Risk assessment and precise prevention and control method and system of port environmental microorganisms

Abstract

The application provides a kind of port environment microorganism risk assessment and precision prevention and control method and system, through sample collection to key area of port, extract total nucleic acid and reverse transcription RNA into complementary DNA;The mixture of DNA and complementary DNA is subjected to multiple displacement amplification, and after purification, a metagenomic sequencing library is constructed and high-throughput sequencing is performed;Bioinformatics analysis is performed on the sequencing data to obtain microbial community composition data;Based on the data, the microbial load intensity index of each sampling point is calculated, the risk level is divided according to the distribution of the index of all sampling points, and the corresponding disinfection management strategy is output.The application solves the problem of insufficient nucleic acid starting amount of environmental samples through the multiple displacement amplification step, enabling metagenomic sequencing to be stably applied to various low biomass environmental samples;It is the first time to convert metagenomic data into microbial load intensity index and divide the risk based on dynamic threshold, realizing the closed-loop decision from environmental monitoring to precise intervention.

Description

Technical Field

[0001] This invention relates to the field of biosafety and public health monitoring technology, and in particular to a method and system for risk assessment and precise control of microorganisms in port environments. Background Technology

[0002] The increasing frequency of global movement of people and goods has exacerbated the risk of cross-border transmission of pathogens. As the first line of defense for border health and quarantine, the effectiveness of port control directly impacts national public health security. In recent years, the frequent emergence of new and re-emerging infectious diseases has intensified the risk of cross-border transmission of pathogens, placing higher demands on port infectious disease control capabilities. Currently, port infectious disease control mainly relies on symptom monitoring, temperature screening, and targeted pathogen detection of inbound travelers. However, this passive, "human-centered" control model has significant shortcomings. On the one hand, infected individuals or carriers can contaminate environmental surfaces and air through contact, droplets, and aerosols during their activities at ports, creating a persistent "environmental pathogen reservoir." These residual pathogens can pose a risk of contact or aerosol transmission to susceptible individuals passing through. The existing control system lacks systematic and broad-spectrum assessment methods for environmental residual risks, resulting in a large number of potential risk points remaining undetected. On the other hand, traditional environmental microbial monitoring mainly relies on culture methods, which are time-consuming, have low throughput, and are only applicable to some culturable bacteria, failing to detect viruses, difficult-to-culture microorganisms, and unknown pathogens. While molecular detection technologies such as polymerase chain reaction (PCR) based on specific primers have high sensitivity, their detection targets are fixed, making unbiased broad-spectrum screening difficult and unable to address unknown or mutated pathogens. Crucially, the nucleic acid content of microorganisms in port environmental samples (such as surface swabs and aerosol filters) is typically extremely low, ranging from picograms to nanograms, making it difficult to directly meet the starting quantity requirements for high-throughput sequencing library construction. This technical bottleneck is a core obstacle limiting the application of metagenomics technology in port environmental monitoring, severely restricting the ability to discover low-abundance pathogens and unknown microorganisms in environmental samples. Furthermore, even when environmental microbial composition data is obtained through detection, current technologies lack methods to scientifically and quantitatively translate this high-dimensional, complex data into risk assessment conclusions. Existing practices often rely on experience-based judgment, making it difficult to achieve refined and differentiated risk-based management. Summary of the Invention

[0003] This invention provides a method and system for risk assessment and precise control of port environmental microorganisms, which addresses the shortcomings of traditional port environmental microorganism risk assessment technologies, such as blind spots in environmental risk, insufficient detection sensitivity and broad spectrum, difficulty in building libraries for low biomass samples, and inability to meet the actual needs of precise risk assessment and control of port environmental microorganisms.

[0004] This invention provides a method for risk assessment and precise control of microorganisms in port environments, comprising: Samples were collected from key areas of the port environment, and total nucleic acid was extracted from the samples. The total nucleic acid contained DNA and RNA. The RNA was reverse transcribed into complementary DNA. The mixture of the DNA and the complementary DNA was subjected to whole-genome pre-amplification, and the amplification products were purified. Metagenomic sequencing libraries were constructed based on the purified amplification products, and high-throughput sequencing was performed on the metagenomic sequencing libraries to obtain raw sequencing data. Bioinformatics analysis was performed on the raw sequencing data to obtain microbial community composition data in the environmental samples; Based on the microbial community composition data, the microbial load intensity index of each sampling point is calculated. According to the distribution of the microbial load intensity index of all sampling points, the sampling points are divided into multiple risk levels, and a corresponding disinfection management strategy is output based on each risk level.

[0005] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the step of collecting samples from key areas in the port environment, extracting total nucleic acid from the samples, wherein the total nucleic acid contains DNA and RNA, and reverse transcribing the RNA into complementary DNA, includes: Environmental samples were obtained by using sterile swabs to perform standardized area sampling on the key areas. Total nucleic acids were extracted from the environmental samples, and the RNA was reverse transcribed using reverse transcriptase to convert it into complementary DNA.

[0006] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the step of performing whole-genome pre-amplification on the mixture of the DNA and the complementary DNA, and purifying the amplification product, includes: The mixture of the DNA and the complementary DNA is mixed with Phi29 DNA polymerase, random primers and reaction buffer to form an amplification reaction system; The amplification reaction system was placed under isothermal conditions for multiple displacement amplification, which resulted in the exponential amplification of the nucleic acid template. The amplified product was purified by magnetic beads to obtain the purified amplified product.

[0007] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the step of constructing a metagenomic sequencing library based on purified amplification products and performing high-throughput sequencing on the metagenomic sequencing library to obtain raw sequencing data includes: The amplification product was fragmented to obtain DNA fragments; The DNA fragments were subjected to end repair, adapter ligation, and PCR amplification to construct a metagenomic sequencing library; The metagenomic sequencing libraries are subjected to quality testing, and libraries that meet the quality requirements are loaded into the high-throughput sequencing platform; The high-throughput sequencing platform employs a paired-end sequencing mode to perform high-throughput sequencing and obtain raw sequencing data.

[0008] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the step of performing bioinformatics analysis on the original sequencing data to obtain microbial community composition data in the environmental sample includes: Low-quality sequences and adapter sequences are removed from the original sequencing data to obtain high-quality sequences; The high-quality sequences are compared with the host reference genome, and the host-derived sequences are removed to obtain the non-host sequences; The non-host sequence is assembled from scratch to obtain the assembled sequence; The assembled sequences were annotated with species classification using taxonomic annotation tools to obtain species composition and relative abundance data. Unclassified sequences were compared with viral nucleic acid and viral protein databases to obtain virome composition and relative abundance data; By integrating the species composition and relative abundance data and the virus composition and relative abundance data, the microbial community composition data in the environmental sample is obtained.

[0009] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the step of calculating the microbial load intensity index of each sampling point based on the microbial community composition data, and classifying the sampling points into multiple risk levels according to the distribution of the microbial load intensity index of all sampling points, includes: Calculate the microbial load intensity index for each sampling point based on the microbial community composition data; Statistical analysis of the frequency distribution of microbial load intensity indicators at all sampling points; Based on the statistical characteristics of the frequency distribution, one or more dynamic thresholds are determined using at least one method; Based on one or more dynamic thresholds, all sampling points are divided into multiple risk levels.

[0010] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the at least one method includes at least one of the percentile method, cluster analysis method, or natural breakpoint method.

[0011] The method for risk assessment and precise control of port environmental microorganisms provided by the present invention further includes: Based on the microbial community composition data, key pathogens with potential public health significance were identified. The genome of the key pathogen is assembled to obtain the genome sequence or characteristic gene sequence of the key pathogen; The genome sequence or characteristic gene sequence is compared with global reference sequences in a public database to construct a phylogenetic tree; By analyzing the clustering position of the key pathogens in the phylogenetic tree, the geographical origin or host origin of the key pathogens can be inferred, and an early warning report of imported infectious disease risk can be generated.

[0012] According to the method for risk assessment and precise control of port environmental microorganisms provided by the present invention, the step of outputting a corresponding disinfection management strategy based on each risk level includes: A visual risk zoning map is generated based on the risk level; Based on the risk level of each sampling point in the risk zoning map, a differentiated disinfection management strategy is automatically matched and output. The disinfection management strategy includes one or more of the following: disinfection frequency, disinfectant strength, and disinfection method priority.

[0013] This invention also provides a risk assessment and precise control system for port environmental microorganisms, comprising: The extraction module is used to collect samples from key areas in the port environment, extract total nucleic acid from the samples, the total nucleic acid containing DNA and RNA, and reverse transcribe the RNA into complementary DNA; An amplification module is used to perform whole-genome pre-amplification of the mixture of the DNA and the complementary DNA, and to purify the amplification products; The acquisition module is used to construct a metagenomic sequencing library based on the purified amplification product and perform high-throughput sequencing on the metagenomic sequencing library to obtain raw sequencing data. The analysis module is used to perform bioinformatics analysis on the raw sequencing data to obtain microbial community composition data in the environmental sample; The output module is used to calculate the microbial load intensity index of each sampling point based on the microbial community composition data, divide the sampling points into multiple risk levels according to the distribution of the microbial load intensity index of all sampling points, and output the corresponding disinfection management strategy based on each risk level.

[0014] This invention provides a method and system for risk assessment and precise control of microorganisms in port environments. The method involves collecting samples from key areas of the port environment, extracting total nucleic acid (total nucleic acid, including DNA and RNA), and reverse transcribing the RNA into complementary DNA. The mixture of the DNA and complementary DNA is then pre-amplified into a whole genome, and the amplification product is purified. A metagenomic sequencing library is constructed based on the purified amplification product, and high-throughput sequencing is performed on the library to obtain raw sequencing data. Bioinformatics analysis is performed on the raw sequencing data to obtain microbial community composition data in the environmental samples. Based on the microbial community composition data, a microbial load intensity index is calculated for each sampling point. According to the distribution of the microbial load intensity index across all sampling points, the sampling points are divided into multiple risk levels, and a corresponding disinfection management strategy is output for each risk level. Through innovative multiple displacement amplification steps, this method overcomes the core challenge of insufficient initial nucleic acid quantity in environmental samples, enabling metagenomic sequencing to be stably applied to various low-biomass environmental samples. It is the first to convert metagenomic data into a "microbial load intensity index" and use a quantitative model based on dynamic thresholds for "risk zoning," achieving a closed-loop decision-making process from environmental monitoring to precise intervention. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 This is a flowchart of the method for risk assessment and precise control of port environmental microorganisms provided in this embodiment of the invention; Figure 2 This is a schematic diagram of the port environment sampling area and sites provided in an embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the principle of multiple substitution amplification provided in an embodiment of the present invention; Figure 4 This is a schematic diagram of a key pathogen phylogenetic tree provided in an embodiment of the present invention; Figure 5 This is a functional structure diagram of the port environmental microbial risk assessment and precise control system provided in the embodiments of the present invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0018] Figure 1 A flowchart of the risk assessment and precise control method for port environmental microorganisms provided in this embodiment of the invention is shown below. Figure 1 As shown in the embodiments of the present invention, the method for risk assessment and precise control of port environmental microorganisms includes: Step 101: Collect samples from key areas in the port environment, extract total nucleic acid from the samples, the total nucleic acid containing DNA and RNA, and reverse transcribe the RNA into complementary DNA; In this embodiment of the invention, key biosafety monitoring areas at ports of entry include, for example, epidemiological investigation areas, medical screening rooms, sampling areas, observation rooms, medical waste storage areas, and laboratories. For high-touch surfaces such as chairs, tabletops, door handles, biosafety countertops, and lab benches, sterile swabs are used to collect standardized area (e.g., 5cm × 5cm) samples. Figure 2 As shown. Total nucleic acids (including DNA and RNA) were extracted from the collected samples. The extracted RNA fraction was reverse transcribed into complementary DNA (cDNA) using a high-fidelity reverse transcriptase (such as Thermo Fisher SuperScript™ IV).

[0019] Step 102: Perform whole-genome pre-amplification on the mixture of the DNA and the complementary DNA, and purify the amplification product; Step 103: Construct a metagenomic sequencing library based on the purified amplification product, and perform high-throughput sequencing on the metagenomic sequencing library to obtain raw sequencing data; Step 104: Perform bioinformatics analysis on the raw sequencing data to obtain microbial community composition data in the environmental samples; Step 105: Based on the microbial community composition data, calculate the microbial load intensity index for each sampling point. According to the distribution of the microbial load intensity index of all sampling points, divide the sampling points into multiple risk levels and output the corresponding disinfection management strategy based on each risk level.

[0020] Traditional port infectious disease control relies primarily on symptom monitoring, temperature screening, and targeted pathogen detection of inbound travelers. This human-centered, passive control model has significant drawbacks. Firstly, there are blind spots in environmental risk. Infected individuals or carriers can contaminate environmental surfaces and air through contact, droplets, and aerosols during their activities, creating a persistent environmental pathogen pool. This poses a risk of contact or aerosol transmission to subsequent susceptible populations, and current technologies lack systematic and broad-spectrum assessment methods for such environmental residual risks. Secondly, detection technologies lack sufficient sensitivity and breadth of information. Traditional environmental microbial monitoring relies on culture methods, which are time-consuming, have low throughput, and cannot detect viruses, difficult-to-culture microorganisms, or unknown pathogens. While molecular detection technologies such as polymerase chain reaction (PCR) based on specific primers have high sensitivity, their detection targets are fixed, preventing unbiased broad-spectrum screening. Furthermore, the nucleic acid content of microorganisms in environmental swabs and aerosol filters is typically in the picogram to nanogram range, making it difficult to directly meet the starting quantity requirements for high-throughput sequencing library construction. This is a core obstacle limiting the application of metagenomics technology in port environmental monitoring. Furthermore, even if environmental microbial composition data is obtained through testing, there is still a lack of mature and standardized decision support models for how to scientifically and quantitatively transform it into risk assessment conclusions and further guide differentiated and precise intervention measures such as disinfection, isolation, and ventilation.

[0021] The method for risk assessment and precise control of port environmental microorganisms provided in this invention involves collecting samples from key areas of the port environment, extracting total nucleic acid (total nucleic acid, including DNA and RNA), and reverse transcribing the RNA into complementary DNA. The mixture of the DNA and complementary DNA is then pre-amplified into a whole genome, and the amplification product is purified. A metagenomic sequencing library is constructed based on the purified amplification product, and high-throughput sequencing is performed on the metagenomic sequencing library to obtain raw sequencing data. Bioinformatics analysis is performed on the raw sequencing data to obtain microbial community composition data in the environmental samples. Based on the microbial community composition data, a microbial load intensity index is calculated for each sampling point. According to the distribution of the microbial load intensity index across all sampling points, the sampling points are divided into multiple risk levels, and a corresponding disinfection management strategy is output for each risk level. Through innovative multiple displacement amplification steps, this method overcomes the core problem of insufficient initial nucleic acid quantity in environmental samples, enabling metagenomic sequencing to be stably applied to various low-biomass environmental samples. It pioneers a quantitative model that transforms metagenomic data into a "microbial load intensity index" and performs "risk zoning" based on dynamic thresholds, achieving closed-loop decision-making from environmental monitoring to precise intervention.

[0022] Based on any of the above embodiments, the step of collecting samples from key areas in the port environment, extracting total nucleic acid from the samples, wherein the total nucleic acid contains DNA and RNA, and reverse transcribing the RNA into complementary DNA includes: Step 201: Use sterile swabs to perform standardized area sampling on the key areas to obtain environmental samples; Step 202: Extract total nucleic acid from the environmental sample, and use reverse transcriptase to reverse transcribe the RNA to convert it into complementary DNA.

[0023] Based on any of the above embodiments, the step of performing whole-genome pre-amplification on the mixture of the DNA and the complementary DNA, and purifying the amplification product, includes: Step 301: Mix the mixture of the DNA and the complementary DNA with Phi29 DNA polymerase, random primers and reaction buffer to form an amplification reaction system; Step 302: Place the amplification reaction system under isothermal conditions for multiple displacement amplification, so that the nucleic acid template is amplified exponentially; In embodiments of the present invention, such as Figure 3 As shown, total DNA and cDNA were combined and subjected to isothermal, random primer-mediated whole-genome amplification using a Phi29 polymerase-based Multiple Displacement Amplification (MDA) kit (such as the QIAGEN REPLI-gSingle Cell Kit). A typical reaction system consisted of 20 μL of nucleic acid product, 29 μL of reaction buffer, and 1 μL of Phi29 DNA polymerase, for a total volume of 50 μL. The reaction conditions were: incubation at 30°C for 4–8 hours, followed by heating at 65°C for 3 minutes to inactivate the enzyme. The amplified products were purified using magnetic beads (such as VAHTS DNA CleanBeads). This step exponentially amplifies low-concentration environmental microbial nucleic acids from picograms to nanograms to micrograms, completely resolving the bottleneck problem of low-biomass samples being unable to be directly sequenced for metagenomics.

[0024] Step 303: Purify the amplified product using magnetic beads to obtain the purified amplified product.

[0025] Based on any of the above embodiments, the construction of a metagenomic sequencing library based on the purified amplification product, and the high-throughput sequencing of the metagenomic sequencing library to obtain raw sequencing data, includes: Step 401: Fragment the amplification product to obtain DNA fragments; Step 402: Perform end repair, adapter ligation, and PCR amplification on the DNA fragment to construct a metagenomic sequencing library; Step 403: Perform quality checks on the metagenomic sequencing libraries, and load libraries that meet the quality requirements into the high-throughput sequencing platform; Step 404: Perform high-throughput sequencing on the high-throughput sequencing platform using paired-end sequencing mode to obtain raw sequencing data.

[0026] In this embodiment of the invention, the purified MDA amplification product was used to construct a sequencing library using a commercial library construction kit (such as the MGISEasy series from BGI Genomics). Paired-end sequencing (PE150) was performed using a high-throughput sequencing platform (such as Illumina NovaSeq 6000 or MGISEQ-2000RS from BGI Genomics) to generate raw sequencing data.

[0027] Based on any of the above embodiments, the step of performing bioinformatics analysis on the raw sequencing data to obtain microbial community composition data in environmental samples includes: Step 501: Remove low-quality sequences and adapter sequences from the original sequencing data to obtain high-quality sequences; Step 502: Align the high-quality sequence with the host reference genome, remove the host-derived sequence, and obtain the non-host sequence; Step 503: Perform de novo assembly on the non-host sequence to obtain the assembled sequence; Step 504: Use taxonomic annotation tools to perform species classification annotation on the assembled sequence to obtain species composition and relative abundance data; Step 505: Compare the unclassified sequences with the viral nucleic acid database and the viral protein database to obtain viral composition and relative abundance data; Step 506: Integrate the species composition and relative abundance data and the virus composition and relative abundance data to obtain the microbial community composition data in the environmental sample.

[0028] In this embodiment of the invention, the raw sequencing data undergoes quality control, and host (human) sequences are removed using tools such as FastP. High-quality non-host sequences are then assembled de novo using tools such as MEGAHIT. Taxonomic annotation tools such as Kraken2, combined with comprehensive databases such as PlusPF, are used to classify the assembled sequences into species, identifying bacteria, fungi, archaea, protozoa, etc.

[0029] Simultaneously, a specific analysis was conducted on the virome: unclassified sequences were compared with viral nucleic acid (using BLASTn) and protein databases (using DIAMOND), and viral taxonomic annotation was performed using MEGAN, thereby comprehensively analyzing the composition and relative abundance of the microbial community in environmental samples.

[0030] Based on any of the above embodiments, the step of calculating the microbial load intensity index for each sampling point based on the microbial community composition data, and classifying the sampling points into multiple risk levels according to the distribution of the microbial load intensity index across all sampling points, includes: Step 601: Calculate the microbial load intensity index for each sampling point based on the microbial community composition data; Step 602: Statistically analyze the frequency distribution of microbial load intensity indicators for all sampling points; Step 603: Based on the statistical characteristics of the frequency distribution, determine one or more dynamic thresholds using at least one method; Step 604: Divide all sampling points into multiple risk levels based on the one or more dynamic thresholds.

[0031] In embodiments of the present invention, the at least one method includes at least one of percentile method, cluster analysis method, or natural breakpoint method.

[0032] In this embodiment of the invention, a microbial load intensity index is calculated for each sampling point. This index is a quantitative value that comprehensively reflects the abundance and diversity of microorganisms at that location. In a preferred embodiment, this index can be the total number of detected microbial populations (n); in other embodiments, this index can be a weighted microbial abundance index or a comprehensive score combining the weights of specific pathogenic microorganisms. Based on the distribution of the microbial load intensity index of all sampling points, one or more dynamic thresholds are determined using statistical methods such as percentile method, cluster analysis method, or natural breakpoint method to divide the sampling points into multiple risk level zones.

[0033] In one specific embodiment, the dynamic threshold divides the sampling points into four levels: a relatively safe zone, a secondary safe zone, a general disinfection zone, and a key disinfection zone. The microbial load intensity index, from low to high, corresponds to the potential risk of infection transmission and the required intensity of disinfection intervention, from low to high.

[0034] Based on any of the above embodiments, the method for risk assessment and precise control of port environmental microorganisms further includes: Step 701: Based on the microbial community composition data, identify key pathogens with potential public health significance; Step 702: Perform genome assembly on the key pathogen to obtain the genome sequence or characteristic gene sequence of the key pathogen; Step 703: Align the genome sequence or characteristic gene sequence with global reference sequences in a public database to construct a phylogenetic tree; Step 704: By analyzing the cluster position of the key pathogen in the phylogenetic tree, the geographical origin or host origin of the key pathogen is inferred, and an imported infectious disease risk warning report is generated.

[0035] In this embodiment of the invention, in-depth molecular tracing is performed on identified key pathogens with potential public health significance (such as influenza virus, coronavirus, monkeypox virus, etc.), specifically including: Assemble or extract the complete genome or key gene (e.g., the HA gene of influenza virus) sequences of the pathogen from sequencing data. Align the obtained sequences with global sequences in public databases (e.g., NCBI GenBank) and construct phylogenetic trees using software such as MEGA. Figure 4 As shown, by analyzing the cluster position of the sequence in the phylogenetic tree, its possible geographical or host origin can be inferred. If the analysis results strongly suggest that it is an imported infectious disease risk warning report, a scientific basis is generated to strengthen targeted quarantine from relevant regions / populations.

[0036] Based on any of the above embodiments, the step of outputting a corresponding disinfection management strategy for each risk level includes: Step 801: Generate a visualized risk zoning map based on the risk level; Step 802: Based on the risk level of each sampling point in the risk zoning map, automatically match and output differentiated disinfection management strategies. The disinfection management strategies include one or more of the following: disinfection frequency, disinfectant strength, and disinfection method priority.

[0037] This invention generates a visualized risk zoning map and, based on the zoning results, automatically matches and recommends differentiated disinfection management strategies for areas with different risk levels, including but not limited to disinfection frequency, disinfectant strength, and priority of disinfection methods.

[0038] The risk assessment and precise control method for port environmental microorganisms provided in this invention is shown in the figure. Specifically, it includes: First, sample collection and nucleic acid preparation. Standardized swab sampling is performed on high-contact surfaces in key port areas to extract total nucleic acids (including DNA and RNA). The RNA component is reverse transcribed into complementary DNA. Subsequently, a mixture of DNA and complementary DNA is pre-amplified using multiple substitution amplification technology, exponentially amplifying micro-volume nucleic acids from picograms to nanograms to micrograms. High-quality amplification products are obtained through nucleotide purification and enrichment. Next, library construction and sequencing are performed. The amplification products are fragmented to construct metagenomic sequencing libraries. Paired-end sequencing is performed on a sequencing platform to obtain raw sequencing data and complete data quality control. Then, bioinformatics analysis is performed. Non-host sequences are obtained through metagenomic assembly and removal of human genomes. Database comparison and species classification annotation are used to analyze the species composition and relative abundance of microorganisms such as bacteria, fungi, and viruses. Data analysis and visualization are then performed to generate a risk assessment report. For viruses identified as having public health significance, further target gene fragment amplification and sequencing are performed. Combined with methods such as virus isolation and culture, observation of cytopathic effects, and viral nucleic acid detection, virus identification and preservation are completed. Subsequently, key pathogens undergo genome enrichment and deep sequencing to obtain whole genome sequences. Finally, in the analysis and validation phase, the pathogen genome sequences are compared with global reference sequences in public databases to construct phylogenetic trees. Cluster analysis is used to infer their geographical or host origin. After cross-confirmation through laboratory validation, an imported infectious disease risk warning report is generated, providing a basis for precise port control decisions.

[0039] Taking the microbial risk assessment and prevention and control application in the complex environment of a certain city's port as an example, in the summer of 2024 (July - September), at three locations including a certain city's port, a certain city's travel health care center, and a certain city's international airport, six key areas such as the epidemiological investigation area and the medical screening room were selected, and five types of high-contact surfaces such as chairs, tabletops, and doorknobs were sampled once a week, obtaining a total of 126 environmental swab samples. Total nucleic acids were extracted using a nucleic acid extraction kit, and RNA was reverse transcribed. Using the QIAGEN REPLI-g Single Cell Kit, a 50 μL MDA reaction system was prepared strictly according to the instructions, incubated at 30 °C for 6 hours, and inactivated at 65 °C for 3 minutes. The amplified products were purified using VAHTS DNA Clean Beads and eluted in 25 μL Elution Buffer. The purified products were sent to a commercial sequencing center for PE150 sequencing using the Illumina NovaSeq 6000 platform. After the off-machine data was quality-controlled and human-derived sequences were removed, Kraken2 (PlusPF database) and a custom virus analysis process were used for species annotation, comprehensively analyzing the bacterial community and virome. The total number of microbial populations (n) at each sampling point was calculated as an indicator of microbial load intensity. Through the frequency distribution analysis of the n values of all 126 samples, three thresholds were dynamically determined based on datasets of time, location, and detection depth (including long-term microbial background monitoring data, seasonal changes, sampling strategies, and sequencing sensitivity, etc.), and the environment was divided into four risk levels. In the specific dataset of this example, the division results were: n ≤ 39 (relatively safe area), 46 < n < 48 (sub-safe area), 50 < n < 53 (general disinfection area), n ≥ 55 (key disinfection area). The tabletops in the airport sampling area, the chair surfaces in the port observation room, etc. were classified as the "key disinfection area". In the samples of the "key disinfection area", influenza A virus (H4N2) was detected. A phylogenetic tree was constructed for its HA gene, showing that this strain clustered with avian-origin H4N2 viruses in Asia, suggesting possible input of avian influenza virus in the environment. Pigeon circovirus was detected in the port observation room, and evolutionary analysis showed the closest genetic relationship with the European strain, suggesting possible cross-border input. According to the zoning results, a visual risk map was generated, recommending implementing high-frequency intensive disinfection once every 2 - 4 hours in the "key disinfection area"; performing routine disinfection 2 - 3 times a day in the "general disinfection area". Regarding the detected risk of imported influenza virus, the early warning report recommended strengthening the screening of febrile passengers from relevant flight routes and checking and optimizing the ventilation systems in relevant areas.

[0040] It should be noted that the specific thresholds (39, 46-48, 50-53, 55) used for partitioning in this embodiment of the invention are specific dynamic thresholds calculated based on the dataset calibration at a specific time, location, and detection depth. In practical applications, these dynamic thresholds can be periodically calibrated and optimized based on long-term microbial baseline monitoring data of the target port, seasonal changes, sampling strategies, and sequencing sensitivity.

[0041] The risk assessment and precise control method for port environmental microorganisms provided in this invention provides a scientific and operable basis for differentiated and precise disinfection and biosafety management by performing highly sensitive and unbiased detection of all microorganisms (including low-abundance and difficult-to-culture pathogens) in high-risk port environments. This method establishes a model from microbial data to quantitative risk assessment, achieves risk-based regional grading, and enables molecular tracing of key pathogens, providing early warning of their cross-border importation and local transmission risks. Specifically, this invention overcomes the core challenge of insufficient initial nucleic acid quantity in environmental samples through an innovative multiple displacement amplification pre-amplification step, enabling metagenomic sequencing to be stably applied to various low-biomass environmental samples, achieving unbiased and highly sensitive detection of all microbial groups such as bacteria, viruses, and fungi. Based on this, it pioneers a quantitative model that transforms metagenomic data into microbial load intensity indicators and performs risk zoning based on dynamic thresholds. This model directly transforms complex omics data into clear and operable "tiered disinfection" and "key control" instructions, achieving a closed loop from monitoring to precise intervention. Meanwhile, by integrating phylogenetic analysis, it can proactively identify and track the potential sources of pathogens in the environment, distinguishing between local circulation and imported cases, and providing early and targeted warning information for preventing imported epidemics. The complete and standardized process from sampling, experimentation, analysis to decision support is particularly suitable for the practical needs of scenarios such as ports, customs, and international airports, and has the advantages of strong systematicity, high practical value, and ease of promotion.

[0042] The risk assessment and precise control system for port environmental microorganisms provided by this invention is described below. The risk assessment and precise control system for port environmental microorganisms described below can be referred to in correspondence with the risk assessment and precise control method for port environmental microorganisms described above.

[0043] Figure 5 A functional structure diagram of the port environmental microbial risk assessment and precise control system provided in this embodiment of the invention is shown below. Figure 5 As shown, the port environmental microbial risk assessment and precise control system provided in this embodiment of the invention includes: Extraction module 501 is used to collect samples from key areas in the port environment, extract total nucleic acid from the samples, the total nucleic acid containing DNA and RNA, and reverse transcribe the RNA into complementary DNA. Amplification module 502 is used to perform whole-genome pre-amplification on the mixture of the DNA and the complementary DNA, and to purify the amplification product; The acquisition module 503 is used to construct a metagenomic sequencing library based on the purified amplification product and perform high-throughput sequencing on the metagenomic sequencing library to obtain raw sequencing data. Analysis module 504 is used to perform bioinformatics analysis on the raw sequencing data to obtain microbial community composition data in environmental samples; The output module 505 is used to calculate the microbial load intensity index of each sampling point based on the microbial community composition data, divide the sampling points into multiple risk levels according to the distribution of the microbial load intensity index of all sampling points, and output the corresponding disinfection management strategy based on each risk level.

[0044] The port environment microbial risk assessment and precise control system provided in this invention collects samples from key areas of the port environment, extracts total nucleic acid (total nucleic acid, including DNA and RNA), and reverse transcribes the RNA into complementary DNA. The mixture of the DNA and complementary DNA is then pre-amplified into a whole genome, and the amplification product is purified. A metagenomic sequencing library is constructed based on the purified amplification product, and high-throughput sequencing is performed on the metagenomic sequencing library to obtain raw sequencing data. Bioinformatics analysis is performed on the raw sequencing data to obtain microbial community composition data in the environmental samples. Based on the microbial community composition data, a microbial load intensity index is calculated for each sampling point. According to the distribution of the microbial load intensity index across all sampling points, the sampling points are divided into multiple risk levels, and a corresponding disinfection management strategy is output for each risk level. Through innovative multiple displacement amplification steps, this system overcomes the core problem of insufficient initial nucleic acid quantity in environmental samples, enabling metagenomic sequencing to be stably applied to various low-biomass environmental samples. It pioneers a quantitative model that transforms metagenomic data into a "microbial load intensity index" and performs "risk zoning" based on dynamic thresholds, achieving closed-loop decision-making from environmental monitoring to precise intervention.

[0045] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs. Those skilled in the art can understand and implement this without any creative effort.

[0046] Through the above description of the embodiments, those skilled in the art can clearly understand that each embodiment can be implemented by means of software plus necessary general-purpose hardware platforms, and of course, it can also be implemented by hardware. Based on this understanding, the above technical solutions, in essence or the parts that contribute to the related technology, can be embodied in the form of software products. This computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, magnetic disk, optical disk, etc., and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in the various embodiments or some parts of the embodiments.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for risk assessment and precise control of microorganisms in port environments, characterized in that, include: Samples were collected from key areas of the port environment, and total nucleic acid was extracted from the samples. The total nucleic acid contained DNA and RNA. The RNA was reverse transcribed into complementary DNA. The mixture of the DNA and the complementary DNA was subjected to whole-genome pre-amplification, and the amplification products were purified. Metagenomic sequencing libraries were constructed based on the purified amplification products, and high-throughput sequencing was performed on the metagenomic sequencing libraries to obtain raw sequencing data. Bioinformatics analysis was performed on the raw sequencing data to obtain microbial community composition data in the environmental samples; Based on the microbial community composition data, the microbial load intensity index of each sampling point is calculated. According to the distribution of the microbial load intensity index of all sampling points, the sampling points are divided into multiple risk levels, and a corresponding disinfection management strategy is output based on each risk level.

2. The method for risk assessment and precise control of port environmental microorganisms according to claim 1, characterized in that, The process involves collecting samples from key areas of the port environment, extracting total nucleic acid from the samples, wherein the total nucleic acid contains DNA and RNA, and reverse transcribing the RNA into complementary DNA, including: Environmental samples were obtained by using sterile swabs to perform standardized area sampling on the key areas. Total nucleic acids were extracted from the environmental samples, and the RNA was reverse transcribed using reverse transcriptase to convert it into complementary DNA.

3. The method for risk assessment and precise control of port environmental microorganisms according to claim 1, characterized in that, The step of performing whole-genome pre-amplification on the mixture of the DNA and the complementary DNA, and purifying the amplification product, includes: The mixture of the DNA and the complementary DNA is mixed with Phi29 DNA polymerase, random primers and reaction buffer to form an amplification reaction system; The amplification reaction system was placed under isothermal conditions for multiple displacement amplification, which resulted in the exponential amplification of the nucleic acid template. The amplified product was purified by magnetic beads to obtain the purified amplified product.

4. The method for risk assessment and precise control of port environmental microorganisms according to claim 3, characterized in that, The metagenomic sequencing library is constructed based on the purified amplification product, and high-throughput sequencing is performed on the metagenomic sequencing library to obtain raw sequencing data, including: The amplification product was fragmented to obtain DNA fragments; The DNA fragments were subjected to end repair, adapter ligation, and PCR amplification to construct a metagenomic sequencing library; The metagenomic sequencing libraries are subjected to quality testing, and libraries that meet the quality requirements are loaded into the high-throughput sequencing platform; The high-throughput sequencing platform employs a paired-end sequencing mode to perform high-throughput sequencing and obtain raw sequencing data.

5. The method for risk assessment and precise control of port environmental microorganisms according to claim 1, characterized in that, The bioinformatics analysis of the raw sequencing data to obtain microbial community composition data in the environmental samples includes: Low-quality sequences and adapter sequences are removed from the original sequencing data to obtain high-quality sequences; The high-quality sequences are compared with the host reference genome, and the host-derived sequences are removed to obtain the non-host sequences; The non-host sequence is assembled from scratch to obtain the assembled sequence; The assembled sequences were annotated with species classification using taxonomic annotation tools to obtain species composition and relative abundance data. Unclassified sequences were compared with viral nucleic acid and viral protein databases to obtain virome composition and relative abundance data; By integrating the species composition and relative abundance data and the virus composition and relative abundance data, the microbial community composition data in the environmental sample is obtained.

6. The method for risk assessment and precise control of port environmental microorganisms according to claim 1, characterized in that, Based on the microbial community composition data, the microbial load intensity index of each sampling point is calculated. According to the distribution of the microbial load intensity index across all sampling points, the sampling points are divided into multiple risk levels, including: Calculate the microbial load intensity index for each sampling point based on the microbial community composition data; Statistical analysis of the frequency distribution of microbial load intensity indicators at all sampling points; Based on the statistical characteristics of the frequency distribution, one or more dynamic thresholds are determined using at least one method; Based on one or more dynamic thresholds, all sampling points are divided into multiple risk levels.

7. The method for risk assessment and precise control of port environmental microorganisms according to claim 6, characterized in that, The at least one method includes at least one of percentile method, cluster analysis method, or natural breakpoint method.

8. The method for risk assessment and precise control of port environmental microorganisms according to claim 1, characterized in that, Also includes: Based on the microbial community composition data, key pathogens with potential public health significance were identified. The genome of the key pathogen is assembled to obtain the genome sequence or characteristic gene sequence of the key pathogen; The genome sequence or characteristic gene sequence is compared with global reference sequences in a public database to construct a phylogenetic tree; By analyzing the clustering position of the key pathogens in the phylogenetic tree, the geographical origin or host origin of the key pathogens can be inferred, and an early warning report of imported infectious disease risk can be generated.

9. The method for risk assessment and precise control of port environmental microorganisms according to claim 1, characterized in that, The disinfection management strategy output based on each risk level includes: A visual risk zoning map is generated based on the risk level; Based on the risk level of each sampling point in the risk zoning map, a differentiated disinfection management strategy is automatically matched and output. The disinfection management strategy includes one or more of the following: disinfection frequency, disinfectant strength, and disinfection method priority.

10. A risk assessment and precise control system for port environmental microorganisms, characterized in that, include: The extraction module is used to collect samples from key areas in the port environment, extract total nucleic acid from the samples, the total nucleic acid containing DNA and RNA, and reverse transcribe the RNA into complementary DNA; An amplification module is used to perform whole-genome pre-amplification of the mixture of the DNA and the complementary DNA, and to purify the amplification products; The acquisition module is used to construct a metagenomic sequencing library based on the purified amplification product and perform high-throughput sequencing on the metagenomic sequencing library to obtain raw sequencing data. The analysis module is used to perform bioinformatics analysis on the raw sequencing data to obtain microbial community composition data in the environmental sample; The output module is used to calculate the microbial load intensity index of each sampling point based on the microbial community composition data, divide the sampling points into multiple risk levels according to the distribution of the microbial load intensity index of all sampling points, and output the corresponding disinfection management strategy based on each risk level.

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