Method for rapidly screening high-frequency cross-border fungi based on high-throughput sequencing
Through a fast screening method based on high-throughput sequencing, the problems of long time, slow speed and inaccurate identification in cross-border fungi detection are solved, and the rapid and accurate screening of high-frequency cross-border fungi are achieved, which improves detection efficiency and accuracy.
Patent Information
- Application Number
- CN202510054561.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art has problems such as long time, slow speed, and inaccurate identification in cross-border fungi detection. Especially when the traditional fungal bacterial activity determination methods are time-consuming and dormant bacteria are inaccurate, there is a lack of traceability technology for molecular phylogenetics and a method for identifying fungi, bacteria and other organisms in one go.
The rapid screening method based on high-throughput sequencing, including DNA extraction, data automation analysis and library preparation, is used to identify common fungi through high-throughput amplicon sequencing, which is suitable for the high-throughput rapid identification of common plant pathogenic fungi in imported plants and plant products.
The rapid screening of high-frequency cross-border fungi was achieved, and the result analysis time was shortened to 1 hour, and thousands of pathogenic fungi were able to be screened at the same time, improving the accuracy and efficiency of detection.
Smart Images
Figure CN119979751A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fungus detection, and in particular relates to a method for rapidly screening high-frequency cross-border fungi based on high-throughput sequencing. Background Art
[0002] Cross-border organisms refer to foreign organisms that enter my country through inbound goods, containers, vehicles, and belongings of inbound passengers. my country intercepts a large number of cross-border fungi, bacteria, viruses, insects, and vectors every year. Among these massive amounts of intercepted cross-border organisms, there are still a large number of species that cannot be identified in a timely and accurate manner. Countries around the world are also facing the same problem, so they have all carried out research on cross-border biological detection technology.
[0003] The technical methods currently used mainly include morphology, immunology and molecular biology, and many technologies have been established, but these technologies still have some shortcomings: they mostly target a single target, take a long time, are slow, and lead to inaccurate identification.
[0004] Moreover, the traditional methods for determining the activity of fungi and bacteria are time-consuming, and the determination of the activity of dormant bacteria is inaccurate; there is a lack of molecular phylogenetic traceability technology, and the sources of the same fungi and bacteria are unclear; there is no method to simultaneously identify fungi, bacteria and other organisms carried by seedlings. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing, including DNA extraction and automated data analysis, as well as library preparation methods, and high-throughput amplicon sequencing for common fungal identification, which is suitable for high-throughput rapid identification of common plant pathogenic fungi in imported plants and plant products, and can simultaneously realize the identification of multiple quarantine or high-frequency cross-border plant pathogenic fungi. The results are analyzed within 1 hour, and thousands of pathogenic fungi can be screened at the same time.
[0006] A method for rapidly screening high-frequency cross-border fungi based on high-throughput sequencing in the present invention to solve the above technical problems comprises the following steps:
[0007] (1) Obtain imported plant samples and extract epiphytic microorganisms;
[0008] (2) DNA extraction;
[0009] (3) PCR amplification: Two rounds of PCR amplification were performed on the internal transcribed spacer ITS1 region of the fungal total DNA using primer pairs;
[0010] (4) Purification: All PCR products were purified using the GeneJET Gel Extraction Kit before sequencing;
[0011] (5) High-throughput sequencing: The obtained cDNA library was sequenced using the Illumina MiSeq system, the sequencing mode was PE300, the library denaturation concentration was 2 nM, the loading concentration was 20 pM, and the high-throughput sequencing results were analyzed by bioinformatics;
[0012] (6) Perform automated data filtering and statistics and automated OTUs clustering analysis on the sequencing results.
[0013] The results of OTUS clustering annotations contain the names of quarantined species. The relevant results are qualitative tests, not quantitative tests.
[0014] In the present invention, Illumina NEBNext, Ultra DNA Library Prep Kit is used for library construction, the constructed library is quantified by Qubit and Q-PCR, and high-throughput amplicon sequencing is used for common fungal identification.
[0015] The extraction steps of epiphytic microorganisms in step (1) are as follows:
[0016] For each sample, a seed sample was taken and placed in a container filled with peptone buffered water, placed in a conical flask, and shaken at 150 rpm for 1 hour; the liquid was then transferred to a high-pressure steam centrifuge tube and centrifuged at 10,000 g / min for 15 min, and the supernatant was removed; finally, all the sediment at the bottom of the centrifuge tube was transferred to a smaller centrifuge tube, and continued to be centrifuged at 4,000 g / min for 20 min, and the final sediment was collected for subsequent DNA extraction; the mass ratio of seed sample to peptone buffered water was 25:225.
[0017] The DNA extraction in step (2) is as follows:
[0018] Transfer all collected epiphytic microorganisms to The Lysing Matrix E tube in the Spin kit (MP Biomedicals, Solon, OH, USA) was added with sodium phosphate buffer and MT buffer to the Lysing Matrix E Tube sample, followed by mixing and centrifugation; the volume ratio of sodium phosphate buffer to MT buffer was 978:122.
[0019] After centrifugation, the epiphytic microorganisms were directly placed on Spin kit, then break and centrifuge, transfer the obtained supernatant to a centrifuge tube, add PPS reagent to mix and centrifuge; transfer the supernatant to a clean 15ml tube, add 1ml of mixed Binding Matrix suspension, vortex or manually mix for 2min to bind DNA, let stand for 3min to precipitate the silica matrix, and then carefully remove the upper 500μL supernatant to avoid touching the precipitate;
[0020] Discard the supernatant, resuspend the remaining Binding Matrix in the tube, transfer the mixture to the SPIN Filter, centrifuge, empty the Catch Tube, and centrifuge and empty the remaining mixture;
[0021] Add SEWS-M to the SPIN Filter, centrifuge and pour off the filtrate, put the SPIN Filter back into the Catch Tube, centrifuge again to remove the remaining SEWS-M, put the SPIN Filter into a new Catch Tube, and air-dry at room temperature;
[0022] Finally, add DES solution and gently resuspend. The resuspending solution is a highly efficient DNA elution solution. Centrifuge, discard the SPINFilter, and store the final solution at -80°C. The volume ratio of SEWS-M and DES solution is 10:1-2.
[0023] In the first round of amplification in step (3), a primer pair and a highly efficient Takara high-fidelity enzyme or Invitrogen Platinum SuperFi DNA polymerase are used for PCR and 10 ng of template DNA is used for the first round of PCR amplification.
[0024] The primer pair in step (3) is a specific primer with a barcode, and the specific sequence is as follows:
[0025] ITS1F primer: 5′-CTTGGTCATTTAGAGGAAGTAA-3′;
[0026] ITS2R primer: 5′-GCTGCGTTCTTCATCGATGC-3′.
[0027] The reaction conditions and procedures of the amplification are as follows: first, pre-denaturation at 95° for 2 minutes, then reaction at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, for a total of 25 cycles; finally, incubation at 72°C for 10 minutes, and termination of the reaction at 4°C.
[0028] The cDNA library construction steps in step (5) are:
[0029] A. Connect the "Y" shaped connector;
[0030] B. Use magnetic beads to screen and remove the self-ligated fragments;
[0031] C. Enrichment of library templates using PCR amplification;
[0032] D. Denaturation with sodium hydroxide produces single-stranded DNA fragments.
[0033] The automated data filtering statistics in step (6) include filtering, removal of connector contamination and downstream processing of data.
[0034] Specifically:
[0035] (1) Filtering: Filter the original sequence to obtain high-quality Clean Reads; the original sequence is filtered using QIIME2 software.
[0036] (2) Remove adapter contamination: For paired-end sequencing, if one end is contaminated by adapters, remove the reads at both ends; remove low-quality reads; remove reads with an N ratio greater than 5%; if for paired-end sequencing, if one end contains an N ratio greater than 5%, remove the reads at both ends; use QIIME2 software to automatically remove.
[0037] The low-quality reads are specifically: the bases with a quality value Q≤19 in the reads account for more than 15% of the total bases. For double-end sequencing, if one end is a low-quality read, the reads at both ends will be removed.
[0038] (3) Downstream data processing: Use the fastq_filter command in USEARCH 10.0 software to screen the quality of paired-end sequences in FASTQ format one by one, filter out low-quality sequences, and then use the fastq_mergepairs command in USEARCH 10.0 software to merge paired-end sequences. The overlapping base length of Read 1 and Read 2 is required to be ≥10 bp, and base mismatches are not allowed.
[0039] The sequence in step (3) requires an average base quality ≥ Q20, that is, an average base sequencing accuracy ≥ 99%.
[0040] The OTUs clustering analysis step in step (6) is as follows: all sequences excluding chimeras are clustered and divided into OTUs using the UPARSE (≥97%) clustering algorithm, and the sequence with the highest abundance in each OTU is selected as the representative sequence of the OTU. Finally, all representative sequences are annotated with species using the RDP classification algorithm. During the annotation process, the UNITE fungal ITS database and the pathogenic fungi core database are selected for comparison, and the final annotation results of the two databases are compared. After the species annotation is completed, the sequences that cannot be clustered into known groups at any classification level are defined as unclassified.
[0041] In the present invention, the fungal internal transcribed spacer (ITS) is a non-functional RNA sequence of the structural RNA in the genome. The ITS sequence is a non-functional fragment in the entire genome, and is subject to less evolutionary selection pressure. It has a large number of copies, making it easy to amplify, and ITS also has high variability in similar fungal groups, which can effectively distinguish similar species. It is also the most widely accepted indicator of fungal phylogeny and classification barcodes.
[0042] The present invention is suitable for high-throughput rapid identification of common plant pathogenic fungi in imported plants and plant products, and can realize the preliminary screening of various plant pathogenic fungi, and is particularly suitable for screening pathogenic fungi on seeds, pathogenic fungi in plant tissues, or fungi in other materials. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 This is a flow chart of high-throughput data analysis in the present invention. DETAILED DESCRIPTION
[0044] The present invention is further described below in conjunction with specific embodiments:
[0045] Main instruments: centrifuge, FastPrep rapid nucleic acid extraction instrument, gel electrophoresis instrument, PCR instrument and Illumina MiSeq system.
[0046] Main reagents: distilled water, peptone, High-Fidelity PCR Master Mix and agarose;
[0047] Reagent test kit: Spin Kit, GeneJET Gel Extraction Kit, Ultra DNA LibraryPrep Kit.
[0048] Example 1
[0049] High-throughput screening consists of the following steps:
[0050] (1) Sample processing: Select imported sorghum seed samples, take 500 grams of each sample, and transport it back to the laboratory.
[0051] And extract epiphytic microorganisms: take 25g of seed sample from each sample and put it into 225ml peptone buffer water, put it into a 500ml conical flask, and shake it at 150 rpm for 1 hour. Then transfer the liquid to a 500ml high-pressure steam centrifuge tube and centrifuge it at 10000g / min for 15min, and remove the supernatant. Finally, transfer all the sediment at the bottom of the 500ml centrifuge tube to a 50ml centrifuge tube, continue centrifugation at 4000g / min for 20min, and collect the final sediment for DNA extraction.
[0052] (2) DNA extraction: All collected epiphytic microorganisms were transferred to The samples were placed in the Lysing Matrix E tube in the Spin kit (MP Biomedicals, Solon, OH, USA), and 978 μL sodium phosphate buffer and 122 μL MT buffer were added to the Lysing Matrix E Tube sample. The samples were then mixed at a speed of 6.0 for 40 s on the FastPrep instrument and centrifuged at 14,000 g for 10 min.
[0053] After centrifugation for 10 min, the epiphytic microorganisms were directly placed on In the Spin kit, the tube was broken 3 times at 6.0 speed for 40s on the FastPrep instrument, followed by centrifugation. The supernatant was transferred to a clean 2ml centrifuge tube, 250μL of PPS reagent was added, and the tube was shaken 10 times to mix, then centrifuged at 14000g for 5 minutes, and the supernatant was transferred to a clean 15ml tube, 1ml of mixed Binding Matrix suspension was added, vortexed or manually mixed for 2min to bind DNA, and allowed to stand for 3min to precipitate the silica matrix. Then the upper 500μL of supernatant was carefully removed to avoid touching the precipitate.
[0054] Discard the supernatant, resuspend the remaining Binding Matrix in the tube, and transfer about 600 μL of the mixture to the SPINFilter, centrifuge at 14000 g for 1 min, empty the Catch Tube, and add the remaining mixture to the SPIN Filter, centrifuge at 14000 g for 1 min, and empty.
[0055] Add 500 μL SEWS-M to the SPIN Filter, centrifuge at 14,000 g for 1 min, pour off the filtrate, put the SPIN Filter back into the Catch Tube, centrifuge again at 14,000 g for 2 min to remove the remaining SEWS-M, put the SPIN Filter into a new Catch Tube, and air-dry at room temperature for 5 min.
[0056] Finally, add 50-100 μL of DES solution and gently resuspend. The resuspending solution is a high-efficiency DNA elution solution. Centrifuge at 14000g for 1 min, discard the SPIN Filter, and store the final solution at -80℃.
[0057] (3) PCR amplification:
[0058] Take an appropriate amount of total DNA of epiphytic microorganisms of sorghum seeds in a centrifuge tube, dilute the sample to 1 ng / μL with sterile water, and use agarose gel electrophoresis to detect the purity and concentration of the DNA. Use the diluted total DNA of epiphytic microorganisms as a template as the sequencing region to reduce the inhibitory effect of the PCR reaction. Then use the primer pair ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3') to perform two rounds of PCR amplification on the internal transcribed spacer ITS1 region of the fungal total DNA. In the first round of amplification, use a specific primer with a barcode ( High-Fidelity PCR Master Mix with GC Buffer, New England Biolabs) and high-efficiency high-fidelity enzymes were used for PCR and 10 ng of template DNA was used for the first round of PCR amplification.
[0059] The amplification reaction conditions were as follows: first, pre-denaturation at 95° for 2 min, then reaction at 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s, for a total of 25 cycles. Finally, the reaction was incubated at 72°C for 10 min, and the reaction was terminated at 4°C.
[0060] In order to reduce the influence of sequencing errors caused by PCR amplification on the results, the present invention performs PCR repetitions 3 times on each sample and then mixes them into one sample.
[0061] The PCR product was detected by 2% agarose gel electrophoresis; equal amounts of samples were mixed according to the concentration of the PCR product, and after sufficient mixing, the PCR product was detected by 2% agarose gel electrophoresis, and the target band was recovered using the gel recovery kit provided by Qiagen.
[0062] Purification: All PCR products were purified using GeneJET Gel Extraction Kit (ThermoScientific) before sequencing. The purification steps were performed according to the kit.
[0063] High-throughput sequencing: Then use Illumina's NEBNext, Ultra DNA Library Prep Kit (New England Biolabs, USA) library construction kit to construct the library, A. Connect the "Y"-shaped adapter; B. Use magnetic beads to screen and remove the adapter self-connected fragments; C. Use PCR amplification to enrich the library template; D. Denature with sodium hydroxide to produce single-stranded DNA fragments. The constructed library is quantified by Qubit and Q-PCR. After the library is qualified, it is sequenced on the Illumina MiSeq system (Illumina Inc., San Diego, CA, USA). Then the bioinformatics analysis of high-throughput sequencing results:
[0064] In the screening method of the present invention, different software processes are integrated together in the bioinformatics analysis step, and automated data analysis can be achieved after scripting.
[0065] The system requirements and installed software list required for the entire data analysis process are shown in Table 1:
[0066] Table 1 Software required for high-throughput data analysis
[0067]
[0068] The data analysis process is shown in the following table:
[0069] Table 2 High-throughput data analysis codes
[0070]
[0071]
[0072]
[0073]
[0074] The results of the Illumina sequencer are initially stored in the computer as raw image data files, which are converted into raw sequencing sequences after base recognition by CASAVA software, generally referred to as Raw Data or Raw Reads. The results are stored in the FASTQ (abbreviated as fq) file format.
[0075] Each sequence in the FASTQ file corresponds to 4 lines of information, including the name of each sequencing sequence (Read), the base sequence, and its corresponding sequencing quality information. In the FASTQ format file, each base corresponds to a base quality character, and the ASCII code value corresponding to each base quality character minus 33 (Sanger quality value system) is the sequencing quality score of the base. Different Phred Quality Scores represent different base sequencing error rates. For example, scores of 20 and 30 represent base sequencing error rates of 1% and 0.1%, respectively.
[0076] The data filtering and statistical steps are as follows:
[0077] (1) Filtering: Since some original sequences carry adapter sequences or contain a small amount of low-quality sequences, in order to ensure the quality of information analysis data, the present invention filters the original sequences to obtain high-quality Clean Reads, and then performs subsequent analysis. Subsequent analysis is based on the filtered clean data.
[0078] (2) Removal of adapter contamination: Adapter contamination refers to the presence of sequencing primer sequences in some read sequences due to the insert being too short. For paired-end sequencing, if one end is contaminated by adapters, the reads at both ends will be removed; remove low-quality reads; remove reads with an N ratio greater than 5%; if for paired-end sequencing, if one end contains an N ratio greater than 5%, the reads at both ends will be removed.
[0079] Low-quality reads are specifically defined as those in which the bases with a quality value of Q≤19 account for more than 15% of the total bases. For paired-end sequencing, if one end is a low-quality read, both ends of the read will be removed.
[0080] All the fungal ITS1 raw data obtained by sequencing were analyzed using USEARCH 10.0 software of the present invention. According to the Index information corresponding to each sample (i.e., Barcode sequence, which is a short base sequence at the beginning of the sequence used to identify the sample), the connected sequence identification was assigned to the corresponding sample (the Index sequence was required to match completely), thereby obtaining the valid sequence of each sample.
[0081] The corresponding Index sequence is a random short sequence, and the sequencing result in this example is the second-generation amplicon sequencing data.
[0082] (3) Downstream data processing: Use the fastq_filter command in USEARCH 10.0 software to screen the quality of paired-end sequences in FASTQ format one by one, filter out low-quality sequences, and then use the fastq_mergepairs command in USEARCH 10.0 software to merge paired-end sequences. The overlapping base length of Read 1 and Read 2 is required to be ≥10 bp, and base mismatches are not allowed.
[0083] The sequence requires the average base quality ≥ Q20, that is, the average sequencing accuracy of the bases ≥ 99%.
[0084] During PCR amplification and high-throughput sequencing, a series of errors or chimera sequences may be generated. In order to ensure the reliability and accuracy of the analysis results, the effective sequences extracted above must be further screened and removed for error sequences and chimera sequences, so as to obtain the correct sequence that can be used for subsequent analysis. First, the UCHIME command in USEARCH 10.0 software is used to compare the UNITE CHIME database to search for chimeras, and then the chimera sequences are eliminated through the chimera sequence information found.
[0085] The processing steps of filtering, removing joint contamination, and downstream processing of data in the present invention are the process of amplicon data analysis. The present invention uses scripted codes to realize the automation of these series of processing processes.
[0086] The steps of automated OTUs clustering analysis are as follows:
[0087] The full name of OTUs is taxonomic operational unit, which refers to a collection of similar sequences. Generally, sequences with a sequence similarity greater than 97% are classified as OTUs internationally, that is, these sequences are considered to belong to the same species in taxonomy. The present invention selects representative sequences as representative sequences of the species to facilitate the next step of species annotation and statistical analysis.
[0088] The present invention uses the UPARSE (≥97%) clustering algorithm to cluster and divide OTUs for all sequences excluding chimeras, and selects the sequence with the highest abundance in each OTU as the representative sequence of the OTU. Finally, the RDP classification algorithm is used to annotate all representative sequences with species. During the annotation process, the present invention selects the UNITEfungal ITS database and the pathogenic fungi core database constructed by the present invention for comparison, and compares the final annotation results of the two databases. After completing the species annotation, the sequences that cannot be clustered into known groups at any classification level are defined as unclassified (Unassigned).
[0089] The result after OTUs clustering analysis is to reduce the dimension of the sequencing results according to the similarity. OTUs clustering analysis is an important process step in the method of the present invention.
[0090] In the present invention, the UPARSE clustering algorithm and the RDP classification algorithm are conventional algorithms, and the UNITE fungal ITS database, a core database of major pathogenic fungi, is a public database of fungal annotations.
[0091] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.
Claims
1. A method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing, characterized in that: The following steps are involved: (1) Obtain imported plant samples and extract epiphytic microorganisms; (2) DNA extraction; (3) PCR amplification: Two rounds of PCR amplification of the internal transcribed spacer ITS1 region of the fungal total DNA were performed using primer pairs; (4) Purification: All PCR products were purified before sequencing; (5) High-throughput sequencing: The obtained cDNA library was sequenced using the Illumina MiSeq system, the sequencing mode was PE300, the library denaturation concentration was 2 nM, the loading concentration was 20 pM, and the high-throughput sequencing results were analyzed by bioinformatics; (6) Automated data filtering statistics and automated OTUs clustering analysis are sufficient.
2. A method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1, characterized in that: The extraction steps of epiphytic microorganisms in step (1) are as follows: Take a seed sample from each sample and put it into a container filled with peptone buffered water, put it into a conical flask, and shake it at 150 rpm for 1 hour; then transfer the liquid to a high-pressure steam centrifuge tube and centrifuge it at 10,000 g / min for 15 minutes, and remove the supernatant; finally, transfer all the sediment at the bottom of the centrifuge tube to a smaller centrifuge tube, continue centrifugation at 4,000 g / min for 20 minutes, and collect the final sediment for subsequent DNA extraction; The mass ratio of seed sample to peptone buffer water is 25:
225.
3. According to claim 1, a method for rapid screening of high-frequency cross-border fungal DNA extraction and automated data analysis based on high-throughput sequencing, characterized in that: In the first round of amplification in step (3), a primer pair, Takara high-fidelity enzyme or Invitrogen Platinum SuperFi DNA polymerase are used for PCR and 10 ng of template DNA are used for the first round of PCR amplification.
4. A method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1 or 2, characterized in that: The primer pair is a specific primer pair with a barcode, and the specific primer pair is: ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3').
5. The method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1, characterized in that: The reaction conditions and procedures for amplification in step (3) are as follows: first, pre-denaturation at 95° for 2 min, then reaction at 95°C for 30 s, reaction at 55°C for 30 s, and reaction at 72°C for 60 s, for a total of 25 cycles; finally, incubation at 72°C for 10 min, and termination of the reaction at 4°C.
6. The method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1, characterized in that: The cDNA library construction steps in step (5) are: A. Connect the "Y" shaped connector; B. Use magnetic beads to screen and remove the self-ligated fragments; C. Enrichment of library templates using PCR amplification; D. Denaturation with sodium hydroxide produces single-stranded DNA fragments.
7. The method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1, characterized in that: The automated data filtering statistics of step (6) include filtering, removal of connector contamination and downstream processing of data.
8. The method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1, characterized in that: The automated OTUs clustering analysis steps in step (6) are as follows: All sequences except chimeras were clustered and divided into OTUs using the UPARSE clustering algorithm, and the sequence with the highest abundance in each OTU was selected as the representative sequence of the OTU. Finally, all representative sequences were annotated with species using the RDP classification algorithm. During the annotation process, the UNITE fungal ITS database and the pathogenic fungi core database were selected for comparison, and the final annotation results of the two databases were compared. After completing the species annotation, sequences that could not be clustered into known groups at any classification level were defined as unclassified.
9. The method for rapid screening of high-frequency cross-border fungi based on high-throughput sequencing according to claim 1, characterized in that: The cross-border fungi are pathogenic fungi on seeds, pathogenic fungi in plant tissues or fungi in other materials.