Method and system for screening inspection samples of imported quarantine species

Abstract

The invention discloses an inspection sample screening method and system for imported quarantine species, and belongs to the technical field of quarantine inspection.The method comprises the steps that S1, gene sequences of the quarantine species are collected in a public database, key DNA bar code sequences are obtained by screening DNA bar code types, and the key DNA bar code sequences are stored as sequence data in a fasta format; s2, inputting morphological characteristics and DNA bar codes of each quarantine species to obtain test specimen data; s3, performing sequence comparison on the sequence data and the test specimen data, and constructing a phylogenetic tree through maximum likelihood analysis; s4, dividing and defining quarantine species by using the phylogenetic tree; and S5, according to the divided and defined information of the specimens to which the quarantine species belong, obtaining the voucher specimens of the quarantine species, and taking the accurate and authoritative voucher specimens as reliable information sources for pest detection and identification, so that important reference can be provided for port detection and identification; meanwhile, important information guarantee can be provided for improvement of epidemic prevention detection quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of quarantine inspection, and in particular, relates to a method and system for screening inspection samples of imported quarantine species. Background Art

[0002] Biological invasion refers to the intentional or unintentional entry of organisms from their original habitats into new ecosystems through human or natural factors, and their rapid colonization and spread in the new ecosystems, causing serious economic, social and ecological losses, and having a significant negative impact on the ecological environment and agricultural development. Biological invasion has become a problem of widespread concern to countries around the world. Therefore, preventing the invasion of alien pests is an important guarantee for achieving sustainable development of agriculture and forestry and maintaining national ecological security. The list of quarantine pests is an important law enforcement basis and work guide for port quarantine. Establishing an accurate and reliable voucher sample screening process is crucial for the accurate and rapid identification of species intercepted at ports. DNA barcodes are a molecular species identification technology that uses a standard, short DNA fragment in the genome to identify species. It has broad application prospects and certain application potential in the field of species identification. With the help of a large-scale database, it can achieve rapid and accurate identification of species in large quantities.

[0003] However, the traditional morphological identification methods of quarantine pests at ports still have many problems, especially the difficulty in confirming and tracing the voucher specimens in the quarantine inspection of imported bulk grains and forest trees. For example: the screening of biological model specimens is not standardized; the voucher sample information of the morphological and molecular characterization of pests is inaccurate; the species information and quality control related to the inspection are inaccurate, etc.

[0004] Based on this, it is urgent to improve the inspection sample screening method for imported quarantine species to meet the sample screening needs for port inspection of imported biological species. Summary of the invention

[0005] In view of this, in order to solve the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a method and system for screening inspection samples of imported quarantine species so as to establish a voucher sample determination procedure with the help of molecular evolution and phylogenetic analysis technology, molecular species definition and other methods; and in combination with biological model specimens, the morphology and molecular characterization of quarantine pests are standardized from the source, and a technical process for scientific screening of voucher specimens is established, authoritative voucher specimen information of pests and relevant information data related to detection quality control are provided, and accurate, authoritative and high-quality voucher sample information is provided for the biological measurement of the detection object, and at the same time, accurate species information and detection-related measurement, standards and other quality control information can be provided for the research and development of detection technology, so as to provide accurate and traceable information resources for improving port detection technology.

[0006] The technical solution adopted by the present invention is: a method for screening inspection samples of imported quarantine species, the method comprising:

[0007] S1: Collect gene sequences of quarantine species in public databases, obtain key DNA barcode sequences by screening DNA barcode types, and store them as sequence data in fasta format;

[0008] S2: Determine the host range of each quarantine species through the public database, and enter the morphological characteristics and DNA barcodes of each quarantine species to obtain the test specimen data;

[0009] S3: After sequence alignment between the sequence data and the test specimen data, a phylogenetic tree was constructed through maximum likelihood analysis and the support rate of each node of the ML tree was obtained;

[0010] S4: Use phylogenetic trees to classify and define quarantine species;

[0011] S5: Obtain the voucher specimen of the quarantine species based on the specimen information of the quarantine species that has been classified and defined.

[0012] Furthermore, in S1, after obtaining the key DNA barcode sequences, DNA barcode sequences without specimen or strain information are eliminated.

[0013] Furthermore, in S3, sequence alignment is performed based on MEGA software, and the sequence alignment method includes:

[0014] S301: Read sequence data in fasta format;

[0015] S302: After selecting the test sample data, set the alignment parameters to perform sequence alignment;

[0016] S303: Export the aligned sequence file.

[0017] Furthermore, the sequence file includes: sequence similarity, gap number, and mismatch number.

[0018] Furthermore, in S3, maximum likelihood analysis is performed by IQ-TREE software, and the support rate of each node of the ML tree is obtained after calculation by the GTR-GAMMA model in the IQ-TREE software.

[0019] Furthermore, in S4, the phylogenetic tree is analyzed based on PTP analysis, the quarantine species are defined, and the specimen information to which the quarantine species belongs is output. The specimen information clarifies the certificate specimen of the quarantine species based on the type specimen.

[0020] The present invention also discloses a test sample screening system for imported quarantine species, which is used to run the above test sample screening method, and the system comprises:

[0021] A data processing module, which is used to collect gene sequences of quarantine species in a public database, obtain key DNA barcode sequences by screening DNA barcode types, and store them as sequence data in fasta format;

[0022] A model specimen input module, which determines the host range of each quarantine species through a public database, and inputs the morphological characteristics and DNA barcode of each quarantine species to obtain test specimen data;

[0023] A data analysis module, which is used to perform sequence comparison between the sequence data and the test specimen data, construct a phylogenetic tree through maximum likelihood analysis, and obtain the support rate of each node of the ML tree;

[0024] A species definition module, wherein the species definition module uses a phylogenetic tree to divide and define quarantine species;

[0025] A voucher specimen generation module, wherein the voucher specimen generation module is used to output the voucher specimen of the quarantine species.

[0026] The beneficial effects of the present invention are:

[0027] 1. The method and system for screening inspection samples of imported quarantine species provided by the present invention apply biosystematics and phylogenetics to determine the voucher specimens of quarantine species, especially to clarify the voucher specimens of some important quarantine fungi and their closely related species, and use accurate and authoritative voucher specimens as a reliable source of information for pest detection and identification, which can not only provide an important reference for port detection and identification, but also provide important information guarantee for improving the quality of epidemic prevention detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is an overall flow chart of the inspection sample screening method for imported quarantine species. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] like Figure 1 As shown, this embodiment specifically provides a method for screening inspection samples of imported quarantine species, the method comprising:

[0032] S1: Collect gene sequences of quarantine species in public databases, obtain key DNA barcode sequences by screening DNA barcode types, remove DNA barcode sequences without specimen or strain information after obtaining the key DNA barcode sequences, process the sequences, and store them in fasta format. Specifically, in S1, the public database refers to the gene sequences of quarantine fungi in public databases such as GenBank and BOLD and references.

[0033] S2: Determine the host range of each quarantine species through public databases, and enter the morphological characteristics and DNA barcodes of each quarantine species to obtain test specimen data. Specifically, in S2, the public database refers to the database obtained through literature research and reference to USDA, GBIF, CABI and other databases.

[0034] S3: After sequence alignment between the sequence data and the test specimen data, the maximum likelihood analysis was performed using the IQ-TREE software and a phylogenetic tree was constructed. After calculation using the GTR-GAMMA model in the IQ-TREE software, a bootstrap test was used. After 1000 repeated sampling tests, the support rate of each node of the ML tree was obtained. Here, a high support rate is related to species confirmation. A support rate greater than 75% generally indicates support, and the species can be confirmed. Specifically, the above is based on sequence alignment using the MEGA software, and the sequence alignment method includes:

[0035] S301: Read sequence data in fasta format;

[0036] S302: After selecting the test sample data, set the alignment parameters to perform sequence alignment, check and cut the sequence alignment results, and perform manual correction if necessary; wherein setting the alignment parameters includes: selecting the alignment algorithm and method, and setting the corresponding parameters;

[0037] S303: Export the aligned sequence file, which is saved as a fasta, Nexus or Phylip format file. The sequence file includes: sequence similarity, number of gaps, and number of mismatches. Among them, the number of gaps refers to the missing area in the sequence, that is, a base or amino acid is missing at a certain position in a sequence. The introduction of gaps is usually to optimize the similarity between sequences, especially in multiple sequence alignments, where gaps can help adjust the alignment between sequences so that they are more matched in specific areas. The number of mismatches refers to the number of mismatches between actual sequencing results and expected results in bioinformatics, especially in genome sequencing and variation detection. The number of mismatches is crucial for evaluating sequencing quality, detecting variations, and subsequent bioinformatics analysis.

[0038] When constructing a phylogenetic tree, the above steps specifically include: (1) defining the root of the tree, adjusting the order of branches, changing the width of the tree, and changing the distance between branches; (2) changing the length of the root of the tree, changing the radius of the corners, and selecting whether the species labels are aligned; (3) selecting the thickness of the branch lines, changing the ladder shape and the length of the branches, and adjusting the branch length to align with solid lines; (4) calculating the support rate of the ML tree through the tree file; and (5) outputting an image.

[0039] S4: Use phylogenetic trees to divide and define quarantine species; specifically, the maximum likelihood tree obtained by IQ-TREE software analysis is used as the most dominant topological structure and combined with the support rate of each node of the ML tree to determine the species and obtain the species.

[0040] S5: According to the specimen information of the quarantine species that has been divided and defined, the voucher specimen of the quarantine species is obtained. Specifically, the phylogenetic tree is analyzed based on PTP analysis (i.e., Poisson tree processes analysis (PTP) of the number of base substitutions), and the specimen information of the quarantine species is output. The specimen information is used as a benchmark to identify the voucher specimen of the quarantine species, that is, samples on the same phylogenetic tree branch as a certain pest model specimen can be identified as the same species, thus identifying the voucher specimen of the quarantine species.

[0041] Example 2

[0042] Based on the inspection sample screening method for entry quarantine species in Example 1, this embodiment further provides an inspection sample screening system for entry quarantine species, which runs the inspection sample screening method provided in Example 1. Specifically, the system includes: a central processor, which is respectively communicated with a data processing module, a model specimen input module, a data analysis module, a species definition module and a voucher specimen generation module, specifically:

[0043] The data processing module is used to collect gene sequences of quarantine species in public databases, obtain key DNA barcode sequences by screening DNA barcode types, and store them as sequence data in fasta format;

[0044] The model specimen input module determines the host range of each quarantine species through a public database, and inputs the morphological characteristics and DNA barcode of each quarantine species to obtain the inspection specimen data;

[0045] After the data analysis module calls the data processing module and the model specimen input module, the sequence data is compared with the test specimen data, and then a phylogenetic tree is constructed through maximum likelihood analysis to obtain the support rate of each node of the ML tree;

[0046] The species definition module uses a phylogenetic tree to divide and define quarantine species;

[0047] After receiving the species definition information from the species definition module, the voucher specimen generation module analyzes it through an online server (the online server is loaded with a Poisson tree process analysis algorithm based on the number of base substitutions), and outputs the voucher specimen of the quarantine species after analysis.

[0048] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for screening samples of quarantine species entering the country, characterized in that: The method includes: S1: Collect gene sequences of quarantine species in public databases, obtain key DNA barcode sequences by screening DNA barcode types, and store them as sequence data in fasta format; S2: Determine the host range of each quarantine species through the public database, and enter the morphological characteristics and DNA barcodes of each quarantine species to obtain the test specimen data; S3: After sequence alignment between the sequence data and the test specimen data, a phylogenetic tree was constructed through maximum likelihood analysis and the support rate of each node of the ML tree was obtained; S4: Use phylogenetic trees to classify and define quarantine species; S5: Obtain the voucher specimen of the quarantine species based on the specimen information of the quarantine species that has been classified and defined.

2. The method for screening samples of quarantine species for entry into the country according to claim 1, characterized in that: In S1, after obtaining the key DNA barcode sequences, DNA barcode sequences without specimen or strain information are eliminated.

3. The method for screening samples of quarantine species for entry into the country according to claim 1, characterized in that: In S3, sequence alignment is performed based on MEGA software, and the sequence alignment method includes: S301: Read sequence data in fasta format; S302: After selecting the test sample data, set the alignment parameters to perform sequence alignment; S303: Export the aligned sequence file.

4. The method for screening samples of quarantine species for entry into the country according to claim 1, characterized in that: The sequence file includes: sequence similarity, gap number, and mismatch number.

5. The method for screening samples of quarantine species for entry into the country according to claim 1, characterized in that: In S3, the maximum likelihood analysis is performed by IQ-TREE software, and the support rate of each node of the ML tree is obtained after calculation by the GTR-GAMMA model in the IQ-TREE software.

6. The method for screening samples of quarantine species for entry into the country according to claim 1, characterized in that: In the S4, the phylogenetic tree is analyzed based on the PTP analysis, the quarantine species are defined, and the specimen information to which the quarantine species belongs is output. The specimen information clarifies the certificate specimen of the quarantine species based on the type specimen.

7. A system for screening samples of quarantine species entering the country, the system running the method for screening samples of quarantine species entering the country as claimed in any one of claims 1 to 6, the system comprising: A data processing module, which is used to collect gene sequences of quarantine species in a public database, obtain key DNA barcode sequences by screening DNA barcode types, and store them as sequence data in fasta format; A model specimen input module, which determines the host range of each quarantine species through a public database, and inputs the morphological characteristics and DNA barcode of each quarantine species to obtain test specimen data; A data analysis module, which is used to construct a phylogenetic tree by maximum likelihood analysis after sequence alignment of the sequence data and the test specimen data, and obtain the support rate of each node of the ML tree; A species definition module, wherein the species definition module uses a phylogenetic tree to divide and define quarantine species; A voucher specimen generation module, wherein the voucher specimen generation module is used to output the voucher specimen of the quarantine species.

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