CN112980974B - Yersinia pestis identification method based on chromosome specific probe - Google Patents

Abstract

The invention discloses a yersinia pestis identification method based on a chromosome specific probe. The invention provides a probe set for identifying or assisting in identifying Yersinia pestis, which consists of 97 probes shown in SEQ ID No.1 to SEQ ID No. 97. The probe provided by the invention is used for detecting the sequencing result (including the second generation or the third generation) of an unknown sample, as many as 97 specific probes are adopted, the probe can be judged to be positive as long as any one probe is detected, the more the probes are detected, the higher the reliability is, and the probes are uniformly distributed on the whole chromosome, so that even if the sequencing quality is poor and the genome sequence with low coverage is ensured, the electronic probes can be used for inquiring, and whether the sample carries the plague bacillus can be quickly, simply and efficiently identified.

Description

Yersinia pestis identification method based on chromosome specific probe

Technical Field

The invention relates to the technical field of biology, in particular to a yersinia pestis identification method based on a chromosome specific probe.

Background

Plague is a zoonotic infectious disease caused by yersinia pestis. Plague bacteria belong to pathogenic bacteria of class A infectious diseases, and people are infected by contacting infected animals, eating contaminated food or being bitten by flea animals, and the like, so that glandular plague, fatal pulmonary plague and septicemia plague are caused. The territory of China is vast, and 12 known natural plague areas of plague basically have plague epidemic situations every year, which causes social panic and influences the normal production and life of people. In the face of the plague epidemic situation which frequently appears and relates to a wide area, the selection of a rapid and accurate plague bacteria identification method is of great importance.

The traditional identification method is a culture identification method, and pathogens are identified by biochemical indexes after being cultured, such as a sugar alcohol glycolysis test, a nitrite generation test, a pigmentation test and the like, but the traditional identification method is time-consuming and labor-consuming. With the rapid development of molecular biology, Polymerase Chain Reaction (PCR) is a conventional technique for detecting plague bacteria. However, the plague bacterium has evolved from yersinia pseudotuberculosis, and the homology between the two DNAs is high. The plague bacterium carries 3 plasmids, namely pCD1, pMT1 and pPCP1, the pCD1 plasmid is shared by plague bacterium and pseudotuberculosis bacterium, and the pMT1 and pPCP1 plasmids are obtained exogenously by the plague bacterium in the evolution process and are closely related to the pathogenicity of the plague bacterium. These factors present a significant obstacle to the molecular identification of plague bacteria. The targets previously identified were sequences on unique plasmids that were not able to be identified efficiently when encountering atypical strains (e.g., strains with lost plasmids). There are also some primers on the chromosome to distinguish the two bacteria, but the number of strains used is too small and the specificity of the primers is limited.

With the increasing number of isolated strains and the rapid development of sequencing technologies, yersinia bacterial genomic libraries become more complex. The plague bacteria genome database in NCBI contains some non-plague bacteria genome data, and the data comes from simple sequence comparison and is mixed into the plague bacteria genome database to misguide related personnel to carry out subsequent analysis. The establishment of a simple, clear, rapid and efficient method for identifying plague bacteria is urgently needed.

Disclosure of Invention

The invention aims to provide a yersinia pestis identification method based on a chromosome specific probe.

In a first aspect, the invention claims a probe set for identifying or aiding in identifying yersinia pestis.

The invention claims a probe set for identifying or assisting in identifying Yersinia pestis, which consists of the following components:

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

and (3) probe 2: single-stranded DNA shown in SEQ ID No. 2;

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

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

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

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

and (7) probe: single-stranded DNA shown in SEQ ID No. 7;

and (3) probe 8: single-stranded DNA shown in SEQ ID No. 8;

and (3) probe 9: single-stranded DNA shown in SEQ ID No. 9;

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

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

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

and (3) probe 13: single-stranded DNA shown in SEQ ID No. 13;

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

and (3) probe 15: single-stranded DNA shown in SEQ ID No. 15;

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

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

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

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

and (3) probe 20: single-stranded DNA shown in SEQ ID No. 20;

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

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

and (3) probe 23: single-stranded DNA shown in SEQ ID No. 23;

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

and (3) probe 25: single-stranded DNA shown in SEQ ID No. 25;

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

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

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

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

and (3) probe 30: single-stranded DNA shown in SEQ ID No. 30;

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

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

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

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

and (3) probe 35: single-stranded DNA shown in SEQ ID No. 35;

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

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

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

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

the probe 40: single-stranded DNA shown as SEQ ID No. 40;

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

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

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

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

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

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

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

and (3) probe 48: single-stranded DNA shown in SEQ ID No. 48;

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

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

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

the probe 52: single-stranded DNA shown in SEQ ID No. 52;

the probe 53: single-stranded DNA shown in SEQ ID No. 53;

the probe 54: single-stranded DNA shown in SEQ ID No. 54;

probe 55: single-stranded DNA shown in SEQ ID No. 55;

the probe 56: single-stranded DNA shown in SEQ ID No. 56;

the probe 57: single-stranded DNA shown in SEQ ID No. 57;

the probe 58: single-stranded DNA shown in SEQ ID No. 58;

the probe 59: single-stranded DNA shown as SEQ ID No. 59;

a probe 60: single-stranded DNA shown as SEQ ID No. 60;

the probe 61: single-stranded DNA shown in SEQ ID No. 61;

the probe 62: single-stranded DNA shown in SEQ ID No. 62;

and (3) probe 63: single-stranded DNA shown in SEQ ID No. 63;

the probe 64: single-stranded DNA shown as SEQ ID No. 64;

a probe 65: single-stranded DNA shown in SEQ ID No. 65;

the probe 66: single-stranded DNA shown in SEQ ID No. 66;

the probe 67: single-stranded DNA shown as SEQ ID No. 67;

the probe 68: single-stranded DNA shown as SEQ ID No. 68;

the probe 69: single-stranded DNA shown in SEQ ID No. 69;

and (3) probe 70: single-stranded DNA shown in SEQ ID No. 70;

the probe 71: single-stranded DNA shown in SEQ ID No 71;

the probe 72: single-stranded DNA shown in SEQ ID No. 72;

the probe 73: single-stranded DNA shown in SEQ ID No. 73;

the probe 74: single-stranded DNA shown in SEQ ID No. 74;

probe 75: single-stranded DNA shown in SEQ ID No. 75;

the probe 76: single-stranded DNA shown in SEQ ID No. 76;

the probe 77: single-stranded DNA shown in SEQ ID No. 77;

the probe 78: single-stranded DNA shown as SEQ ID No. 78;

the probe 79: single-stranded DNA shown as SEQ ID No. 79;

a probe 80: single-stranded DNA shown as SEQ ID No. 80;

a probe 81: single-stranded DNA shown in SEQ ID No. 81;

the probe 82: single-stranded DNA shown in SEQ ID No. 82;

the probe 83: single-stranded DNA shown in SEQ ID No. 83;

the probe 84: single-stranded DNA shown in SEQ ID No. 84;

probe 85: single-stranded DNA shown in SEQ ID No. 85;

the probe 86: single-stranded DNA shown in SEQ ID No. 86;

the probe 87: single-stranded DNA shown in SEQ ID No. 87;

the probe 88: single-stranded DNA shown in SEQ ID No. 88;

and (3) probe 89: single-stranded DNA shown in SEQ ID No. 89;

and (3) probe 90: single-stranded DNA shown as SEQ ID No. 90;

a probe 91: single-stranded DNA shown in SEQ ID No. 91;

the probe 92: single-stranded DNA shown in SEQ ID No. 92;

the probe 93: single-stranded DNA shown in SEQ ID No. 93;

the probe 94: single-stranded DNA shown in SEQ ID No. 94;

and (3) probe 95: single-stranded DNA shown in SEQ ID No. 95;

the probe 96: single-stranded DNA shown as SEQ ID No. 96;

the probe 97: single-stranded DNA shown in SEQ ID No. 97.

In a second aspect, the invention claims probes for identifying or aiding in identifying yersinia pestis.

The probe for identifying or assisting in identifying yersinia pestis claimed by the present invention may be any one of the probe 1 to the probe 97 of 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 or probes as described in the second aspect.

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

p1, identification or assisted identification of yersinia pestis;

p2, preparing a product for identifying or assisting in identifying Yersinia pestis;

p3, detecting or detecting in an auxiliary way whether the strain to be detected is Yersinia pestis;

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

p5, detecting or assisting to detect whether the target individual carries Yersinia pestis;

p6, preparing a product for detecting or assisting in detecting whether the target individual carries Yersinia pestis.

In a fifth aspect, the invention claims a method for detecting or assisting in detecting whether a strain to be detected is yersinia pestis.

The method for detecting or assisting in detecting whether the strain to be detected is yersinia pestis claimed by the invention can be the method A or the method B.

The method A can comprise the following steps:

(A1) carrying out whole genome sequencing on the strain to be detected;

(A2) aligning the sequencing result with each probe in the probe set of the first aspect using bioinformatics software;

(A3) determining whether the strain to be detected is Yersinia pestis according to the sequence alignment result of (A2) as follows: if any probe sequence in the probe set exists in the sequencing result, the strain to be tested is or is candidate to be Yersinia pestis; if none of the probe sequences in the probe set are present in the sequencing result, the test strain is not or is not candidate for Yersinia pestis.

The method B may comprise the steps of:

(B1) hybridizing each probe in the probe set of the first aspect with the genome of the strain to be tested;

(B2) determining whether the test strain is Yersinia pestis according to the result obtained in (B1) as follows: if any probe in the probe set has a positive signal after hybridizing with the genome of the strain to be detected, the strain to be detected is or is selected as yersinia pestis; and if any probe in the probe set does not generate a positive signal after hybridizing with the genome of the strain to be detected, the strain to be detected is not Yersinia pestis.

In the method, the more probes are detected, the more reliable the result is, and the plurality of electronic probes can avoid the wrong identification caused by poor sequencing quality or artificial modification of bacteria.

The method can be a non-disease diagnosis and treatment method, such as a method for simply detecting whether the strain to be detected is Yersinia pestis, and does not relate to any disease diagnosis purpose.

In a sixth aspect, the invention claims a method of detecting or aiding in detecting whether a target individual carries yersinia pestis.

The method for detecting or assisting in detecting whether the target individual carries yersinia pestis claimed by the invention can be the method C or the method D.

The method C may comprise the steps of:

(C1) carrying out bacteria whole genome extraction on a sample to be tested of a target individual, and then sequencing;

(C2) aligning the sequencing result with each probe in the probe set of the first aspect using bioinformatics software;

(C3) determining whether the target individual carries yersinia pestis according to the sequence alignment result of (C2) as follows: (ii) if any one of the probe sequences in the probe set is present in the sequencing result, the target individual carries or is candidate to carry yersinia pestis; if none of the probe sequences in the probe set are present in the sequencing result, the target individual does not carry or is candidate not to carry Yersinia pestis.

The method D may comprise the steps of:

(D1) performing bacterial whole genome extraction on a sample to be tested of a target individual, and then hybridizing each probe in the probe set of the first aspect with the extracted genome respectively;

(D2) determining whether the target individual carries yersinia pestis according to the result obtained in (D1) as follows: if any probe in the probe set has a positive signal after hybridization with the genome extracted in the step (D1), the target individual carries or is candidate to carry yersinia pestis; if none of the probes in the panel hybridize to the genome extracted in step (D1) to produce a positive signal, then the target individual does not carry or is candidate for carrying yersinia pestis.

In the method, the more probes are detected, the more reliable the result is, and the plurality of electronic probes can avoid the wrong identification caused by poor sequencing quality or artificial modification of bacteria.

The method may be a non-disease diagnostic treatment, such as for simple detection of whether a target individual carries yersinia pestis (e.g. for customs inspection and quarantine), without involving any disease diagnostic purpose.

In each of the above aspects, the test strain may be any strain, including but not limited to a yersinia strain, such as a bacillus strain.

Further, the strain to be tested can be Yersinia pestis or Yersinia pseudotuberculosis.

According to the invention, through deep research on the whole genome sequences of the plague bacteria and the non-Yersinia pestis in the NCBI database, a python program is written by means of local blast, and a large number of screening comparisons are carried out, so that a conserved DNA region in the plague bacteria genome sequence is determined. The invention screens out specific probe sequences, the probes relate to reagents for in vitro diagnosis in medical examination instruments and services, and can be used for detecting the sequencing result (including the second generation or the third generation) of an unknown sample.

Drawings

FIG. 1 is a diagram of a specific tag acquisition concept according to the present invention.

FIG. 2 is a cluster tree of the entire genomes of Yersinia pestis and Yersinia pseudotuberculosis.

FIG. 3 shows the results of the Blast test of the probe of SEQ ID No.1 in NCBI's library.

FIG. 4 is a specific result example of Blast in a single strain of SEQ ID No. 1.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 establishment of Yersinia pestis identification method based on chromosome-specific probes

The Yersinia pestis DNA sequence described in the present invention is the chromosome sequence of the standard strain Yersinia pestis CO92 (GenBank: NC-003143, 13-DEC-2020), and the position of the nucleic acid sequence is also calculated using Yersinia pestis CO92 chromosome (GenBank: NC-003143, 13-DEC-2020) as a standard.

1. Yersinia pestis (539 strains, Table 1) genomes and Yersinia pestis (comprising 20 572 strains, Table 1) genomes in Yersinia genus (for convenience of description, hereinafter abbreviated as: Yersinia pestis and non-Yersinia pestis, respectively) are downloaded from NCBI genome database.

2. A python script was written to truncate a Yersinia pestis CO92 chromosome sequence (GenBank: NC-003143, 13-DEC-2020) fragment (step size 1 bp; length 100bp) and to truncate a total of 4829756 fragments 100bp long, as shown in FIG. 1.

3. 4191017 tags appearing in non-plague bacteria were removed by local blast (blast-2.7.1+), leaving 638739 tags.

4. A Python script is written, the 638739 labels are inquired about the labels appearing in all the plague bacteria (Yersinia pestis 539 strain), only 1341 (0.21%) of 638739 labels are detected in the genome of one strain of the bacteria (Assembly ID: GCA _902387395.1, UHGG _ MGYG-HGUT-02476), and the label detection rate of other 538 strains of the plague bacteria is 62.36% -100%, and then the strain is found to be not the plague bacteria (figure 2) but Yersinia pseudotuberculosis (Yersinia pseudouberculosis) which is close to the plague bacteria through whole genome cluster tree analysis, so the strain is removed from the genome training set. As a result, 35680 tags were obtained in the present invention which were found in all of the plague bacillus (538 strain).

5. The 35680 labels are merged according to the overlapping of the fragments to obtain 833 specific fragments, and the length of the fragments is between 100bp and 411 bp.

6. Using 833 DNA fragments, local Blast is carried out to inquire non-plague bacillus (572 strains), and a DNA sequence corresponding to each strain is intercepted according to Blast results and made into fasta format.

7. And (3) comparing the files obtained in the step (6) by using megaX software, searching SNP sites capable of distinguishing the plague bacteria from all other Yersinia nervosa, and finding that 483 fragments contain the SNP sites.

8. In order to facilitate application and improve detection specificity, we select a sequence containing at least 2 (including more than 2) SNP sites from the 483 fragments, and intercept the sequence containing the SNP sites with the length of 100-150bp as an electronic probe (Table 2), and find that 97 probes can be used for rapid identification of plague bacteria.

9. In order to test the specificity of the 97 electronic probes in a larger database, the 97 electronic probes are subjected to online blast Query (https:// blast. ncbi. nlm. nih. gov/blast. cgi), the test results are shown in fig. 3 by taking SEQ ID No.1 as an example, and the results show that the electronic probes can only achieve 100% of Query Cover and per.identity (percent of Identity) values in Yersinia pestis, specifically show that the sequences are completely consistent as shown in a in fig. 4, and cannot meet 100% of the Query Cover and per.identity values in other strains, as shown in B and C in fig. 4.

TABLE 1 Yersinia species and numbers used

Note: indicates that the strain is a misidentified plague bacterium, actually yersinia pseudotuberculosis.

TABLE 2 specific electron probe sequences

Note: the start and end positions in the table were calculated using Yersinia pestis CO92 chromosome (GenBank: NC-003143, 13-DEC-2020) as a standard. The bases in brackets [ ] in the table indicate SNPs, the first base refers to a nucleotide in the genome of plague bacteria, and the second base refers to a nucleotide in the genome of other yersinia bacteria. The probe sequences correspond to sequences in the genome of the plague bacterium, and therefore the bases in brackets [ ] in each probe sequence are the first bases.

11. By using the 97-strip plague bacterium specific probe and the method, whether the strain to be detected is the plague bacterium or not and whether the target individual carries the plague bacterium or not can be simply and quickly identified.

The method comprises the following steps:

(1) collecting a strain to be detected or a sample from a target individual, extracting the genome of the strain, and extracting the whole genome of the bacterium according to the steps of a Bacterial Genomic DNA Extraction kit;

(2) sequencing the genome sample by using a sequencer (the second generation or the third generation can be both);

(3) comparing and analyzing the sequencing result with the probe sequence by using bioinformatics software;

if the sequencing result is 100% identical to any one of the probes (namely, the sequence is completely identical) (not only a blast method can be adopted, but also a python script can be written for realization), the detection result is positive, the sample carries the plague bacteria, the more the detected probes are, the more reliable the result is, the plurality of electronic probes can avoid the error identification caused by poor sequencing quality or artificial modification of the bacteria, and if all the probes are not detected (namely, the sequences are not completely identical), the negative result is obtained.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

<110> military medical research institute of military science institute of people's liberation force of China

<120> Yersinia pestis identification method based on chromosome specific probe

<130> GNCLN210502

<160> 97

<170> PatentIn version 3.5

<210> 1

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

ttgttctgga ttatcggtaa cgttctcact gtttcaacaa tgaattcaat ggattgtctg 60

atgatgatca gcattaaaag gaagtaagct gatgactatt 100

<210> 2

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

acgatgcggt agctgctgga aaccatggtg ccgagtagca acatcagcat catgaatagc 60

aggttgctgg cattctgaat tatcagcata gctgtcttat 100

<210> 3

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

tagctgcttt tattctggtt gccagccagc gaggggagga aagcccaatg accaagcaag 60

tgacatttat ctgcgggctt cttaatggcg ttcatgcccg 100

<210> 4

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cctgcgagcc atatagaaag ggtatgcaac cgcttccaat cgcgtttcca ctggtataat 60

ccgcgttctg gcatcgtggg agatggcaag agcgtacttt 100

<210> 5

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

aatcaaggtg ccgaatccat tggcttgttt cgtactgaaa tattgtatat ggatcgtgca 60

gccgcgcctt cagaagagga gctttatacc ctctacgctc aggcacttgg cgctgccaag 120

ggcaaaccga ttattattcg caccatcgat 150

<210> 6

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

tttttccaaa ctggcgcgtc ggatcatgca cgaggggttc cgcgatgcgc tggttagcgc 60

tgggacggcc cagcaagtag aaatgctgct gaaacacgag 100

<210> 7

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ttttggttaa aacataatgg cagtggattg agtgcggaac aggtaaaagt gcttaatcgc 60

ctattagatg gcggtgagaa tggcttttct gctgggataa atgcgtcaca atatcagaaa 120

gtcgcaaagg tcagtaaagc aaccgccacg 150

<210> 8

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

tttctcggcc catggtgtgt cacaggccgt tcgggcagag gcccgttcac gtaatttgac 60

gatgctgttt gatgcaacct gtccattggt taccaaagtc 100

<210> 9

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

aaaatgcggc tttgagtgag atgggatgct gtgcagtatt atcaatacta aacttacttt 60

ctccccgtga cagcagtgct tgaataccaa tccagatcag gtagcaggcc ccggcatatt 120

tcagcaaatt gaataaccat ggtgtggtcg 150

<210> 10

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

gatctttctg gccacagagc aaagcctctt cctgctgttg aagatcgtaa attccttact 60

ttcgatcagg ataatttcga tgcccggatg agagcaatta 100

<210> 11

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

gccgatattg cggtgtactt gttcctcaac aatatcgata agttacctgg cggcgaaacc 60

ttcctctcgt taatgaacga cgaacttctg gccggagggc ttgggattag catgacccat 120

aatgaaaagg ggcagaacaa tttcgttgcc 150

<210> 12

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

gaaatttgag ttccagagga aaatttccgg tgttggaagc ggtacgcaaa acataatcaa 60

tggatacaga aatacaggga ggtctgcgat acgactcgca 100

<210> 13

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

attcacttgc cgtatgactg caacctcgat ggcttttggt atttataagc attctacttc 60

ccaccccgaa tcagcccacc taaaccacgg cgctccataa 100

<210> 14

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gggggcggcc tcttgcccta accgacgacc aaaccagatc tcagcggtta aatcccggat 60

ccggttaaac agcacgctgt ggttatgagt ctcatcgctt ttaaccagaa tcaacaccgc 120

ttctggctcg ttgaagcccg tcagatagct 150

<210> 15

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

gcccgtcagt tagtgcagca agccgcttcc ggggcttgtg ctctggtcga ggctaaccca 60

cacgtgccac tttcggcctt gcgagagcag gtcacgtcca 100

<210> 16

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

cagccgtcag tgctgctgat agcgatcagc gggaaaatat cgatagccgc gcgctgttca 60

ttcaagactc gatgcgccta aacgagcact ggctattact cggtggcctg cgctacgaca 120

gcttcgatgt gatggcaggt aaaggccgcc 150

<210> 17

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

tggtgcccga gcacagtttg ctgcggtgcc attgaacttg cttggtgacc gcaacagcgc 60

tgactatatc gatggtggct attgggcgca cagtgcgatt 100

<210> 18

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gcgaaatgga aaagcgcaat caggcgaaag ctgaactatt gtacggtgcc atcgaccgca 60

caggcttcta tcgcaatcag gttgctatta ctaaccgctc 100

<210> 19

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

gtcattcaag gtattcgggt agttacgggc gcggaagcgg aataacaggg cctccaaccg 60

tgggtcctga aatgtcaaat ccagtgctgg cagattttgt gactcagttt gctggataat 120

ctttatggtg gcgcgatcag catcactgaa 150

<210> 20

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

gttgcgcccg atcacttaaa tcttgcacca acttcaattt ggttggccct tccaaacggt 60

aaatctgtgg ttgccgttgt aacagcccaa tcaaatacac 100

<210> 21

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

tagttgtagt gatatcacta ttatctatca attatacgag gctattgagg ttcgattggt 60

tttgttaaag gtttgtatat gatatgtgtt gtgtattaac 100

<210> 22

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

tggctcttta attgcgtctg gatagaacgc aatgtgccat ccccgactaa tgcaccatta 60

ctcgtggatt gatcctggcc tgtctctaca acggtatatt 100

<210> 23

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

ttttggccgg gttgagtaat cgtgatcgtg cgtgttgcat cactgctacc caatttatct 60

gtagcgctag gcacatctgc cgataacagc gattgtgctt 100

<210> 24

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

aatataaagg tgaagtggat gctactaatg gaaaagtaac atttgcagat gatgctaatg 60

gcgatccaat tgatgacgct accaagctgg aagcagcggc 100

<210> 25

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

acgaaaggcg atcgctctat gtacccattc gcaacagcga actccatcat cgcggcaaaa 60

caagaaaggt agacgtttac cgttctgaca gaccgacctt 100

<210> 26

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

caaaggagta acggtcaact ttggttaaat ctgcaacata gcggctaccg ggttcgcaaa 60

tacgcgctag atcgttacga gccagatcca ccaacattaa atgttctgcc atctctttat 120

ggtcagtacg catttccaac tcgatacggc 150

<210> 27

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

aagaataccc gcaagattct gcgtagcggt gaagttgcac caccgaaaga agatccggta 60

ccattgttag agttgccttg cgagaaatca gatgcttatt tcgtcctacg agatggcgca 120

gctggcgtgt tcttggcagc caatacattc 150

<210> 28

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

agatcaacgg catcagtccc gacaccaaat attgagatga tctgcacttc aggcaataac 60

gccaggacct cattagtgac gccaatatca cctctagtca 100

<210> 29

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

ttatcattac cgaagctgaa ttgtttcagc tacagaatag gcgttaacaa tctctgtcgc 60

accttagggg ggaatgttat tggttgatac gcattgttat acgttggcgt cacctgttaa 120

caggcgataa ttgatgagcc gatgctctac 150

<210> 30

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

ggcttcgcta tccgccgccg gtgcgtacat aaacaagcgc atcactacgg caaagatagc 60

aatcttgctg gcggtcgcca ggaacgtgga aactggcgcg ggtgcgccct gatacacatc 120

gggtgtccac aattggaacg gtaccaacga 150

<210> 31

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

cctattcctg aaaggattcg gttgggcacc gttcttaaaa taccaaatag tgaaccgacg 60

aagaaggcga tcacccaagc acataatgat aatgcgaccg 100

<210> 32

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

cccccttgat gaacctcatg gctctggtct gcacattcac ggtattcact cccacgaaga 60

aggccatagt catggtgatc acgatcatga ccacagccat 100

<210> 33

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

tcgggaccag tcgctggctg acagcacggc aacgccagag tgaaccaatc aataaaccca 60

cctgcaattt tcatcatttt cattaccacc attggtgaaa 100

<210> 34

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

ccccctcaat gggcgtgatc caacttaagc caaatctgaa ggaaggagat ttgactcaag 60

ggtattgtgt tcccaggggc agctcgttct tcacccatat cgcacctggc gagacaaccc 120

gctgcgagtt ccgcagtggt caggatgtgg 150

<210> 35

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

ggctccttat ggcgatagct tgcgctacct ctctattgaa gctcaggtga ctaatcggga 60

tcttccgcgc ctgatttccg ccagtggtaa ttttgaattg accatgtcgg attcagcgcc 120

cgttcacggt gcccggtttg tcttccaacc 150

<210> 36

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

ccccatcccc acagttgcag ccgttacagt tgcaatcggc acagttgcaa cctgtaccga 60

tacagtcaca gccggtgcct gtccagcagc aagggatcca 100

<210> 37

<211> 120

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

cgtatcctgc aacttcacac ttcagattcg aactctataa aattaaagat acgttgaatt 60

taactgatac caacggcgaa cgtgttctgc cgggtggcac aatagcttat acctgggcga 120

<210> 38

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

ccgataataa ttacatcaag gtaaaagaca gccagaacca agaagaccgg ttgcttatca 60

aaatcagcga caccaacggt caacagatga aagtgaatgg 100

<210> 39

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

cccccaagtg tcgactccca ttccgcagga gacagcggaa cctgtagaac aggagagtac 60

ggttatttta attctcggtc cctatgctga gaaatggttt 100

<210> 40

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

ccccctcccc tgcgtatttg ctatttacgc gcagttgagt aatgaacgcc ttagccataa 60

ttctgataac gcatattgga ctggaaatat taatcttgcg aaccagcagc ctatcgagat 120

tacgcaagcc ctacagtcac tgagccaacg 150

<210> 41

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

tgagccaacg acttgagtta caagacatca atcactcaag atttgcaact cagcgtaatg 60

ttatggcgca taatttattt acttggttga atgaatcggg 100

<210> 42

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

atctctggcg cgggagtatt ggcttccagc aaacatccgc agcaagccca gaaattcgtg 60

caatgggtca ccggcaaagc ggggcaggat ttcttgcgcg agagcaatag cgcctttgaa 120

tatgcggttg gcgtgaatgc cgcttccaat 150

<210> 43

<211> 140

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

ttgattacct ttgaataccc ttggcccttg acgttgcaac ggtgttcacc gctctcatga 60

acccgaatga ctgacgcctg tcagctcatc ggggtttgcg cgttagcggc cttgctgcaa 120

caacaatgac ttgggtcggt 140

<210> 44

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

tagtccgcca gcaatttcag gtttaggtgg gtcgtcaggt ttcgatatgg aattacagga 60

ccatggcggc catggccatg ataaattgat ggtcgcccgc aaccaattgt tgcagatggc 120

gtcgcaggaa cccgcgttaa cccgagtgcg 150

<210> 45

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

gcacaagcgt gccaatcaga gaagaataga tcttatgatt atcgattaac ggctcagcta 60

aacctagtga ctagcaaacg tgttacagct attaggatca 100

<210> 46

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

aaagcagtta ctcgatagct tgggtttggc aactgcgccg tggcaattgt tatccagcgc 60

cagtgaatgg cctgaggtgt tcgccacgtt gggtgagcta 100

<210> 47

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

accaacctgt ttcaacatcg caccgatgtc taacaaactc agtgttggga actggcgatt 60

taactgaacc aaagcctctc catcaccgtg atagcggaag 100

<210> 48

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

ccccaccagc aacgaaatca atactaatga tcccattgac cacacccatg gccatagccc 60

ggcagtgggt aactctgcgg gcaataccgg cgctaataac caaaccaata gcctctcaaa 120

taagagacca atgacactgc cgcagaggcc 150

<210> 49

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

tttccaaata attatgtagg tgacaattgt atacctgcat aattaattag caatcaacca 60

gactcgctcc acacattaca tacattacca atcaaccccc 100

<210> 50

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

accagtgacg gggtttgcgt tccatcaagt acatgatggg cgacgtcctg atacaacgct 60

tcacgactag ccaacacatc cagtatttct tcaacgatgg 100

<210> 51

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

ttaaggaagt tatttcgctc gaatcgcaat gggtgcgagc acccatttcc ggcttcatta 60

aggttagtga tcacatgatc ggcatggcca aaagtcgatg 100

<210> 52

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 52

gcaatgaaaa cacaactggt gcctctacca cgggtgttaa ttcaactgac ttatccgcca 60

ttacccgatc tccctatcag ctcagggctt accctctatt 100

<210> 53

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

tttacccacg tcgatgccgg tgccatggac cgtgtcgcca tgcaccacgc tgccatggtt 60

accggacaga ttggccgcca gatccacgcc cttgccctcg gtactgttgc cattgagcgt 120

agcgttatcc agcgtgctct cgccatcgat 150

<210> 54

<211> 120

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 54

agtgcctgtt gtcgccacac cttgtagaat caacaagtca gcttcactgg tcccggtaac 60

gtttgaattt tcgccaatat caataccgta gcccgtgcca gtagtcgttc cgttaatcga 120

<210> 55

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

gctgggtatc ccaaatgacg cccttccatc gccgctgcta cggtacccga atagataaca 60

tcatccccta aattagggcc agcattaata ccagagacaa 100

<210> 56

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 56

gcactaccaa gccaatcaca agtagaagcc gagcaactta aacaaatcgg cgccaaagat 60

tgtctgctga aacccatatc gtctatcagg cttatccctt tactcttggc cgaagacacg 120

cacagtaaag taccatcaga ggagcaacca 150

<210> 57

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

taacgccaac ttgccaccct ttagcgccaa cttactgtca gtgaactcag attcgcggtc 60

aattagccct aattcgccac cgactcacgc caaatcgctg 100

<210> 58

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 58

attttgatgg ccgcctggtt gggtaaagct cgtctgattg ataaccaact ggttgatttg 60

cgccactgat attttaccga tcaggcggtt gctcattccc ctcaagccgc gagttggccc 120

aaaatcaacc aggaacgcca aggtaaaagt 150

<210> 59

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

ggggaagcca gattgggcat aaagaatcaa tcaaggatac cgcccgtgta ttaggccgta 60

tgtatgacgg cattcaatat cgtggctatg ggcaacgcgt 100

<210> 60

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 60

aacactcgca gaatttgccg gcgtaccggt ctggaatggc ctgaccgatg agtttcatcc 60

aacccaactc ctggctgatt tgctgactat gcgggaacac ctgcccaata aatcgctgaa 120

taaaatgacg ttggcgtacc tcggtgatac 150

<210> 61

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

agcggccgca ctggtaggaa tggatttacg cctagtcgca ccgaaagcct gttggccaga 60

ggaagccttc gttatcagtt gccaggcact ggcacagaaa 100

<210> 62

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 62

aaaagggtca ggtcatcccg caagccgaca acaattggcg tctggcaatt cttgatagca 60

agcaaccttt ggatattcgt tttgaaacct ctccaactgc 100

<210> 63

<211> 130

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 63

gcttcgccgt ttatcacatc gatttacaga gaaagtaatc tctggcggca acgccttaca 60

ctgtacgctg ctgtcacccc ataataccca cttggagtta tatcacaatc tataacatgc 120

atttattatg 130

<210> 64

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 64

catcacagcg cctaacttgt ggtgatgggg cctttgtgga agaagaacaa cacctagtaa 60

ttaaagcgat aaccccactg cgcgccctct tgattgattt 100

<210> 65

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 65

tcgagccaaa cttcataacc ttgttgttga taactgctca gcaaatccag aacggcatcg 60

ggcacacaat ccgggcgagt ccccacgcac aggcccacaa 100

<210> 66

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 66

ttttgatgca gtattgaccc gtgaacgcat gggtagcacc gggattggca gtggtatcgc 60

aatacctcat ggcaagttgg aagaggatac actgcgcgcg 100

<210> 67

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 67

tatgatatcc ctttccgcat tacgtaaatc aggagtcaag atgttacgtg ctatcaaaac 60

taaactcgcc cctgctgcca ttggccctta tgttcaaggt gtcgatctgg gcagcatgat 120

catgacttcc ggtcagatcc cgatcgatcc 150

<210> 68

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 68

ttcctttgcc agccaggagt acggggaaat acgggcttta tcgcggcgtg gataattgat 60

tgtcacactg tgtagtgggt gaccatattc attgcaacgc agcacaatct gctggctgca 120

ttgcgggtct tgcgcgatac gttcgtagtg 150

<210> 69

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 69

ttttcatcca accagaattg gacagaactg cggtaaagca actgactctc tgcgcccata 60

ttattgttgg tggtgcacag taaatagggt ttactggcga 100

<210> 70

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 70

atgcctgttg gcaacgcgat agccagtggt tcggcaaagc ggttaccact ctggttgcgg 60

taaatgagca gttggctggt ggtggcataa ataaggtcaa tggtaccgga gccatcaata 120

tctgccagat agatcgctag tggattaaat 150

<210> 71

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 71

aagccatagt taaagtgtga gaaggcgtcc tgacggaaat tccaggattt tccttttatg 60

attaccggca cttgctccgc gatattactg tattcgccgt 100

<210> 72

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 72

aagcttgatc gccttggcta ataatcaagt ctaaggtacg cataaagatc gccagcttat 60

attgcatcgg atcttgcatg gcaataacat ccggatcatc 100

<210> 73

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 73

attgtacaaa aatctctgtt agactcagac ctatggcgtt ctctatttta tccgtataag 60

tcgtcgtatt actacgtcgc cacggcactt ttctgaacga 100

<210> 74

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 74

tcatcaccac ggcgaatagc aacaatcaca caaggagcaa tttgcggata atagcgctcc 60

cgacagtggt tacataagct ggcccattcc gtccggctgg 100

<210> 75

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 75

ccatcccggt ctatgacacc tccggtccct atggtgaccc acaagctaaa ctggatgttc 60

ataacgggct gcctaaactg cgtgccgctt gggtcgcaga tcgccaagat actgaagcgc 120

tggcatctgt cagttccggc tttacccaac 150

<210> 76

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 76

ccgttatcga gagcaaagct aacggattga ttagctaata ggttgtcatg agcatcggta 60

accaacgctt gtgcaatgtt accggcttta ccattcgcaa gctggttatc caagaacgac 120

gttagcttaa gctgggctgt cgaaacatcg 150

<210> 77

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 77

cacccgagac ggtatgcgtc agtgtggtac tggcagtacc attagctcct gttcttgcct 60

ctgtcgtact taatgttgcc tgagtattaa ccgcaaagtt gatgagctgc ccgataatcg 120

gttggccgtc gccatcggtt actgtggctt 150

<210> 78

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 78

cctgtaattg gcatgtcgat agttgcacgc tgaccaaagt tgtcagaagc agcgagtacc 60

gcaggatttg acgtcactcc agcttatctt tttcacgtgg cccaagagag gaagcctgag 120

tatgatgaac ctgtctttca cgcatgttga 150

<210> 79

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 79

ggtaaccatc attaaattca tactcacggc aaacccgtgt ccaggcagca taggcgctga 60

ctatacgttc atactcttcg gggcgaagat tatctctgga taagttgtac aaatagatgc 120

cgagaaggct taggctcatc tcaactcctg 150

<210> 80

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 80

aattacatca ggctcatccg ggccgggttc ttccaaggcg tcaaattggc ttctcaataa 60

atcgcttggc ataaaatggc cggaacgtgc ttgcaaccgc 100

<210> 81

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 81

cattgccacc attttcaacg ccgatatggg caaaacgctg ctgtacggca cgttgctggc 60

aatcccaacc gtgattttgg caggcccggt gtacgcccgc tttctaaaag gaattgataa 120

gcctgttcct gaagggttgt ataacccgaa 150

<210> 82

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 82

aaaataatgt tcattcggcg cggtaatggt ttatttaact gttctcacga ataaacacgt 60

ctaattccgc cagtggcttt gcaccatcaa tagcattggc 100

<210> 83

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 83

tttattcata acgcggtgcc tgtgggtgaa tgtcgatagc cgctagtcaa tcacaagccg 60

gtaaatgcaa aatgtcagaa gccgttcata attgtcataa agttgaatag ttaaccgaga 120

tcacattccc gatagggaaa tcatcgctag 150

<210> 84

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 84

aacttgctcg tggtggcggt caccctgatg gtgagtggtg tcgcggtgct gggcttcgag 60

caggcaagcc gcttacaaaa gcaggtccag gagcggacac 100

<210> 85

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 85

tcagcccaga tgaccgcggc ggtctctatc ggtattgcca gttataccgg catgccagta 60

tcgaccactc aagtgctctc ttctgctgtt gccgggacca tgttggtcga tggcggtggc 120

gtgcagagta aaacggtgaa gagcattatg 150

<210> 86

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 86

tattagtgta ggaaatgaaa tggaaaatag cctgataatc tcgcatttaa gcctatcttg 60

ccgtttgttc aataccgatt aatgttaacg tctcattggt attgattgtt atcaccgtat 120

ttctcgcgct gatcaccgtg ctcaaacatt 150

<210> 87

<211> 120

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 87

tatgcctatt atcctaatcc aggtagcttc agcccaatat ggattaatta cagtttcagc 60

gataaccaac atcccagcag attaaatgat ggtggcggag tattaacaca tgagatcggc 120

<210> 88

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 88

ctcagcaagg cagtgcggct tcagtggcat cgatggcagc ctatttagga ttagacctca 60

cactgccacg cattgcgctg atcgtcagtc tgcgtgaaca 100

<210> 89

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 89

agagcacact cagcagcggg atctggtcga aatattaagc aaccacagcc gtgaaacatt 60

ggtcacctta catgggtttg agcgggttgt ggtgctattg ccgctgaacc ttgcacacag 120

tgacgatcgt gaagtgacgg ccagaaaggc 150

<210> 90

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 90

actggcactg gcagttgcga cgatgctgct gacagcggtc ggggtgttta tcgatgtcgc 60

ggtgattacc gtttccccaa tcgctctggc cattgcgcac 100

<210> 91

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 91

tgatactgcg ctgcctgcct ttggtgcagc aatggcggca cctctggtcg ccatcttcct 60

actggcatta cgccctatag ctggcattgc cattgacccc 100

<210> 92

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 92

tacaaccggg gattgagata gtcaccgaag cgctaaaatt agcggctacc gtgcaaaatg 60

ctgatttagt cattaccggc gaagggcgta tcgatagcca aaccatttac ggcaaaacac 120

cggtcggggt ggcgcgagtc gcgaagcgtt 150

<210> 93

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 93

gtcgcatttg agccacccgc tgaaatatag atacccgcac ccttggtcgc atgagtacta 60

ttaatggtgt tattcgcgcc cgttaagctc cctccggaac catccagata aatgccatag 120

ctaccactgc cactggtatt gagggtggta 150

<210> 94

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 94

cgttacggtc tggcaataac cgcactaccc tctggggatt ttttactgct ggccaacaaa 60

gcaagcctga taacccagga tcgcgcctca gcagaggagc tgcaagatca cacctgtcag 120

gtgctacctc acctcccggt gcaataacac 150

<210> 95

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 95

tcccggataa gaccaattgt agatccagcc ccgccagatt tccgggatcg gttaaggtcc 60

cgacgacggc caccagagta gaaccgtagc gcatatccgc 100

<210> 96

<211> 150

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 96

caaaaggccg ttgtaactcg gcggagactt tttggttaat cgttggcttc aagcgattcc 60

agtcaaaaga agagacaaat atcaccaccg ccaccaatga caacaggata aacccactgg 120

cgcccaccaa tacttttccg gttcttgtca 150

<210> 97

<211> 100

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 97

ttttgccacc cagataatgc agtgaaatga ttcgagtcta ccaacccgac gatctcaatg 60

ccctgatgca actgtggcta accagtacta ttgacgcgca 100

Claims (12)

1. A set of probes for identifying yersinia pestis consisting of:

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

and (3) probe 2: single-stranded DNA shown in SEQ ID No. 2;

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

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

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

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

and (7) probe: single-stranded DNA shown in SEQ ID No. 7;

and (3) probe 8: single-stranded DNA shown in SEQ ID No. 8;

and (3) probe 9: single-stranded DNA shown in SEQ ID No. 9;

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

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

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

and (3) probe 13: single-stranded DNA shown in SEQ ID No. 13;

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

and (3) probe 15: single-stranded DNA shown in SEQ ID No. 15;

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

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

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

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

and (3) probe 20: single-stranded DNA shown in SEQ ID No. 20;

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

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

and (3) probe 23: single-stranded DNA shown in SEQ ID No. 23;

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

and (3) probe 25: single-stranded DNA shown in SEQ ID No. 25;

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

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

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

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

and (3) probe 30: single-stranded DNA shown in SEQ ID No. 30;

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

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

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

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

and (3) probe 35: single-stranded DNA shown in SEQ ID No. 35;

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

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

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

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

the probe 40: single-stranded DNA shown as SEQ ID No. 40;

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

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

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

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

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

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

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

and (3) probe 48: single-stranded DNA shown in SEQ ID No. 48;

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

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

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

the probe 52: single-stranded DNA shown in SEQ ID No. 52;

the probe 53: single-stranded DNA shown in SEQ ID No. 53;

the probe 54: single-stranded DNA shown in SEQ ID No. 54;

probe 55: single-stranded DNA shown in SEQ ID No. 55;

the probe 56: single-stranded DNA shown in SEQ ID No. 56;

the probe 57: single-stranded DNA shown in SEQ ID No. 57;

the probe 58: single-stranded DNA shown in SEQ ID No. 58;

the probe 59: single-stranded DNA shown as SEQ ID No. 59;

a probe 60: single-stranded DNA shown as SEQ ID No. 60;

the probe 61: single-stranded DNA shown in SEQ ID No. 61;

the probe 62: single-stranded DNA shown in SEQ ID No. 62;

and (3) probe 63: single-stranded DNA shown in SEQ ID No. 63;

the probe 64: single-stranded DNA shown as SEQ ID No. 64;

a probe 65: single-stranded DNA shown in SEQ ID No. 65;

the probe 66: single-stranded DNA shown in SEQ ID No. 66;

the probe 67: single-stranded DNA shown as SEQ ID No. 67;

the probe 68: single-stranded DNA shown as SEQ ID No. 68;

the probe 69: single-stranded DNA shown in SEQ ID No. 69;

and (3) probe 70: single-stranded DNA shown in SEQ ID No. 70;

the probe 71: single-stranded DNA shown in SEQ ID No. 71;

the probe 72: single-stranded DNA shown in SEQ ID No. 72;

the probe 73: single-stranded DNA shown in SEQ ID No. 73;

the probe 74: single-stranded DNA shown in SEQ ID No. 74;

probe 75: single-stranded DNA shown in SEQ ID No. 75;

the probe 76: single-stranded DNA shown in SEQ ID No. 76;

the probe 77: single-stranded DNA shown in SEQ ID No. 77;

the probe 78: single-stranded DNA shown as SEQ ID No. 78;

the probe 79: single-stranded DNA shown as SEQ ID No. 79;

a probe 80: single-stranded DNA shown as SEQ ID No. 80;

a probe 81: single-stranded DNA shown in SEQ ID No. 81;

the probe 82: single-stranded DNA shown in SEQ ID No. 82;

the probe 83: single-stranded DNA shown in SEQ ID No. 83;

the probe 84: single-stranded DNA shown in SEQ ID No. 84;

probe 85: single-stranded DNA shown in SEQ ID No. 85;

the probe 86: single-stranded DNA shown in SEQ ID No. 86;

the probe 87: single-stranded DNA shown in SEQ ID No. 87;

the probe 88: single-stranded DNA shown in SEQ ID No. 88;

and (3) probe 89: single-stranded DNA shown in SEQ ID No. 89;

and (3) probe 90: single-stranded DNA shown as SEQ ID No. 90;

a probe 91: single-stranded DNA shown in SEQ ID No. 91;

the probe 92: single-stranded DNA shown in SEQ ID No. 92;

the probe 93: single-stranded DNA shown in SEQ ID No. 93;

the probe 94: single-stranded DNA shown in SEQ ID No. 94;

and (3) probe 95: single-stranded DNA shown in SEQ ID No. 95;

the probe 96: single-stranded DNA shown as SEQ ID No. 96;

the probe 97: single-stranded DNA shown in SEQ ID No. 97.

2. A probe for identifying yersinia pestis, comprising:

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

3. A kit comprising a set of probes according to claim 1 or a probe according to claim 2.

4. Use of a set of probes according to claim 1 or probes according to claim 2 or a kit according to claim 3 in any one of:

p1, identifying Yersinia pestis;

p2, preparing products for identifying Yersinia pestis;

p3, detecting whether the strain to be detected is Yersinia pestis;

p4, preparing a product for detecting whether the strain to be detected is Yersinia pestis;

p5, detecting whether the target individual carries Yersinia pestis;

p6, preparing a product for detecting whether the target individual carries Yersinia pestis;

the applications indicated by said P1, said P3 and said P5 are for non-disease diagnostic purposes.

5. The use according to claim 4, wherein: the strain to be detected is a yersinia strain.

6. Use according to claim 5, characterized in that: the strain to be detected is Yersinia pestis or Yersinia pseudotuberculosis.

7. A method for detecting whether a strain to be detected is Yersinia pestis for non-disease diagnosis purposes comprises the following steps:

(A1) carrying out whole genome sequencing on the strain to be detected;

(A2) aligning the sequencing result to each probe in the probe set of claim 1;

(A3) determining whether the strain to be detected is Yersinia pestis according to the sequence alignment result of (A2) as follows: and if any probe sequence in the probe set exists in the sequencing result, the strain to be detected is Yersinia pestis.

8. A method for detecting whether a strain to be detected is Yersinia pestis for non-disease diagnosis purposes comprises the following steps:

(B1) hybridizing each probe in the probe set of claim 1 with the genome of the strain to be tested;

(B2) determining whether the test strain is Yersinia pestis according to the result obtained in (B1) as follows: and if any probe in the probe set has a positive signal after being hybridized with the genome of the strain to be detected, determining that the strain to be detected is Yersinia pestis.

9. A method for detecting whether a target individual carries yersinia pestis for non-disease diagnostic purposes, comprising the steps of:

(C1) carrying out bacteria whole genome extraction on a sample to be tested of a target individual, and then sequencing;

(C2) aligning the sequencing result to each probe in the probe set of claim 1;

(C3) determining whether the target individual carries yersinia pestis according to the sequence alignment result of (C2) as follows: if any one of the probe sequences in the probe set is present in the sequencing result, the target individual carries Yersinia pestis.

10. A method for detecting whether a target individual carries yersinia pestis for non-disease diagnostic purposes, comprising the steps of:

(D1) performing bacterial whole genome extraction on a sample of a target individual, and then respectively hybridizing each probe in the probe set of claim 1 with the extracted genome;

(D2) determining whether the target individual carries yersinia pestis according to the result obtained in (D1) as follows: if any probe in the probe set has a positive signal after hybridization with the genome extracted in the step (D1), the target individual carries Yersinia pestis.

11. The method according to claim 7 or 8, wherein: the strain to be detected is a yersinia strain.

12. The method of claim 11, wherein: the strain to be detected is Yersinia pestis or Yersinia pseudotuberculosis.

Images