A method and system for designing sequencing primers targeting pathogenic microorganisms

Abstract

The present invention discloses a method and system for designing primers for sequencing targeted pathogenic microorganisms, the method comprising the following steps: step 1) obtaining genome data of pathogenic microorganisms and establishing a pathogenic microorganism genome database; step 2) obtaining the conserved regions of pathogenic microorganisms according to the pathogenic microorganism genome database; step 3) designing multiple primers according to the conserved regions in step 2); step 4) evaluating the specificity and inclusiveness of the multiple primers designed in step 3), and selecting multiple primers with specificity and inclusiveness of ≥90%. The primer set obtained in the present invention can detect multiple strains of pathogens at the same time, and the primer detection results are more accurate than those obtained in the prior art.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a design method and system for sequencing primers targeting pathogenic microorganisms. Background Art

[0002] Targeted pathogen genome sequencing is an emerging technology used in clinical pathogen infection detection. It has the advantages of wide range of pathogen detection, high sensitivity, high accuracy, and fast timeliness. It is gradually becoming a frontline clinical pathogen infection detection method. One of the core technologies of targeted pathogen genome technology is the primer design of pathogenic microorganisms. The quality of primers directly affects the number of pathogenic microorganisms detected, accuracy, and analytical performance of targeted pathogen genome detection.

[0003] At present, most targeted genome primer design processes are to select one strain as the representative genome of the species randomly or through clustering analysis, or to include all strains in the database without selection. The advantage of the method of selecting only one strain as a representative is that the amount of data in the constructed database is relatively small and the analysis time is fast, but the disadvantage is also prominent. Because microbial genomes evolve rapidly, there are differences in the genomes of strains of the same species. The genomes sequenced from different regions, different times, or even different strains isolated at the same time may differ. The genome differences between strains of some rapidly evolving species can reach more than 3%.

[0004] The method of incorporating all strain genomes of the same species into the database can effectively avoid this kind of missed detection, but the disadvantages of this method are also particularly obvious. On the one hand, after incorporating all strain genomes, the amount of database data becomes very large, resulting in a long analysis time, even requiring more than one day, which is unacceptable for the timeliness requirements of clinical applications. At the same time, the resource requirements for computing servers or clusters are also greatly increased, and the analysis cost is greatly increased; on the other hand, the sequencing quality of strain genomes from public databases varies, and some strains contain contaminated sequences or even misclassified strains. If they are not screened and filtered, it is easy to cause false positive results, which brings great trouble to clinical diagnosis and treatment. Summary of the invention

[0005] The present application proposes a method and system for designing primers for sequencing targeted pathogenic microorganisms to solve the problems of low efficiency and insufficient accuracy of primer design methods in the prior art.

[0006] A method for designing primers targeting pathogenic microorganism sequencing comprises the following steps:

[0007] Step 1) Obtaining genome data of pathogenic microorganisms and establishing a pathogenic microorganism genome database;

[0008] Step 2) obtaining the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database;

[0009] Step 3) designing multiple primers based on the conserved regions in step 2);

[0010] Step 4) Evaluate the specificity and inclusiveness of the multiplex primers designed in step 3), and select multiplex primers with specificity and inclusiveness ≥ 90%.

[0011] Optionally, the method for establishing a pathogenic microorganism genome database in step 1) includes:

[0012] For each targeted pathogenic microorganism species for which primers need to be designed, screen out genomes that meet the following criteria:

[0013] The N50 value of the genome is greater than or equal to the median of the N50 values ​​of all genome data;

[0014] The N content in the genome is less than or equal to the median of the N content in all genomes;

[0015] The results calculated by the genome fastANI program were ≥95;

[0016] Filtering: Perform ANI analysis and checkM analysis on the selected genomes, remove genomes with at least one score less than 95, remove genomes with incorrect annotation information, incomplete chromosome assembly, and incorrect classification, and obtain high-quality genomes of the species;

[0017] Construct a genome index: Merge the high-quality genomes of each targeted pathogenic microorganism species for which primers need to be designed, and then create an index for the merged data set.

[0018] Optionally, use bwa and makeblast to index the merged dataset;

[0019] Optionally, the step 1) further comprises a library: for each pathogenic microorganism for which primers are to be designed, repeating the above steps.

[0020] Optionally, the method for obtaining the conserved region sequence of the pathogenic microorganism in step 2) comprises:

[0021] Extract the common conserved regions in the pathogenic microorganism genomes from the original microorganism genome database;

[0022] Specificity assessment: blast was used to align the conserved regions to the NT database, specificity analysis was performed on the conserved regions, and regions with specificity scores less than 95 were filtered out;

[0023] Inclusiveness assessment: blast was used to align the conserved regions to the high-quality merged gene set, and inclusiveness analysis was performed on the conserved regions, filtering out regions with inclusiveness scores less than 95.

[0024] Optionally, in step 3), a fragment with a length of the conserved region in the range of 200 to 1000 bp is selected to design primers;

[0025] The setting parameters are: primer length: 18-28bp; GC content: 30%-70%; annealing temperature: 60-66℃;

[0026] Preferably, primer length: 22 bp, GC content: 50%, temperature: 63°C.

[0027] Optionally, if primers cannot be generated within the set parameter range, the parameters are set as follows: primer length: 17-30 bp; GC content: 20%-80%; annealing temperature: 58-67°C.

[0028] Optionally, the specificity evaluation method in step 4) is: using the NT library to compare and screen the primers, the screening conditions are that the last base of the sequence is aligned, and the total number of unmatched bases and mismatched bases is less than or equal to 2; the specificity is calculated using the following formula:

[0029] Specificity = the number of species of the pathogenic microorganisms screened / the number of all species screened in the NT library;

[0030] The method of inclusiveness evaluation is: the pathogenic microorganism genome database established in step 1) is used to screen the primer pairs composed of each primer, and the amplicon results are obtained with the number of mismatches not exceeding 2 and no mismatch at the 3' end as the screening conditions; inclusiveness is calculated using the following formula:

[0031] Inclusiveness = the number of amplicon result genomes obtained / the number of all genomes in the pathogen genome database.

[0032] The present invention also provides multiple primers obtained according to the above method.

[0033] Optionally, the multiple primers are used for targeting pathogen genome sequencing of Clostridium tetani, and the multiple primers include at least one of the following primer pairs:

[0034] Primer pair 1, the nucleotide sequence of the upstream primer of primer pair 1 is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer of primer pair 1 is shown as SEQ ID NO.2;

[0035] Primer pair 2, the nucleotide sequence of the upstream primer of primer pair 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer of primer pair 2 is shown in SEQ ID NO.4;

[0036] Primer pair 3, the nucleotide sequence of the upstream primer of primer pair 3 is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer of primer pair 3 is shown as SEQ ID NO.6.

[0037] Optionally, the multiple primers are used for targeted pathogen genome sequencing of Klebsiella aerogenes, and the multiple primers include at least one of the following primer pairs:

[0038] Primer pair 4, the nucleotide sequence of the upstream primer of primer pair 4 is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer of primer pair 4 is shown as SEQ ID NO.8;

[0039] Primer pair 5, the nucleotide sequence of the upstream primer of primer pair 5 is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer of primer pair 5 is shown in SEQ ID NO.10;

[0040] Primer pair 6, the nucleotide sequence of the upstream primer of primer pair 6 is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer of primer pair 6 is shown in SEQ ID NO.12.

[0041] Optionally, the multiple primers are used for targeting pathogen genome sequencing of Klebsiella oxytoca, and the multiple primers include at least one of the following primer pairs:

[0042] Primer pair 7, the nucleotide sequence of the upstream primer of primer pair 7 is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer of primer pair 7 is shown in SEQ ID NO.14;

[0043] Primer pair 8, the nucleotide sequence of the upstream primer of primer pair 8 is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer of primer pair 8 is shown in SEQ ID NO.16;

[0044] Primer pair 9, the nucleotide sequence of the upstream primer of primer pair 9 is shown as SEQ ID NO.17, and the nucleotide sequence of the downstream primer of primer pair 9 is shown as SEQ ID NO.18.

[0045] A design system for multiple primers targeting pathogenic microorganisms, comprising:

[0046] A pathogenic microorganism genome database establishment module is used to obtain the genome data of pathogenic microorganisms and establish a pathogenic microorganism genome database;

[0047] The conserved region prediction module is used to obtain the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database; and the specificity and inclusiveness of the obtained common conserved regions are evaluated respectively, and the regions with specificity and inclusiveness ≥ 95% are selected;

[0048] Multiple primer design module, used to design multiple primers based on conserved regions;

[0049] The multiplex primer evaluation module is used to evaluate the specificity and inclusiveness of multiplex primers and select multiplex primers with specificity and inclusiveness ≥ 90%.

[0050] Compared with the prior art, the beneficial effects of the present invention include:

[0051] The present invention downloads microbial genomes, screens high-quality genomes, removes annotated and misclassified genomes, filters low-quality or contaminated genome fragments, removes redundancy between species and strains, and finally combines all species genomes to obtain a pathogenic microorganism genome database. Then, the bed file is generated by annotating the genome, pan-genome analysis, and conservative region prediction, and the specificity and inclusiveness evaluation are evaluated to finally obtain a conservative prediction interval that meets the requirements. Finally, the best primers that meet the requirements are obtained by specificity and inclusiveness evaluation. The method is used to design primers, which has the advantages of high accuracy, short analysis time, and cost saving. The primer set obtained in the present invention can detect multiple strains of pathogens at the same time, and the primer detection results are accurate compared to those obtained in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0053] Figure 1 It is a flowchart of database construction;

[0054] Figure 2 It is a conservative region prediction flow chart;

[0055] Figure 3 It is a flowchart of multiplex targeted primer design;

[0056] Figure 4 is a flowchart of primer evaluation. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical scheme and advantages of the implementation of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed, but merely represents the implementation of the present invention.

[0058] The present invention proposes a method for designing sequencing primers targeting pathogenic microorganisms, comprising the following steps:

[0059] Step 1) Obtaining genome data of pathogenic microorganisms and establishing a pathogenic microorganism genome database;

[0060] Step 2) obtaining the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database;

[0061] Step 3) designing multiple primers based on the conserved regions in step 2);

[0062] Step 4) Evaluate the specificity and inclusiveness of the multiplex primers designed in step 3), and select multiplex primers with specificity and inclusiveness ≥ 90%.

[0063] like Figure 1 As shown, the method for establishing a pathogenic microorganism genome database in step 1) includes:

[0064] Screen out genomes that meet the following criteria:

[0065] The genome N50 is greater than or equal to the median N50 of all genome data;

[0066] The N content of the genome is less than or equal to the median of the N content of all genomes;

[0067] The fastANI program was used to calculate the genome, and the result was greater than or equal to 95%.

[0068] Filtering: Perform ANI analysis and checkM analysis on the selected genomes, remove genomes with scores less than 95, remove strains with incorrect annotation information, incomplete chromosome assembly, and incorrect classification, and obtain high-quality genomes of the species;

[0069] Construct genome index: merge the above high-quality genomes, and then use bwa and makeblast to index the merged data set;

[0070] Group library: Repeat the above steps to obtain the predetermined pathogenic microorganism genome database.

[0071] The method for establishing the above database is to download microbial genomes, screen high-quality genomes, remove incorrectly labeled and classified genomes, filter low-quality or contaminated genome fragments, remove redundancy between genomes of species and strains and merge them, and finally compile all species genomes to obtain a pathogenic microbial genome database.

[0072] like Figure 2As shown, in step 2), the common conserved regions in the pathogenic microorganism genome in the original microorganism genome database are extracted;

[0073] Specificity assessment: blast was used to align the conserved regions to the NT database, specificity analysis was performed on the conserved regions, and regions with specificity scores less than 95 were filtered out;

[0074] Then, an inclusiveness assessment was performed: the conserved regions were aligned to the high-quality merged gene set using blast, and an inclusiveness analysis was performed on the conserved regions, filtering out regions with an inclusiveness score of less than 95.

[0075] The above-mentioned method for predicting conserved regions is to generate bed files through annotating genomes, performing pan-genome analysis, predicting conserved regions, and performing specificity and inclusiveness evaluations to finally obtain a conservative prediction interval that meets the requirements.

[0076] like Figure 3 As shown, the method for designing multiple primers in step 3) comprises the following steps:

[0077] Conserved region: select fragments with a length of 200~1000bp in the conserved region for primer design;

[0078] Primer design: obtain bed interval;

[0079] Set primer length: 18-28bp, optimal length: 22bp; GC content: 30%-70%, optimal GC: 50%; annealing temperature: 60-66℃, optimal temperature: 63℃.

[0080] Run the program: If primers can be generated, perform primer evaluation

[0081] If no primers are generated, modify the parameters: primer length: 17-30bp; optimal length: 23bp; GC content: 20%-80%, optimal GC: 45%; annealing temperature: 58-67℃, optimal temperature: 62℃.

[0082] Run the program and repeat the above steps.

[0083] like Figure 4 As shown, the method for multiple primer evaluation in step 4) comprises the following steps:

[0084] The specificity evaluation method in step 4) is as follows: using blast software to compare and screen the primers, using the NT library for comparison, and saving the results; using taxonkit software to obtain all sub-taxids under the species, and the screening conditions are that the last base of the sequence is matched, the total number of unmatched bases and mismatched bases is less than or equal to 2; and the specificity is calculated using the following formula:

[0085] Specificity = the number of species of the pathogenic microorganisms screened / the number of all species screened in the NT library;

[0086] The method of inclusiveness evaluation is: using seqkit software, using the pathogenic microorganism genome database established in step 1) to screen the primer pairs (upstream primer + downstream primer) composed of each primer, and using the number of mismatches not exceeding 2 and no mismatch at the 3' end as the screening conditions to obtain the amplicon results; the inclusiveness is calculated using the following formula:

[0087] Inclusiveness = the number of amplicon result genomes obtained / the number of all genomes in the pathogen genome database.

[0088] Primer result judgment: if the specific frequency P>=90% and the inclusive frequency P>=90%, the primer pair is marked as YES, otherwise it is marked as NO. The screened multiple primer set is obtained.

[0089] The present invention also proposes a design system for multiple primers targeting pathogenic microorganisms based on the above method, comprising:

[0090] A pathogenic microorganism genome database establishment module is used to obtain the genome data of pathogenic microorganisms and establish a pathogenic microorganism genome database;

[0091] The conserved region prediction module is used to obtain the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database; and the specificity and inclusiveness of the obtained common conserved regions are evaluated respectively, and the regions with specificity and inclusiveness ≥ 95% are selected;

[0092] Multiple primer design module, used to design multiple primers based on conserved regions;

[0093] The multiplex primer evaluation module is used to evaluate the specificity and inclusiveness of multiplex primers and select multiplex primers with specificity and inclusiveness ≥ 90%.

[0094] Example 1

[0095] A pathogenic microorganism primer design system based on the method of the present invention and an application example are as follows:

[0096] 1. Data acquisition and processing

[0097] Data acquisition: The genome data of a specific pathogen (Clostridium tetani) were downloaded from public databases such as NCBI, and a total of 500 strain genomes were obtained.

[0098] Genome screening: The genome N50 is greater than or equal to the median of the N50 of all genome data, the genome N content is less than or equal to the median of the N content of all genomes, and the fastANI result is greater than or equal to 95. Select the species strain genome.

[0099] Genome filtering: Genomes with incomplete chromosome assembly, incorrect annotations, or containing contaminating sequences were removed, and finally 150 high-quality genomes were obtained.

[0100] Genome index construction: bwa and makeblastdb software were used to construct an index for the genomes of 150 strains to facilitate subsequent primer design and evaluation.

[0101] 2. Conservative regional forecast

[0102] Genome annotation: Annotate each genome and generate a gff file.

[0103] Pan-genome analysis: All GFF files were analyzed using Roary software to obtain the common conserved regions and generate BED files.

[0104] Specificity and inclusiveness evaluation: By comparing with the NT database and all strain genomes, conserved regions with specificity greater than 95 and inclusiveness greater than 95 were screened out.

[0105] 3. Multiplex Primer Design

[0106] Primer design: By running the software, primers with a length of 18-28 bp, a GC content of 30%-70%, and an annealing temperature of 60-66°C were designed in the conserved region.

[0107] 4. Screen out primers that meet the standards:

[0108] Primer evaluation: The specificity and inclusiveness of primers were evaluated using the NT database and the self-built species database. Primers with specificity frequency P ≥ 90% and inclusive frequency P ≥ 90% were marked as YES.

[0109] Table 1:

[0110]

[0111] Experimental verification

[0112] Experimental verification: The designed primers were applied to targeted pathogen genome sequencing of clinical samples. The results showed that all primers were detected in all 5 samples, with an overall detection rate of 100%.

[0113] Table 2:

[0114]

[0115] Comparative Example 1

[0116] The primers for tetanus clostridium bacilli were designed using the NCBI primer design software Primer-BLAST. The specific steps are as follows:

[0117] 1. Input target sequence

[0118] Directly paste the nucleic acid sequence of Clostridium tetani downloaded from NCBI in the "Enter Target Sequence" box.

[0119] 2. Set primer design parameters

[0120] Design primers with a length of 18-28 bp, a GC content of 30%-70%, and an annealing temperature of 60-66°C.

[0121] 3. Submit a request

[0122] Click the "Get Primers" button and Primer-BLAST will start calculating and generating primers for Clostridium tetani.

[0123] Table 3:

[0124]

[0125] Experimental verification

[0126] Experimental verification: The designed primers were applied to targeted pathogen genome sequencing of clinical samples. The results showed that only 2 samples were detected out of a total of 5 samples, with an overall detection rate of 40%, indicating that the primer effect was poor.

[0127] Table 4:

[0128]

[0129] Example 2

[0130] Another pathogenic microorganism primer design system based on the method of the present invention and its application example are as follows:

[0131] 1. Data acquisition and processing

[0132] Data acquisition: The genome data of target pathogens (Klebsiella aerogenes and Klebsiella oxytoca) were downloaded from the public databases NCBI and GenBank, totaling 1,328 strain genomes.

[0133] Genome screening: According to predetermined screening rules (such as sequencing coverage and genome completeness), 820 high-quality genomes were screened out.

[0134] Genome filtering: Genomes with annotation errors, incomplete genomes, and genomes containing a high proportion of repeated sequences were removed, and finally 755 high-quality genomes were retained.

[0135] Construct genome index: Use bwa and makeblastdb software to construct indexes for these genomes in preparation for subsequent analysis.

[0136] 2. Conservative regional forecast

[0137] Genome annotation: Prokka software was used to annotate each genome and generate GFF files.

[0138] Pan-genome analysis: All GFF files were pooled and analyzed to identify common conserved regions and generate bed files.

[0139] Specificity and inclusiveness evaluation: By comparing with the NT database and all strain genomes, conserved regions with good specificity and inclusiveness were screened out.

[0140] 3. Multiplex Primer Design

[0141] Primer design: By running the software, primers with a length of 20-30 bp, a GC content of 40%-60%, and an annealing temperature of 58-68°C in the conserved region were selected.

[0142] 4. Screen out primers that meet the standards

[0143] Primer evaluation: The specificity and inclusiveness of primers were evaluated using the NT database and the self-built database. Primers with specificity frequency P ≥ 90% and inclusive frequency P ≥ 90% were marked as YES.

[0144] Table 5:

[0145]

[0146] Experimental verification

[0147] Experimental verification: The designed primers were applied to targeted pathogen genome sequencing of samples with different bacterial combinations. The test results showed that both the specificity and sensitivity reached 100%, and the detection efficiency was significantly improved.

[0148] Table 6:

[0149]

[0150] Comparative Example 2

[0151] The primers for Klebsiella aerogenes and Klebsiella oxytoca were designed using the NCBI primer design software Primer-BLAST. The specific steps are as follows:

[0152] 1. Input target sequence

[0153] Paste the nucleic acid sequences of Klebsiella aerogenes and Klebsiella oxytoca downloaded from NCBI in the "Enter Target Sequence" box respectively.

[0154] 2. Set primer design parameters

[0155] Design primers with a length of 18-28 bp, a GC content of 30%-70%, and an annealing temperature of 60-66°C.

[0156] 3. Submit a request

[0157] Click the "Get Primers" button and Primer-BLAST will start calculating and generating primers for Klebsiella aerogenes and Klebsiella oxytoca.

[0158] Table 7:

[0159]

[0160] Experimental verification

[0161] Experimental verification: The designed primers were applied to targeted pathogen genome sequencing of samples of different bacterial combinations. The test results showed that the sensitivity of Klebsiella aerogenes was 66.7% and the specificity was 100%; the sensitivity of Klebsiella acidophilus was 50% and the specificity was 50%. The overall detection effect was poor.

[0162] Table 8:

[0163]

[0164] In addition, it should be noted that the above are only preferred embodiments of the present invention and are not limited to the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions recorded in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for designing primers targeting pathogenic microorganism sequencing, characterized in that: The steps include: Step 1) obtaining genome data of pathogenic microorganisms and establishing a pathogenic microorganism genome database; the method for establishing a pathogenic microorganism genome database comprises: For each targeted pathogenic microorganism species for which primers need to be designed, screen out genomes that meet the following criteria: The N50 value of the genome is greater than or equal to the median of the N50 values ​​of all genome data; The N content in the genome is less than or equal to the median of the N content in all genomes; The calculation results of the genome fastANI program are ≥95; Filtering: Perform ANI analysis and checkM analysis on the selected genomes, remove genomes with at least one score less than 95, remove genomes with incorrect annotation information, incomplete chromosome assembly, and incorrect classification, and obtain high-quality genomes of the species; Constructing a genome index: Merge the high-quality genomes of each targeted pathogenic microorganism species for which primers need to be designed, and then create an index for the merged data set; Step 2) Obtain the common conserved regions in the pathogenic microorganism genome based on the pathogenic microorganism genome database; evaluate the specificity and inclusiveness of the obtained common conserved regions, and select regions with specificity and inclusiveness ≥ 95%; Step 3) designing multiple primers based on the conserved regions in step 2); Step 4) Evaluate the specificity and inclusiveness of the multiplex primers designed in step 3), and select multiplex primers with specificity and inclusiveness ≥ 90%.

2. The method for designing sequencing primers targeting pathogenic microorganisms according to claim 1, characterized in that: The method for obtaining the conserved region sequence of pathogenic microorganisms in step 2) comprises: Extract the common conserved regions in the pathogenic microorganism genomes from the original microorganism genome database; Specificity assessment: the conserved regions were aligned to the NT database, specificity analysis was performed on the conserved regions, and regions with specificity scores less than 95 were removed; Inclusiveness assessment: The conservative regions were compared to the pathogenic microorganism genome database, and inclusiveness analysis was performed on the conservative regions, and regions with inclusiveness scores less than 95 were removed; That is, the region where the specificity and inclusiveness are both ≥ 95% is obtained.

3. The method for designing sequencing primers targeting pathogenic microorganisms according to claim 1, characterized in that: In the step 3), a fragment of the conserved region with a length ranging from 200 to 1000 bp is selected to design primers; The setting parameters are: primer length 18-28bp; GC content 30%-70%; annealing temperature 60-66℃; If primers cannot be generated within the set parameter range, the parameters are set as follows: primer length 17-30bp; GC content 20%-80%; annealing temperature 58-67°C.

4. The method for designing sequencing primers targeting pathogenic microorganisms according to claim 1, characterized in that: The specificity evaluation method in step 4) is: using the NT library to compare and screen the primers, the screening conditions are that the last base of the sequence is aligned, the total number of unmatched bases and mismatched bases is less than or equal to 2; the specificity is calculated using the following formula: Specificity = the number of species of the pathogenic microorganisms screened / the number of all species screened in the NT library; The method of inclusiveness evaluation is: the pathogenic microorganism genome database established in step 1) is used to screen the primer pairs composed of each primer, and the amplicon results are obtained with the number of mismatches not exceeding 2 and no mismatch at the 3' end as the screening conditions; inclusiveness is calculated using the following formula: Inclusiveness = the number of amplicon result genomes obtained / the number of all genomes in the pathogen genome database.

5. A multiplex primer obtained according to the method of any one of claims 1 to 4.

6. The multiple primer according to claim 5, characterized in that: The multiple primers are used for targeting pathogen genome sequencing of Clostridium tetani, and the multiple primers include at least one of the following primer pairs: Primer pair 1, the nucleotide sequence of the upstream primer of primer pair 1 is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer of primer pair 1 is shown as SEQ ID NO.2; Primer pair 2, the nucleotide sequence of the upstream primer of primer pair 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer of primer pair 2 is shown in SEQ ID NO.4; Primer pair 3, the nucleotide sequence of the upstream primer of primer pair 3 is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer of primer pair 3 is shown as SEQ ID NO.

6.

7. The multiple primer according to claim 5, characterized in that: The multiple primers are used for targeted pathogen genome sequencing of Klebsiella aerogenes, and the multiple primers include at least one of the following primer pairs: Primer pair 4, the nucleotide sequence of the upstream primer of primer pair 4 is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer of primer pair 4 is shown as SEQ ID NO.8; Primer pair 5, the nucleotide sequence of the upstream primer of primer pair 5 is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer of primer pair 5 is shown in SEQ ID NO.10; Primer pair 6, the nucleotide sequence of the upstream primer of primer pair 6 is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer of primer pair 6 is shown in SEQ ID NO.

12.

8. The multiple primer according to claim 5, characterized in that: The multiple primers are used for targeting pathogen genome sequencing of Klebsiella oxytoca, and the multiple primers include at least one of the following primer pairs: Primer pair 7, the nucleotide sequence of the upstream primer of primer pair 7 is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer of primer pair 7 is shown in SEQ ID NO.14; Primer pair 8, the nucleotide sequence of the upstream primer of primer pair 8 is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer of primer pair 8 is shown in SEQ ID NO.16; Primer pair 9, the nucleotide sequence of the upstream primer of primer pair 9 is shown as SEQ ID NO.17, and the nucleotide sequence of the downstream primer of primer pair 9 is shown as SEQ ID NO.

18.

9. A design system for multiple primers targeting pathogenic microorganisms, characterized in that: include: A pathogenic microorganism genome database establishment module is used to obtain the genome data of pathogenic microorganisms and establish a pathogenic microorganism genome database; The method for establishing a pathogenic microorganism genome database comprises: For each targeted pathogenic microorganism species for which primers need to be designed, screen out genomes that meet the following criteria: The N50 value of the genome is greater than or equal to the median of the N50 values ​​of all genome data; The N content in the genome is less than or equal to the median of the N content in all genomes; The results calculated by the genome fastANI program were ≥95; Filtering: Perform ANI analysis and checkM analysis on the selected genomes, remove genomes with at least one score less than 95, remove genomes with incorrect annotation information, incomplete chromosome assembly, and incorrect classification, and obtain high-quality genomes of the species; Constructing a genome index: Merge the high-quality genomes of each targeted pathogenic microorganism species for which primers need to be designed, and then create an index for the merged data set; The conserved region prediction module is used to obtain the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database; and the obtained common conserved regions are evaluated for specificity and inclusiveness, and regions with specificity and inclusiveness ≥ 95% are selected; Multiple primer design module, used to design multiple primers based on conserved regions; The multiplex primer evaluation module is used to evaluate the specificity and inclusiveness of multiplex primers and select multiplex primers with specificity and inclusiveness ≥ 90%.

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