often highly embedded and rich in collagen and lipids, making it difficult for conventional lysis systems to effectively release nucleic acids. Enzymatic digestion using a ratio of proteinase K and lysozyme simultaneously disrupts the structure of both animal tissue fragments and bacterial cell walls, exposing microbial nucleic acids to the solution environment. The magnetic bead capture strategy further selectively enriches exogenous nucleic acids in this complex background, significantly improving the quality of subsequent metagenomic library construction. This mechanism effectively preserves nucleic acids from low-abundance pathogens in leather samples, providing a foundation for effective coverage in high-throughput sequencing and alleviating the previous industry dilemma of "unable to collect and extract."

[0025] In the metagenomic sequencing stage, this method does not simply produce sequences. Instead, it addresses the challenges of high background noise and significant host contamination in animal hide samples by controlling the inserted fragments and setting data volume thresholds, combined with a dedicated "imported animal hide pathogen metagenomic data analysis platform." The analysis platform integrates high-quality databases covering three major groups: bacteria, viruses, and fungi. It also optimizes species annotation and community analysis algorithms based on the unique microecological composition of hides, enabling the simultaneous identification of known animal disease pathogens, zoonotic pathogens, and potential emerging microorganisms within the same dataset. From an industry perspective, this model represents a leap from "targeted detection" to "comprehensive scanning" in port quarantine, significantly reducing the risk of missed detections and overcoming the critical weakness of traditional methods in detecting unknown pathogens.

[0026] To address how metagenomic results can serve regulatory decision-making, this invention constructs a risk assessment system closely integrated with the actual workflow of port quarantine. By automatically comparing detected pathogens with the "List of Quarantine Diseases for Imported Animals" and the monitoring technical table for non-edible animal products, the system enables tiered management of pathogens, allowing regulatory personnel to obtain the risk level of samples and recommended handling pathways in a very short time. The core of this mechanism lies in transforming metagenomic results into actionable quarantine strategies, rather than remaining merely a "species list" for scientific research. This connection from data to regulatory action is precisely the most lacking yet crucial link in industrial applications. This invention achieves a smooth closed loop between detection and enforcement through a rule-based and standardized risk grading system.

[0027] After an early warning is triggered, how to quickly and accurately confirm high-risk pathogens is another pain point in port laboratory testing. While metagenomic results are comprehensive, as "discovery evidence," they require precise identification using targeted methods. This invention proposes, based on early warning, the development of rapid nucleic acid validation systems such as CRISPR / Cas, recombinant RPA, and digital PCR for high-risk pathogens. By selecting specific functional genes such as VP1 and pagA as targets, targeted confirmation can be achieved within minutes to hours. This combined strategy of "metagenomic screening + targeted rapid detection confirmation" effectively solves the long-standing contradiction in the industry of "the inability to simultaneously achieve detection speed and detection coverage," achieving a coupling of high-throughput discovery capabilities and high-precision confirmation capabilities.

[0028] Ultimately, this method not only enables rapid early warning of pathogens in animal hide samples at ports of entry, but also promotes the evolution of the regulatory system towards data-driven and intelligent approaches. By constructing a unified data platform, a standardized nucleic acid enrichment process, and an automatically triggered risk grading mechanism, this invention transforms the complex scenario of animal hide quarantine into a replicable and scalable regulatory model. Its working principle essentially couples microbial ecology, pathogen molecular biology, and cross-scale data analysis into a continuous detection chain, enabling quarantine departments to identify potential epidemic imports at an early stage and achieve closed-loop judgment through rapid confirmation methods. This solves the technical bottlenecks faced by the traditional quarantine system in the context of global trade expansion, sample complexity, and the diversification of pathogen spectrums, providing a practical and systematic solution for the safety supervision of imported animal hide products.

[0029] like Figure 1 As shown, this embodiment of the invention provides a rapid early warning method for pathogenic microorganisms in the skin of imported animals based on metagenomic sequencing. The method includes: S1: Optimized pretreatment of imported animal hide samples: Imported animal hide samples were aseptically ground into a homogenate, and then nucleic acid enrichment was performed using a combination of proteinase K and lysozyme and magnetic beads to obtain pathogen nucleic acid templates. S2: Metagenomic sequencing and bioinformatics analysis: Construct a metagenomic library from the nucleic acid templates obtained in step S1 and perform high-throughput sequencing. Upload the raw sequencing data to the "Metagenomic Data Analysis Platform for Pathogens of Skin of Imported Animals" to complete biological category annotation, microbial diversity analysis and community structure analysis. S3: Biosafety Risk Assessment and Early Warning: Based on the analysis results of step S2, and combined with the monitoring technical table for imported non-edible animal products, pathogens are classified according to risk and early warning information is generated; S4: Highly efficient and accurate detection of target pathogens: For high-risk pathogens identified in step S3, standardized detection reagents are developed using at least one of the following technologies: CRISPR / Cas, recombinant RPA, and digital PCR, to perform targeted verification detection on samples.

[0030] The sampling amount of the imported animal hide sample in S1 is 2-5g, the final concentration of proteinase K is 15-25mg / mL, the final concentration of lysozyme is 40-60mg / mL, and the enzymatic hydrolysis conditions are incubation at 35-39℃ for 25-35min.

[0031] The length of the inserted fragment in the metagenomic library in S2 is 200-600 bp, and the amount of raw data from high-throughput sequencing is not less than 3 Gb.

[0032] The "Metagenomic Data Analysis Platform for Pathogens of Imported Animal Skin" mentioned in S2 includes databases that cover at least bacterial, viral, and fungal databases.

[0033] The risk classification in S3 includes: Level I (High Risk): Corresponds to major animal disease pathogens listed in the "List of Quarantine Diseases for Imported Animals"; Level II (Medium Risk): Corresponding zoonotic pathogens; Level III (Low Risk): Corresponds to newly emerging or unknown pathogens.

[0034] The high-risk pathogens mentioned in S4 include at least one of foot-and-mouth disease virus, anthrax bacillus, Brucella, and Q fever rickettsia, and the target of the standardized detection reagent is the specific functional gene of the pathogen.

[0035] The specific functional gene of the foot-and-mouth disease virus is the VP1 gene, and the specific functional gene of the anthrax bacillus is the pagA gene.

[0036] The CRISPR / Cas detection reagent in S4 contains guide RNA targeting pathogen-specific genes, the recombinant RPA detection reagent contains primer pairs targeting pathogen-specific genes, and the digital PCR detection reagent contains primers and probes targeting pathogen-specific genes.

[0037] The application of the rapid early warning method for pathogenic microorganisms in imported animal hides based on metagenomic sequencing in the port quarantine of imported animal hides.

[0038] This invention constructs a rapid pathogen early warning system for imported animal hides and pelts, capable of operating in complex sample environments. Its applications primarily cover cross-border trade, port quarantine supervision, biosafety monitoring, and quality and safety assessment of animal-derived products. In port supervision operations, this method can be directly embedded into the quarantine processes of customs, border inspection, and other departments for high-throughput pathogen scanning of imported animal hides, fur products, and primary processed animal hide materials. This identifies potential major animal disease pathogens, zoonotic pathogens, and unknown pathogens, enabling rapid risk warning and classified handling.

[0039] Beyond port scenarios, this invention is also applicable to pathogen risk screening before animal-derived raw material processing enterprises, leather manufacturing enterprises, fur traders, and hide storage and transportation enterprises. By performing metagenomic testing on hide batches, high-risk pathogens can be prevented from entering the industrial chain, reducing the risk of animal disease transmission. For biosafety research institutions and regional animal disease monitoring networks, this invention can also serve as a foundational method for large-scale monitoring and tracing, establishing a baseline for the microbial distribution of hide samples, thereby improving the early identification capability of cross-border epidemic importation. In terms of product form, this invention can be transformed into three types of application products: a nucleic acid pretreatment kit for hide samples, a metagenomic analysis software system for hide pathogens, and a high-risk pathogen targeted detection reagent, realizing the overall engineering, standardization, and industrialization of the method.

[0040] This invention provides a complete evidence system for its pathogen capture efficiency, metagenomic sequencing resolution, and risk warning accuracy in complex hide samples through systematic experiments. In the sample pretreatment stage, a combination of proteinase K and lysozyme digestion, along with magnetic bead enrichment, was used to validate hide samples from different animal species and with different curing processes. The results showed that the total amount of microbial nucleic acid was increased by approximately one order of magnitude compared to the traditional lysis method, and the detection rate of low-abundance pathogens was significantly increased, demonstrating that the combined enzymatic digestion strategy can effectively solve the problems of difficult lysis and low nucleic acid yield in hide samples.

[0041] During the metagenomic sequencing phase, the library construction and data volume control strategies employed in this invention ensured stable and resolvable data even under high background noise conditions in the hide samples. In validation across multiple samples, the proportion of effective microbial sequences recovered remained high, and the identification coverage of viral and bacterial sequences was significantly superior to conventional random amplification methods. By comparing datasets of hide samples from different processing sources, the examples consistently identified key pathogenic gene fragments such as VP1, pagA, and BCSP31, which are listed in the disease catalogue, demonstrating that the method of this invention can accurately locate pathogenic functional genes in highly contaminated environments.

[0042] In risk assessment, the pathogen classification system constructed in this invention was cross-validated with the monitoring technology tables currently used by regulatory authorities. After automatically mapping metagenomic results to risk levels, multiple historical positive reference samples were correctly identified as Level 1 risk, demonstrating the reliability of the risk classification rules. For emerging or unknown microorganisms not listed in the monitoring catalog, this method can also provide indications through metagenomic sequence characteristics, offering traceable signals for "occult pathogens."

[0043] In the targeted confirmation phase, the examples demonstrate the development of CRISPR, RPA, or digital PCR detection reagents based on metagenomic early warning results to validate high-risk pathogens. Experimental results show that targeted detection of VP1 and pagA yielded clear positive signals within a short time and were completely consistent with the metagenomic results. The speed and high specificity of targeted validation prove that the "broad-spectrum screening and targeted confirmation" combined strategy of this invention effectively supports the rapid response needs at ports of entry.

[0044] In summary, the technical effectiveness of this invention is verified by the experimental results of three major stages: nucleic acid pretreatment, metagenomic sequencing analysis, and targeted confirmatory detection. The evidence is sufficient, and it can significantly improve the comprehensiveness, accuracy, and timeliness of pathogen screening for imported animal skins.

[0045] Taking a certain batch of imported cowhides as an example, the specific implementation process of the method of the present invention is as follows: First, following step S1, randomly select several pieces of animal hide from each batch of imported animals, with a total weight of no less than 50 g. In a biosafety cabinet, cut the hides into small pieces and add pre-cooled PBS buffer. Use a tissue homogenizer to thoroughly homogenize the hides. Add 5–10 mL of the homogenate to a lysis system containing proteinase K and lysozyme, and incubate at 37–56 °C to lyse bacteria, fungi, and some viral particles attached to the hide surface, releasing nucleic acids. Then, add pre-equilibrated magnetic beads coated with nucleic acid-binding groups and incubate on a shaker under high-salt conditions. This allows free pathogenic nucleic acids in the sample to selectively adsorb onto the magnetic bead surface. After separation using a magnetic rack, wash away impurities with washing buffer, and finally elute with low-salt elution buffer to obtain the pathogenic nucleic acid template. Detect the nucleic acid quality and concentration using a quantitative fluorescence analyzer or a bioanalyzer to ensure it meets the requirements for library construction.

[0046] Secondly, according to step S2, a general metagenomic library construction kit is used to fragment, end-repair, and ligate adapters to the above nucleic acid template. After purification, a paired-end library with an inserted fragment length of approximately 300 bp is obtained. The qualified library is loaded onto a high-throughput sequencing platform for paired-end sequencing to obtain raw sequencing data. Through the analysis platform for metagenomic data of pathogens in imported animal skins developed for this invention, quality control, adapter removal, and host sequence filtering are performed on the raw reads, and species alignment and abundance estimation are carried out based on the built-in reference database of pathogenic microorganisms, and the biological classification annotation results and community structure composition of bacteria, fungi, parasites, and key monitored viruses in the sample are output.

[0047] Furthermore, according to step S3, the abundance, virulence genes, and drug resistance gene information of each pathogenic species output by the analysis platform are compared with the monitoring technical requirements for imported non-edible animal products. Based on the pathogenicity of the pathogen, whether it is a legally quarantined pathogen, previous monitoring data, and epidemiological information, the detected pathogens are automatically risk-graded, and the pathogens suspected of being highly pathogenic or related to major animal diseases are marked as high-risk, and warning information including the pathogen name, abundance, risk level, and recommended disposal measures is generated in the system. At the same time, it is fed back to on-site quarantine personnel and the laboratory management system in the form of a report or online push.

[0048] Finally, according to step S4, for the target pathogens determined to be high-risk, specific primers and crRNA of the CRISPR / Cas system are designed from their conserved gene sequences, or specific primer probes for RPA or digital PCR are designed. Taking the nucleic acid template purified by magnetic beads or the nucleic acid rapidly extracted from the same batch of samples subsequently as the detection object, a CRISPR / Cas fluorescence reading, isothermal RPA amplification, or digital PCR quantitative detection reaction is established, and targeted nucleic acid detection is carried out on a portable fluorescence reader or a digital PCR platform. By comparing with the negative and positive controls and the standard curve, the presence or absence and approximate load of high-risk pathogens are quickly confirmed, and the results are linked with the metagenomic warning information to achieve rapid and accurate warning and verification of pathogenic microorganisms in imported animal skins.

[0049] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope disclosed by the present invention, as long as they are made within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. A rapid early warning method for pathogenic microorganisms in the skin of imported animals based on metagenomic sequencing, comprising: S1. Pre-processing of imported animal hide samples: grind the hide samples into a homogenate, and obtain pathogen nucleic acid templates by enzymatic digestion with proteinase K and lysozyme combined with magnetic bead enrichment. S2, construct a metagenomic library from the nucleic acid template and perform high-throughput sequencing. Input the sequencing data into the metagenomic data analysis platform for pathogens of imported animal skin to complete the annotation of biological categories and the analysis of microbial community structure. S3. Based on the results of step S2 and combined with the technical requirements for monitoring imported non-edible animal products, the pathogens are risk-classified and early warning information is generated. S4. For pathogens classified as high-risk in step S3, perform targeted nucleic acid detection using at least one of CRISPR / Cas, recombinant RPA, or digital PCR technologies.

2. The method according to claim 1, wherein in step S1, the amount of hide sample taken is 2 to 5 grams, the final concentration of proteinase K is 15 to 25 mg / mL, the final concentration of lysozyme is 40 to 60 mg / mL, the enzymatic hydrolysis temperature is 35 to 39 degrees Celsius, and the enzymatic hydrolysis time is 25 to 35 minutes.

3. The method according to claim 1, wherein the fragment length of the metagenomic library in step S2 is 200 to 600 base pairs, and the raw data volume of the high-throughput sequencing is not less than 3 gigabytes.

4. The method according to claim 1, wherein the database of the metagenomic data analysis platform in step S2 includes a bacterial database, a viral database, and a fungal database.

5. The method of claim 1, wherein the risk classification in step S3 includes: Level 1 corresponds to the major animal disease pathogens listed in the list of quarantine and quarantine diseases for imported animals; Level 2 corresponds to zoonotic pathogens; Level 3 corresponds to newly emerging pathogens or pathogens of unknown origin.

6. A targeted detection system for rapid identification of pathogens in the skins of imported animals, comprising: The pathogen target gene identification module is used to analyze the functional gene information of pathogens in metagenomic sequencing results; The detection reaction construction module is used to design CRISPR guide RNA, RPA primers, or digital PCR primers and probes based on the target gene; The nucleic acid detection module is used to perform targeted amplification or shearing reactions on hide samples and output the detection results. The data comparison module is used to match the detection results with the early warning information to complete the pathogen confirmation.

7. The system according to claim 6, wherein the pathogen target gene includes at least one of the following: foot-and-mouth disease virus VP1 gene, Bacillus anthracis pagA gene, Brucella-specific gene, and Q-heat Rickettsia-specific gene.

8. A metagenomic data analysis platform for pathogens on the skin of imported animals, comprising: The data import module is used to receive metagenomic sequencing data from animal skin samples; The species annotation module is used to complete the classification annotation of bacteria, viruses and fungi based on the database; The microbial diversity analysis module is used to calculate community diversity indices and structural composition. The risk assessment module is used to automatically generate pathogen risk classification information based on the annotation results.

9. The platform according to claim 8, wherein the database adopts a hierarchical index structure and is indexed according to gene sequence similarity and pathogen risk level.

10. An application of the method, system or platform described in any one of claims 1 to 9 for the quarantine of imported animal hides at ports of entry, including nucleic acid pretreatment, metagenomic sequencing, bioinformatics analysis, risk warning and targeted confirmatory detection of imported hide samples.

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