CN113462799B

CN113462799B - Bacillus anthracis identification method based on chromosome specific probe

Info

Publication number: CN113462799B
Application number: CN202110895338.8A
Authority: CN
Inventors: 吕宇飞; 刘先凯; 王恒樑; 王东澍; 朱力; 潘超; 冯尔玲
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2023-06-20
Anticipated expiration: 2041-08-05
Also published as: CN113462799A

Abstract

The invention discloses a bacillus anthracis identification method based on chromosome specific probes. The invention provides a probe set for identifying or assisting in identifying bacillus anthracis, which consists of 51 probes shown in SEQ ID No.1 to SEQ ID No. 51. The probe detects the sequencing result (comprising the second generation or the third generation) of the unknown sample, and as the number of the probes is up to 51, the positive can be judged as long as any probe is detected, the more the detected probes are, the higher the reliability is, and the probes are uniformly distributed on the whole chromosome, so that even if the genome sequence with poor sequencing quality and low coverage is ensured, the electronic probes can be used for inquiring, and whether the sample carries bacillus anthracis can be rapidly, simply, conveniently and efficiently identified.

Description

Bacillus anthracis identification method based on chromosome specific probe

Technical Field

The invention relates to the technical field of biology, in particular to a bacillus anthracis identification method based on chromosome specific probes.

Background

Anthrax is a zoonosis caused by bacillus anthracis. Bacillus anthracis (Bacillus anthracis, bacillus anthracis for short) is an important virulent pathogen and potential biological warfare agent. Humans are infected by contact with a bacteria-bearing animal or animal product and are classified into three types, skin anthrax, lung anthrax and intestinal anthrax, according to different infection pathways. Bacillus anthracis has the characteristics of global popularity, environmental tolerance, infection destructiveness and the like. In the face of anthrax epidemic situations that are frequently occurring and involve a wide range of areas, it is critical to select a rapid, accurate method of identifying bacillus anthracis.

The conventional identification method is a culture identification method, and obviously, the practical requirements cannot be met. Bacillus cereus, bacillus thuringiensis and Bacillus anthracis have very high genomic homology. Researchers consider that bacillus anthracis evolved from bacillus cereus and bacillus thuringiensis in 1-2 thousands of years ago, and obtain two virulence related plasmids of pXO1 and pXO2 in the evolution process, and respectively guide the synthesis of exotoxins and polypeptide capsules, so that the exotoxins and the polypeptide capsules become virulent pathogenic bacteria. The unique sequences on plasmids are used as identification targets and cannot be effectively identified when atypical strains (such as those with plasmid loss) are encountered. There are also primers on the chromosome that are used to distinguish between these two bacteria, but the number of strains is too small and the specificity of the primers is limited.

The rapid development of sequencing technology, more and more strains acquire complete genomic sequences, but NCBI's bacillus anthracis genomic database still contains some non-bacillus anthracis genomic data. The bacillus cereus mixed with the bacillus anthracis genome database is easy to misguide related personnel to carry out subsequent analysis, and a simple, rapid and efficient method for distinguishing bacillus anthracis is needed.

Disclosure of Invention

The invention aims to provide a bacillus anthracis identification method based on chromosome specific probes.

In a first aspect, the invention claims a probe set for the identification or assisted identification of bacillus anthracis.

The probe set for identifying or assisting in identifying bacillus anthracis claimed in the invention can be composed of the following components:

probe 1: single-stranded DNA shown in SEQ ID No. 1;

probe 2: single-stranded DNA shown in SEQ ID No. 2;

probe 3: single stranded DNA shown in SEQ ID No. 3;

probe 4: single-stranded DNA shown in SEQ ID No. 4;

probe 5: single stranded DNA shown in SEQ ID No. 5;

probe 6: single stranded DNA shown in SEQ ID No. 6;

probe 7: single stranded DNA shown in SEQ ID No. 7;

probe 8: single stranded DNA shown in SEQ ID No. 8;

probe 9: single stranded DNA shown in SEQ ID No. 9;

probe 10: single stranded DNA shown in SEQ ID No. 10;

probe 11: single stranded DNA shown in SEQ ID No. 11;

probe 12: single stranded DNA shown in SEQ ID No. 12;

probe 13: single stranded DNA shown in SEQ ID No. 13;

probe 14: single stranded DNA shown in SEQ ID No. 14;

probe 15: single stranded DNA shown in SEQ ID No. 15;

probe 16: single stranded DNA shown in SEQ ID No. 16;

probe 17: single stranded DNA shown in SEQ ID No. 17;

probe 18: single stranded DNA shown in SEQ ID No. 18;

probe 19: single stranded DNA shown in SEQ ID No. 19;

probe 20: single stranded DNA shown in SEQ ID No. 20;

probe 21: single stranded DNA shown in SEQ ID No. 21;

probe 22: single stranded DNA shown in SEQ ID No. 22;

probe 23: single stranded DNA shown in SEQ ID No. 23;

probe 24: single stranded DNA shown in SEQ ID No. 24;

probe 25: single stranded DNA shown in SEQ ID No. 25;

probe 26: single stranded DNA shown in SEQ ID No. 26;

probe 27: single stranded DNA shown in SEQ ID No. 27;

probe 28: single stranded DNA shown in SEQ ID No. 28;

probe 29: single stranded DNA shown in SEQ ID No. 29;

probe 30: single stranded DNA shown in SEQ ID No. 30;

probe 31: single stranded DNA shown in SEQ ID No. 31;

probe 32: single stranded DNA shown in SEQ ID No. 32;

probe 33: single stranded DNA shown in SEQ ID No. 33;

probe 34: single stranded DNA shown in SEQ ID No. 34;

probe 35: single stranded DNA shown in SEQ ID No. 35;

probe 36: single stranded DNA shown in SEQ ID No. 36;

probe 37: single stranded DNA shown in SEQ ID No. 37;

probe 38: single stranded DNA shown in SEQ ID No. 38;

probe 39: single stranded DNA shown in SEQ ID No. 39;

probe 40: single stranded DNA shown in SEQ ID No. 40;

probe 41: single stranded DNA shown in SEQ ID No. 41;

probe 42: single stranded DNA shown in SEQ ID No. 42;

probe 43: single stranded DNA shown in SEQ ID No. 43;

probe 44: single stranded DNA shown in SEQ ID No. 44;

probe 45: single stranded DNA shown in SEQ ID No. 45;

probe 46: single stranded DNA shown in SEQ ID No. 46;

probe 47: single stranded DNA shown in SEQ ID No. 47;

probe 48: single stranded DNA shown in SEQ ID No. 48;

probe 49: single stranded DNA shown in SEQ ID No. 49;

probe 50: single stranded DNA shown in SEQ ID No. 50;

probe 51: single-stranded DNA shown in SEQ ID No. 51.

In a second aspect, the invention claims a probe for identifying or aiding in the identification of bacillus anthracis.

The probe for identifying or assisting in identifying Bacillus anthracis claimed in the present invention may be any one of the probe 1 to the probe 51 in the first aspect described above.

In a third aspect, the invention claims a kit comprising a set of probes as described in the first aspect above or a probe as described in the second aspect above.

In a fourth aspect, the invention claims the use of a set of probes as described in the first aspect hereinbefore or a probe as described in the second aspect hereinbefore or a kit as described in the third aspect hereinbefore, in any of the following:

p1, identifying or assisting in identifying bacillus anthracis;

p2, preparing a product for identifying or assisting in identifying bacillus anthracis;

p3, detecting or assisting in detecting whether the strain to be detected is bacillus anthracis;

p4, preparing a product for detecting or assisting in detecting whether the strain to be detected is bacillus anthracis;

p5, detecting or assisting in detecting whether the target individual carries bacillus anthracis;

p6, preparing a product for detecting or assisting in detecting whether a target individual carries bacillus anthracis.

In a fifth aspect, the invention claims a method for detecting or aiding in the detection of whether the strain to be tested is Bacillus anthracis.

The method for detecting or assisting in detecting whether the strain to be detected is bacillus anthracis or not, which is claimed in the invention, can be a method A

Or method B.

The method a may comprise the steps of:

(A1) Performing whole genome sequencing on the strain to be tested;

(A2) Aligning the sequencing result with the sequence of each probe in the probe set according to the first aspect by bioinformatics software;

(A3) Determining whether the strain to be tested is bacillus anthracis according to the comparison result (A2) as follows: if any probe sequence in the probe group exists in the sequencing result, the strain to be tested is or is candidate to be bacillus anthracis; if any one of the probe sequences in the probe set is not present in the sequencing result, the strain to be tested is not or is not a candidate for Bacillus anthracis.

The method B may include the steps of:

(B1) Hybridization is carried out on each probe in the probe set in the first aspect and the genome of the strain to be detected;

(B2) Determining whether the strain to be tested is Bacillus anthracis according to the result obtained in (B1) as follows: if a positive signal exists after hybridization between any one probe in the probe set and the genome of the strain to be detected, the strain to be detected is or is candidate to be bacillus anthracis; if no positive signal is generated after hybridization between any one probe in the probe set and the genome of the strain to be detected, the strain to be detected is not bacillus anthracis.

In this method, the more probes detected, the more reliable the results, the more probes can avoid misidentification due to poor sequencing quality, or artificially modified bacteria.

The method may be a non-disease diagnostic treatment, such as for the simple detection of whether the strain to be tested is bacillus anthracis, without any disease diagnostic purpose being involved.

In a sixth aspect, the invention claims a method for detecting or aiding in the detection of whether a target individual carries Bacillus anthracis.

The method for detecting or assisting in detecting whether the target individual carries bacillus anthracis can be a method C or a method D.

The method C may include the steps of:

(C1) Extracting the whole genome of bacteria from a sample of a target individual, and then sequencing;

(C2) Aligning the sequencing result with each probe in the probe set according to the first aspect by bioinformatics software;

(C3) Determining whether the target individual carries bacillus anthracis according to the sequence comparison result (C2) as follows: if any one probe sequence in the probe group exists in the sequencing result, the target individual carries or candidates to carry bacillus anthracis; if none of the probe sequences in the probe set is present in the sequencing result, the target individual does not carry or is candidate to not carry B.anthracis.

The method D may include the steps of:

(D1) Extracting the whole genome of the bacteria from the sample of the target individual, and then hybridizing each probe in the probe set according to the first aspect with the extracted genome;

(D2) Determining from the results obtained in (D1) whether the target individual carries bacillus anthracis as follows: if any one probe in the probe set has a positive signal after hybridization with the genome extracted in the step (D1), carrying or candidate carrying bacillus anthracis by the target individual; if no positive signal is generated after hybridization of any one of the probes in the probe set with the genome extracted in the step (D1), the target individual does not carry or is candidate to carry bacillus anthracis.

The method may be a non-disease diagnostic treatment, such as for purely detecting whether the target individual carries bacillus anthracis (e.g. for customs inspection and quarantine), without any disease diagnostic purpose being involved.

In the above aspects, the strain to be tested may be any strain including, but not limited to, a bacillus cereus family strain.

Further, the bacillus cereus family strain may specifically be bacillus anthracis, bacillus cereus or bacillus thuringiensis.

The inventor carries out intensive study on the whole genome sequences of anthrax and non-anthrax bacillus strains in NCBI database, writes a python program by means of local blast, carries out a large number of screening and comparison, and determines a conserved DNA region in the genome sequences of the anthrax bacillus. The invention adopts up to 51 specific probes, and can judge positive if any one probe is detected, and the more the detected probes are, the higher the reliability is, so that even if the genome sequence with poor sequencing quality and low coverage is detected, the electronic probes can be used for inquiring, and whether the sample carries bacillus anthracis can be rapidly, simply, conveniently and efficiently identified.

Drawings

FIG. 1 shows the concept of specific tag interception according to the present invention.

FIG. 2 is a cluster tree of Bacillus anthracis and Bacillus cereus across the genome of Bacillus thuringiensis. Bacillus anthracis adopts BAXXX uniformly, bacillus cereus adopts BCXXXX, and Bacillus thuringiensis adopts BTXXX.

FIG. 3 shows the Blast test results of the BaF_1 probe (SEQ ID No. 1) in NCBI library.

FIG. 4 shows an example of Blast specific results of BaF_1 probe (SEQ ID No. 1) in a single strain, with boxes marked as deleted suspicious B.anthracis. The strains corresponding to the portions marked a and B in the figure are the strains corresponding to a and B, respectively, marked in fig. 3.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 establishment of chromosome-specific Probe-based Bacillus anthracis identification method

The Bacillus anthracis genomic sequence described herein is the Bacillus anthracis standard strain Bacillus anthracis Ames Ancestor chromosomal sequence (GenBank: NC-007530, 25-OCT-2020), and the nucleic acid sequence positions are also calculated using the Bacillus anthracis AmesAncestor chromosome (GenBank: NC-007530, 25-OCT-2020) as standard.

1. The genome of Bacillus anthracis (252 strains in total, table 1) and the genome of a strain other than Bacillus anthracis (4695 strains in total, 83 species of bacteria, table 1) in the genus Bacillus (hereinafter abbreviated as "Bacillus anthracis" and "Bacillus anthracis" respectively for convenience of description) were downloaded from NCBI genome database.

TABLE 1 Bacillus species name and species number information Table (4947 strain)

Note that: bacillus anthracis it represents suspected bacillus anthracis.

2. A Bacillus anthracis Ames Ancestor chromosomal sequence (GenBank: NC-007530, 25-OCT-2020) fragment (step size 1bp step; length 100 bp) was cut out, and a total of 5227320 fragments 100bp long were cut out, as shown in FIG. 1.

3. 4665794 tags present in Bacillus non-Bacillus anthracis were removed by local blast (blast-2.7.1+), leaving 561526 tags.

4. The Python program was written and the 561526 tags were queried for the tags that appeared in all Bacillus anthracis (Bacillus anthracis total 252 strains, corresponding nomenclature to that in the public database is shown in Table 2), and in this process, it was found that the detection rate of these tags was only 0.07% -2.02% in 12 strains, while the detection rate of these tags was 59.28% -100% in the other 240 strains of Bacillus anthracis. After the analysis of the whole genome cluster tree, none of the 12 strains with very low fragment detection rate was bacillus anthracis (fig. 2). They are eliminated from the genome training set. As a result, we obtained 95823 tags which appeared in all Bacillus anthracis (240 strains).

TABLE 2 Bacillus anthracis Assemble ID and rename correspondence table

5. These 95823 tags were combined according to the overlap between the fragments, resulting in 1355 specific fragments, which were between 101bp-915bp in length.

6. Local blast was performed with these 1355 fragments and Bacillus cereus (1118 strain) and Bacillus thuringiensis (622 strain), and the DNA sequences corresponding to each strain were cut out according to the blast result, and made into fasta format.

7. And (3) comparing the files obtained in the step (6) by adopting megaX software, searching SNP loci capable of distinguishing bacillus anthracis from all other near bacteria, and finding that 610 fragments contain the SNP loci.

8. For convenient application and improved detection specificity, we selected 51 sequences containing at least 2 (including 2) SNP sites from the 610 fragments, and cut out the sequences with the lengths of 100-200bp containing the SNP sites as electronic tags (Table 3) for rapid identification of bacillus anthracis.

TABLE 3 specific electronic probe sequences

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Note that: the starting and ending positions in the table were calculated using the chromosomal sequence of Bacillus anthracis standard strain Bacillus anthracis Ames Ancestor (GenBank: NC-007530, 25-OCT-2020) as standard. The probe sequence corresponds to the sequence in the Bacillus anthracis genome.

9. In order to test the specificity of the 51 electronic probes in a larger database, the 51 electronic probes are subjected to online blast inquiry (https:// blast. Ncbi. Nlm. Nih. Gov/blast. Cgi), and the result shows that the 51 electronic probes can only reach 100% of the values of Query Cover and Per. Ident (Percentage of Identity) in Bacillus anthracis, the sequences are completely consistent, the detection in other strains cannot be realized, the values of Query Cover and Per. Ident cannot be 100%, and the sequences are not completely consistent.

Taking Ba_F1 (SEQ ID No. 1) as an example, the test result is shown in FIG. 3, and the result shows that the electronic probe can only reach the values of Query Cover and Per.Ident (Percentage of Identity) of 100% in Bacillus anthracis, specifically shows that the sequences are completely consistent in FIG. 4A, and can not be found in other strains, as shown in FIG. 4B, the values of Query Cover and Per.Ident of 100% can not be satisfied, and the sequences are not completely consistent.

10. In summary, it can be seen that: by using the 51 bacillus anthracis specific electronic probe and the method, whether the strain to be detected is bacillus anthracis and whether a target individual carries bacillus anthracis can be simply and rapidly identified.

The steps are as follows:

(1) Collecting a sample of the strain to be tested or from the target individual, extracting the genome thereof, and extracting the whole genome of the bacteria according to the Bacterial Genomic DNA Extraction kit kit steps.

(2) The genomic samples are sequenced using a sequencer, either second generation sequencing or third generation sequencing.

(3) The sequencing results were aligned and analyzed with the probe sequences using bioinformatics software.

(4) If the sequencing result is 100% identical (i.e., the sequences are completely identical) to any one of the probes, the detection result is positive, the sample carries bacillus anthracis, the more the detected probes are, the more reliable the result is, and the plurality of electronic probes can avoid false identification caused by poor sequencing quality or artificially modified bacteria, and if all the probes are not detected (the sequences are not completely identical), the detection result is negative.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

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aacgcatacc ccaacctgtg gcacgtccgt ctatctggaa tgtaaatgaa cgccttgacc 60

aatcaccagc agggaattca gtttgtagat tttctgtcca tgatccgttg ataaattgga 120

atacaagaaa agtaacgtaa acatcatttt gaagatcgac actgaatgtc atgtctttac 180

ctttttcaaa attacccatc 200

<210> 27

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

ttttgaacac cgactaattg tccagctatt tcttgcgctt taccttccac actttgaact 60

tttagattta attctgtttt tgtggattca atatctttgc tgatttgctc tattgtttct 120

tttttaaccg attccacatc aggaacaacc 150

<210> 28

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

tatagaaatc taaaagccca cgtaaattga ctgttaagac taggttagtt gcggctgcgt 60

ttggaagtac gctacgagca tcctcagcag gaattcctaa tgttctaagt agatcataac 120

cactttggag tttgtacatc atttcgttgt 150

<210> 29

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

tcatctcgaa atacattcat ttcagcttct aacttatgat tgtcttccgc caacttacat 60

accggacaaa ctacttcacc atttatcttc atgaatcgaa cagtacgttt tcgttctttt 120

ttacaaacct cacaagtatc agaaaggaag 150

<210> 30

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

tttaaaataa ggattttgca ttaattccct ctaactgcat ttacggctgc tgcacaaaca 60

gcaattccaa acttttcttc aataatgtaa tcattgtcat ctatgttaac ttgccaatca 120

ccagattcgt ttttaatact cacgtcacga 150

<210> 31

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

cctttccatc tttcccccaa tgagtttcta ccttttccca tctaagcact tcatgagcaa 60

ttacatcatt taatttttca acctcattca aatcctcaaa tgagatccaa ctctcttcaa 120

aatgagctac aaagttttca tcaataacgc 150

<210> 32

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

aagctaggat gctcatttat tccttttagc cattctgtaa ctaccttatt aatttgttct 60

tgaagctttt ccccttcgtc catcggcaca cctgaaagcc aatattccgc aacctctcca 120

cattcatcgg ttgctcgttc atacaattca 150

<210> 33

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

acgtgcagca aagcttggga ttactgcagt cgtttccaat ggaaatgatg gtccaaaacc 60

ttggtctgtt gatgcacctg gaaatgcaag tagcgttata tcagttggag catctacagt 120

ttctattccg tttccaacat tccaagtagc tggttccagc aaaacatacc aaggattatc 180

gttatcaaaa tcagatttcc 200

<210> 34

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

tcttaatcga ccctttaaca agcatttggt ctagtataac tgttactctt tcactaagta 60

ttttttatac gttttcacat tggatacacc aacaatccaa atataaaatg aaactatagt 120

atgtggccct tttggtagtt taaattttgc 150

<210> 35

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

ttcttttgac tttcttctaa cttcgcaatc atattttcaa ttttatttgt atcggtacta 60

atgtggttac gagcttgatc aatgacgcga ttagataatc caagacgttt cgaaatttca 120

aaggcattac tacgtcctgg tacaccgatt 150

<210> 36

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

cggcgttgac cgtgagcctg gactaatgac aacatcacat acatttagaa gcgtcagttc 60

atccacgaac gcgcctaaat aatatgataa tgcttctata tggttgttca tttcattaat 120

tcctccaaag caccaagcat cggtctaaac 150

<210> 37

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

tacatggatt gcattagact atcgaaatgt tgctgaaaat cctcccgtta tatttatcga 60

taatgaggtt gagcaaatta ttgaattagc acctaatttt gaaagcttct tacaaaactt 120

aactacgtac aattatgacg aagagtaatt ctctctaaag gagtacgcaa tagtgcaaga 180

tttcattatt agtttacaca 200

<210> 38

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

ctgctttaaa accacttgga tttaagaaaa aaagaaacac attttccaaa gctgaaaacg 60

gattttataa gctcattaac atacaaaaaa gccagtttgg agatgctttc tatataaata 120

taggagttca tcctatcggt ctcccgcagc 150

<210> 39

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

cgtcccatct aataattctt ctattacttc ttcctcgctt gaaaaactat acaaatgata 60

agtttcttcg tacgtccacg tatttatctc atttgcttct tctatcgtta acttatgtat 120

atccatcctt ctctccccta atcaaatatg ctatatatac tcattcattt ttcgttcctc 180

atttccctct aattgaaata 200

<210> 40

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

ttgtaatgga tcagcaattg gatagcatta ctctcaaaaa agcaccacgc tttttcaagg 60

gagtagtctg cccattccca ttacaaagga acgtattgca tctgtagtat gatgtttacg 120

gaggttcctc tttggggaac ctccttttat ttttgtcttt ttctcatgtc ttcatactat 180

aagacatcca aatagctgta 200

<210> 41

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

tgatctgtaa atggtatagg tatatctata cccgcttcat atcttgctaa agaggcctcc 60

tccccaagtt caaaaccatt tttatctatt ttatcagcaa cttctgcatt ccccatactt 120

aaactagctt ctacataaaa aaataagaag cctcgtttac aggctccctc atcctctatt 180

caaataatct gcaattgttt 200

<210> 42

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

gcctgaaagc atggttaacg caacaattgg caaacagtac gtttcactgg agcaaaatca 60

attatatctc ttaaagaata ctctatctcc aatcatcgtt gctatggaaa gctcgatgga 120

caaagcactt ttgcttgagt ctgaaaaaga caaaggttat ttcttcagat ttgatacttc 180

agagcttatc cgctcgactg 200

<210> 43

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

gaatgtcttc tcgaacgcca gcacgtaagc cagcgatttt gacgatgttc tcaatctgta 60

cggtacagat tggtttgcca gctctcgata aaaagtaatt atcatgttcc aatgctttct 120

tttcgaaata cttcgcttta atccgttcgt aacgtatcat gtattttttc aacattaagc 180

tcatgggaac atgtcgccat 200

<210> 44

<211> 110

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

ttcttcacga aataagcgaa atgatcttct acaacctctt ctgccaatcc attccgcctt 60

tgtaagcgct tcaattgcaa ggttagacat tccttaatat tccgcgcccc 110

<210> 45

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

tgttcaaata ttgaacaacc tctctcatat taacgagttg gattagatat ttcctctgac 60

ttaagcgtat tattcgctcc aagagaatgg tacaatatct ttccatttcc attcacagta 120

acgatatgtt cattattaaa gcttttgaac 150

<210> 46

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

tatcttctgt gttactttct ggtgtcgtgc tcgttgactt ctcttctttt tgagatttgt 60

catcagcttt tttaccagag gcattcgttg cctttgcagc 100

<210> 47

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

tcgactacat acacttcaaa taagtgaagc acatgagcga ttcctttcgt gataggaatg 60

actgtcatat tgtatagttc atccacataa tatttttctt gcaaaatttt ataaagaggt 120

gcttcctcgc caccagccca atctctagca 150

<210> 48

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

ctttaaatgc tacatctttc gtaagccaac ctccgagaaa cgttccaaac cacggtgtat 60

ttatataacc tgccacgact gcaaggacgc caaggacaat catcggatat gtcatgatgc 120

gaggcgattc atgcacatct accttcgtct 150

<210> 49

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

ctagcatatt gatgctttcc catttcactc cagcaactcc gcccgcattc cacatactca 60

cctcttgggc aagattgaag tacagtgcaa tgccagatga 100

<210> 50

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

acagttaact accttttcta tttataaatt tggaatttcc caatcaattt ctccctcgcc 60

catattagct aaaatcgtat ttactttaga gtatggctta ctcccaaaga aacctcgcct 120

tgctgatagt ggacttggat gtacagattc gataatttga tgatttggat tcgtaattaa 180

cttcttcttc gcctgtgcat 200

<210> 51

<211> 200

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

caagttcgcg tcattgctca tttaacctta aattttctat cacgaaggaa atatgatttg 60

ttattgcttg aagtacatct atcgtattca catcaaattc accatgattt gaactaacac 120

ataaaacacc aaatatgtga gtttgaatct gtattggtgt ggcaataacc gttctattag 180

aggctgcctt atgactgatt 200

Claims

1. A set of probes for the identification or assisted identification of bacillus anthracis, consisting of:

probe 1: single-stranded DNA shown in SEQ ID No. 1;

probe 2: single-stranded DNA shown in SEQ ID No. 2;

probe 3: single stranded DNA shown in SEQ ID No. 3;

probe 4: single-stranded DNA shown in SEQ ID No. 4;

probe 5: single stranded DNA shown in SEQ ID No. 5;

probe 6: single stranded DNA shown in SEQ ID No. 6;

probe 7: single stranded DNA shown in SEQ ID No. 7;

probe 8: single stranded DNA shown in SEQ ID No. 8;

probe 9: single stranded DNA shown in SEQ ID No. 9;

probe 10: single stranded DNA shown in SEQ ID No. 10;

probe 11: single stranded DNA shown in SEQ ID No. 11;

probe 12: single stranded DNA shown in SEQ ID No. 12;

probe 13: single stranded DNA shown in SEQ ID No. 13;

probe 14: single stranded DNA shown in SEQ ID No. 14;

probe 15: single stranded DNA shown in SEQ ID No. 15;

probe 16: single stranded DNA shown in SEQ ID No. 16;

probe 17: single stranded DNA shown in SEQ ID No. 17;

probe 18: single stranded DNA shown in SEQ ID No. 18;

probe 19: single stranded DNA shown in SEQ ID No. 19;

probe 20: single stranded DNA shown in SEQ ID No. 20;

probe 21: single stranded DNA shown in SEQ ID No. 21;

probe 22: single stranded DNA shown in SEQ ID No. 22;

probe 23: single stranded DNA shown in SEQ ID No. 23;

probe 24: single stranded DNA shown in SEQ ID No. 24;

probe 25: single stranded DNA shown in SEQ ID No. 25;

probe 26: single stranded DNA shown in SEQ ID No. 26;

probe 27: single stranded DNA shown in SEQ ID No. 27;

probe 28: single stranded DNA shown in SEQ ID No. 28;

probe 29: single stranded DNA shown in SEQ ID No. 29;

probe 30: single stranded DNA shown in SEQ ID No. 30;

probe 31: single stranded DNA shown in SEQ ID No. 31;

probe 32: single stranded DNA shown in SEQ ID No. 32;

probe 33: single stranded DNA shown in SEQ ID No. 33;

probe 34: single stranded DNA shown in SEQ ID No. 34;

probe 35: single stranded DNA shown in SEQ ID No. 35;

probe 36: single stranded DNA shown in SEQ ID No. 36;

probe 37: single stranded DNA shown in SEQ ID No. 37;

probe 38: single stranded DNA shown in SEQ ID No. 38;

probe 39: single stranded DNA shown in SEQ ID No. 39;

probe 40: single stranded DNA shown in SEQ ID No. 40;

probe 41: single stranded DNA shown in SEQ ID No. 41;

probe 42: single stranded DNA shown in SEQ ID No. 42;

probe 43: single stranded DNA shown in SEQ ID No. 43;

probe 44: single stranded DNA shown in SEQ ID No. 44;

probe 45: single stranded DNA shown in SEQ ID No. 45;

probe 46: single stranded DNA shown in SEQ ID No. 46;

probe 47: single stranded DNA shown in SEQ ID No. 47;

probe 48: single stranded DNA shown in SEQ ID No. 48;

probe 49: single stranded DNA shown in SEQ ID No. 49;

probe 50: single stranded DNA shown in SEQ ID No. 50;

probe 51: single-stranded DNA shown in SEQ ID No. 51.

2. A kit comprising the probe set of claim 1.

3. Use of the probe set of claim 1 or the kit of claim 2 in any of the following:

p1, identifying or assisting in identifying bacillus anthracis;

4. A use according to claim 3, characterized in that: the strain to be tested is any strain including, but not limited to, a bacillus cereus family strain.

5. The use according to claim 4, characterized in that: the bacillus cereus family strain is bacillus anthracis, bacillus cereus or bacillus thuringiensis.

6. A method for detecting or assisting in detecting whether a strain to be detected is bacillus anthracis, comprising the steps of:

(A1) Performing whole genome sequencing on the strain to be tested;

(A2) Aligning the sequencing result with the sequence of each probe in the probe set of claim 1;

7. The method according to claim 6, wherein: the strain to be tested is any strain including, but not limited to, a bacillus cereus family strain.

8. The method according to claim 7, wherein: the bacillus cereus family strain is bacillus anthracis, bacillus cereus or bacillus thuringiensis.

9. A method for detecting or aiding in the detection of a target individual for non-disease diagnostic purposes of bacillus anthracis, comprising the steps of:

(C2) Aligning the sequencing result with each probe in the probe set of claim 1;