CN109762915B - Detection method of bacterial drug resistance gene and special kit thereof - Google Patents
Detection method of bacterial drug resistance gene and special kit thereof Download PDFInfo
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Abstract
The invention discloses a detection method of a bacterial drug resistance gene and a special kit thereof. The kit provided by the invention comprises 296 single-stranded DNA shown in a sequence 1-296 in a sequence table. The method for detecting the drug-resistant gene of the bacteria comprises the following steps: (1) extracting the genome DNA of the bacteria to be detected, and amplifying the genome DNA by using the set of single-stranded DNA to obtain an amplification product; (2) constructing a sequencing library by using the amplification product based on an Ampliseq technology; (3) and sequencing the sequencing library to obtain a sequencing result, and comparing the sequencing result with a drug-resistant gene sequence to determine whether the bacteria to be detected carry the drug-resistant gene. The detection method and the kit can detect a plurality of drug-resistant genes of a sample to be detected simultaneously with high flux, rapidness and accuracy, and have good application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection method of a bacterial drug resistance gene and a special kit thereof.
Background
The detection of the drug-resistant gene of the bacteria has important significance in clinical diagnosis. At present, the widely applied commercial bacterial drug resistance gene detection method mainly comprises the following four methods: vitek2 for automated detection system, MALDI-TOF for flight mass spectrometry, rapid biochemical detection and real-time image monitoring. The Vitek2 shows better consistency when clinical samples are tested, but the test samples need to be positive cultures, and the time for reporting positive by the blood samples needs at least 24 hours, thereby greatly delaying the treatment time. The same problem exists with real-time image monitoring. The flight mass spectrum and biochemical detection can only determine the pathogens producing carbapenemase and beta lactamase, and the accurate detection is difficult to be carried out on other drug-resistant bacteria. In addition, the methods for detecting the bacterial drug resistance based on the polymerase chain reaction are widely applied, such as the pro it, the FilmArray, the Verigene and the like, but the methods can only detect a plurality of common drug resistance genes and have certain limitations.
The Ampliseq technology refers to a method for specifically amplifying a target fragment in a sequencing process and finally sequencing the target fragment to determine the base composition. Different from the traditional second-generation sequencing method, the method does not adopt an ultrasonic mode to break the genome into short segments, and in the same reaction system, primers are placed in advance, and target segments of interest are amplified by utilizing the specificity of the primers, so that the short segments which accord with the reading length of subsequent sequencing are generated. The method can be used for amplifying a target fragment by placing as many as 24000 pairs of primers in the same reaction system under the same reaction condition.
Currently, the Ampliseq technology is widely applied to screening of common human diseases, such as screening of cancers and screening of genetic diseases. In the aspect of pathogenic diagnosis, the technology is applied to diagnosis of Ebola virus and diagnosis of drug resistance genes of Mycobacterium tuberculosis. The Ampliseq technology shows excellent identification capability and identification capability of mutation sites in multiple diagnoses, and shows strong advantages in multiple detections. The technology becomes a main technical method for disease screening, pathogen screening and drug resistance screening at home and abroad.
Disclosure of Invention
The first object of the present invention is to provide a set of single-stranded DNA.
The complete set of single-stranded DNA provided by the invention consists of 296 single-stranded DNAs shown in sequences 1-296 in a sequence table.
The second object of the present invention is to provide a novel use of the above-mentioned set of single-stranded DNA.
The invention provides the application of the above-mentioned single-stranded DNA set in any one of a1) -a 6):
a1) detecting or assisting in detecting bacterial drug resistance genes;
a2) preparing a product for detecting or assisting in detecting the drug resistance genes of bacteria;
a3) detecting or assisting to detect whether the bacteria to be detected carry the drug-resistant genes and/or carry the types of the drug-resistant genes;
a4) preparing a product for detecting or assisting in detecting whether the bacteria to be detected carry the drug-resistant genes and/or carry the types of the drug-resistant genes;
a5) detecting or detecting in an auxiliary way whether a sample to be detected contains bacteria carrying a drug-resistant gene;
a6) preparing a product for detecting or assisting in detecting whether a sample to be detected contains bacteria carrying the drug resistance gene.
The third purpose of the invention is to provide a kit for detecting the drug resistance gene of bacteria.
The kit provided by the invention comprises the above-mentioned single-stranded DNA set.
The function of the kit is 1) or 2) or 3) as follows:
1) detecting or assisting in detecting bacterial drug resistance genes;
2) detecting or detecting in an auxiliary way whether the bacteria to be detected carry the drug-resistant genes and/or carry the types of the drug-resistant genes;
3) and detecting or assisting to detect whether the sample to be detected contains bacteria carrying the drug-resistant gene.
The kit may also include reagents required for library construction, reagents required for PCR amplification, reagents required for preparation of sequencing templates, and/or reagents required for sequencing.
The reagents required for constructing the Library may be those in Ion Ampliseq Library Kit 2.0(Thermo Fisher Co., Ltd., No. 4480441). The reagents required for preparing the sequencing Template can be Ion PGM OT 2200 Template Kit (Thermo Fisher Co., Ltd., No. 448094). The reagent required for sequencing can be Ion PITM Hi-QTMReagents from the Sequencing 200 Kit (Thermo Fisher Co., Ltd., No. 26433).
The last object of the present invention is to provide a method for detecting or assisting in detecting whether a bacterium to be detected carries a drug-resistant gene and/or carries a species of a drug-resistant gene.
The method for detecting or assisting in detecting whether the bacteria to be detected carry the drug-resistant genes and/or carry the types of the drug-resistant genes comprises the following steps:
(1) extracting the genome DNA of bacteria to be detected, and amplifying the genome DNA by using the complete single-stranded DNA to obtain an amplification product;
(2) constructing a sequencing library by using the amplification product based on an Ampliseq technology;
(3) and sequencing the sequencing library to obtain a sequencing result, and comparing the sequencing result with a drug-resistant gene sequence to determine whether the bacteria to be detected carry the drug-resistant genes and the types of the drug-resistant genes.
In the above application or kit or method, the drug resistance gene is at least one of: aac (2 ') -lb, aadA1, aadA3, aadB, aadD, adeb, ant (4' -llb), aph3-la, armA, blaZ, catB3, catB6, catB8, cmlA5, CTX-M-14, dfRA19, dfrG, E.coli _ gyrA, E _ coli _ parC, IMP-1, KPC-1, mecA, mecC, mcr-1, mtrC, mtrD, mtrE, mtrR, NDM-1, oprD, oqxA, oqxB, blaOXA-10、blaOXA-23、blaOXA-24、blaOXA-1、blaOXA-51、blaOXA-58、blaOXA-66、blaOXA-69、blaOXA-82、PAO_gyrA_parC、patA、patB、pbp1a、pbp2b、pbp2x、phoP、phoQ、pmrA、pmrB、qacA、qnrA1、qnrB4、rpsJ、sat-1、blaSHV-60、strA、strB、sul1、blaTEM-12、tetA、tetD、tetM、tetR、vanA、vanB、VIM-2。
The bacteria are at least one of the following: acinetobacter baumannii, Enterococcus faecalis faecium, Enterococcus faecium, Klebsiella pneumoniae, Klebsiella pneumoniae with beta-lactam resistance, vancomycin resistance, Enterococcus vancomycin-resistant Enterobacter Enterococcus, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus serohilus, Staphylococcus aureus resistant Staphylococcus aureus.
The sample to be tested may be a blood sample.
The invention provides a detection method of a bacterial drug resistance gene and a special kit thereof. The kit provided by the invention comprises 296 single-stranded DNA shown in a sequence 1-296 in a sequence table. The method for detecting the drug-resistant gene of the bacteria comprises the following steps: (1) extracting genome DNA of bacteria to be detected, and amplifying the genome DNA by using the complete single-stranded DNA to obtain an amplification product; (2) constructing a sequencing library by using the amplification product based on an Ampliseq technology; (3) and sequencing the sequencing library to obtain a sequencing result, and comparing the sequencing result with a drug-resistant gene sequence to determine whether the bacteria to be detected carry the drug-resistant genes and the types of the drug-resistant genes. The detection method can detect 68 drug-resistant genes of a sample to be detected at the same time in a one-time reaction in a high-throughput, rapid and accurate manner, conjecture the drug-resistant phenotype, provide guidance for clinical accurate medication, reduce antibiotic abuse and generated superbacteria, and have good application prospect.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The beta-lactam resistant Klebsiella pneumoniae of the following examples is described in the literature: feng J, Yin Z, ZHao Q, et al, genomic characterization of novel IncFII-type multidrug resistant plasmids p0716-KPC and p12181-KPC from Klebsiella pneumoniae [ J ] Sci Rep,2017,7(1):5830.
Methicillin-resistant staphylococcus aureus in the following examples are described in the literature: li L, Pian Y, Chen S, et al, phenol-soluble module alpha4 media, Staphylococcus aureus-associated vascular leak by stimulating heparin-binding protein release from neutrophiles [ J ]. Sci Rep,2016,6:29373.
Example 1 acquisition of bacterial drug resistance genes and design of primers for detection thereof
First, obtaining drug resistance gene
According to The types of antibiotics commonly used in clinic and The types of pathogens commonly used in clinic, relevant fragments of drug-resistant genes are downloaded in The Comprehensive antibacterial Resistance Database (CARD; http:// apcard. mcmaster. ca) Database and The ARDB Database (http:// ARDB. cbcb. umd. edu), and are arranged according to The types of pathogens, antibiotics and drug-resistant genes, and The arrangement method is as follows: carrying out cd-hit clustering on related segments of the drug resistance genes downloaded in an ARDB database and a CARD database, and removing redundant sequences by taking 95% as a threshold value; and performing interspecies comparison on the obtained fragments by using BLAST, and screening specific fragments of the drug-resistant sites by taking 97 percent as a threshold value to remove redundant sequences. Finally, 68 drug resistance genes and sequences thereof for clinically commonly used antibiotics are obtained, wherein the 68 drug resistance genes are as follows: aac (2 ') -lb, aadA1, aadA3, aadB, aadD, adeb, ant (4' -llb), aph3-la, armA, blaZ, catB3, catB6, catB8, cmlA5, CTX-M-14, dfRA19, dfrG, E.coli _ gyrA, E _ coli _ parC, IMP-1, KPC-1, mecA, mecC, mcr-1, mtrC, mtrD, mtrE, mtrR, NDM-1, oprD, oqxA, oqxB, blaOXA-10、blaOXA-23、blaOXA-24、blaOXA-1、blaOXA-51、blaOXA-58、blaOXA-66、blaOXA-69、blaOXA-82、PAO_gyrA_parC、patA、patB、pbp1a、pbp2b、pbp2x、phoP、phoQ、pmrA、pmrB、qacA、qnrA1、qnrB4、rpsJ、sat-1、blaSHV-60、strA、strB、sul1、blaTEM-12tetA, tetD, tetM, tetR, vanA, vanB, VIM-2, whose GenBank and filing date are shown in Table 1, respectively.
TABLE 1 GenBank of drug resistance genes and filing date
Note: i AF078527| + |3770-4303| represents that the sequence of the aadB drug-resistant gene is 3770-th 4303 th site of the gene sequence shown in GenBank AF078527, and the other same is carried out.
The antibiotic and drug resistance genes produced by different pathogens are specifically shown in table 2.
TABLE 2 antibiotic and drug resistance genes produced by different pathogens
Second, design of primers
Submitting the 68 drug-resistant gene sequences obtained in the first step to an Ampliseq primer design website Ion Ampliseq Designer (http:// www.ampliseq.com/brown. action), performing primer design on the returned primer sequences, comparing the returned primer sequences on NCBI, evaluating the specificity of the primer sequences, and performing appropriate adjustment and optimization to finally obtain 148 pairs of primers for detecting the 68 drug-resistant genes (Table 3).
TABLE 3 primer sequences for detecting 68 drug resistance genes
Example 2 application of primers for detecting bacterial drug resistance genes
(I) detection of drug-resistant genes of pathogens to be detected
Extraction of pathogen nucleic acid
The magnetic bead method was used to extract the pathogen nucleic acids to be detected in Table 4 using the MagAttract HMW DNA Kit from Qiagen. The specific extraction steps were performed according to the instructions of the kit.
TABLE 4 pathogens to be detected
Second, construction of Gene library
Library construction was performed using Ion Ampliseq Library Kit 2.0(Thermo Fisher Scientific, Cat. No.4480441) based on Ampliseq technology. The method comprises the following specific steps:
1. DNA target amplification reaction for establishing 2 primer pools
The Ampliseq primers were divided into a primer Pool 1(primer Pool 1) and a primer Pool 2(primer Pool 2), the primers in the primer Pool 1 consisted of the primers labeled "Pool ═ 1" in table 2, the primers in the primer Pool 2 consisted of the primers labeled "Pool ═ 2" in table 2, and the concentration of each primer in the primer Pool 1 or the primer Pool 2 was 100 nM.
In a 96-well plate, each sample has two reaction wells, and the primers are respectively in a primer pool 1Primers and primers in primer pool 2. The system was 10 microliters per reaction tube: 2 × primer pool: 5 microliter, 5 Xion AmpliseqTMHiFi Mix: 2. mu.l, nucleic-free Water: 0.5. mu.l, DNA: 2.5 microliter. After the above system was prepared as required, the reaction was carried out under the reaction conditions shown in Table 5 (cycle number 19).
TABLE 5 reaction conditions
| Step | Temperature of | Time |
| Pre-denaturation | 99℃ | 2 minutes |
| Denaturation of the material | 99℃ | 15 seconds |
| Annealing and stretching | 60℃ | 4 minutes |
| - | 10℃ | Hold |
2. Merging the target amplification reactions
After step 1 was completed, the reaction product obtained from the 10. mu.l primer pool 1 and the reaction product obtained from the 10. mu.l primer pool 2 were combined for each sample, and the total volume of each sample was 20. mu.l.
3. Partially digested product
(1) To each sample tube was added 2 microliters of FuPa reagent (brown cap) at which time the total volume was 22 microliters.
(2) The 96-well plate was placed on a PCR reaction apparatus, and the procedure shown in Table 6 was performed.
TABLE 6 reaction procedure
| Temperature of | Time |
| 50℃ | 10 minutes |
| 55℃ | 10 minutes |
| 60℃ | 20 minutes |
| 10℃ | (Up to 1 hour) |
4. Joint connection
An adaptor (Adapter) was ligated to the amplicon and purified. The method comprises the following specific steps:
(1) ligation reaction
Carefully remove the 96-well plate sealing membrane, and then add the reagents in table 7 in order to each reaction well.
TABLE 7 reagents
| Order of addition | Composition (I) | Volume of |
| 1 | Switch Solution (yellow cover) | 4 microliter |
| 2 | Diluted Ion Xpress barcode adapter mix | 2 microliter |
| 3 | DNA ligase (blue cap) | 2 microliter |
| - | Total volume | 30 microliter |
The 96-well plate was placed on a PCR reaction apparatus, and the procedure shown in Table 8 was performed.
TABLE 8 reaction procedure
| Temperature of | Time |
| 22℃ | 30 minutes |
| 68℃ | 5 minutes |
| 72℃ | 5 minutes |
| 10℃ | (Up to 24 hours) |
(2) Purification of the library
Add 45. mu.l (1.5X sample volume) of AgencourtTMAMPure Reagent (beckmancourulter, cat. a63880) into the library. Blow 5 times up and down to mix, incubate for 5 minutes at room temperature, place 96-well plate on magnetic rack, incubate for 2 minutes until solution is clear, carefully remove and discard supernatant without touching sediment. Add 150 μ l of 70% ethanol in the new configuration, wash the beads, then carefully remove and discard the supernatant; repeatedly washing with 70% ethanol for the second time; ensure that the ethanol in each reaction well was removed, the 96-well plate was placed on a magnetic rack and left at room temperature for 5 minutes.
5. Quantification of unamplified libraries using qPCR
The unamplified Ion Ampliseq (TM) Library was diluted before passage through Ion Library using qPCR
Quantification Kit (Thermo Fisher Scientific, Cat. No.4468802) quantitated the concentration of the library. The concentration of the unamplified library is typically 100-500 pM. After quantification, the library was diluted to 100pM for subsequent template preparation using the Ion template Kit.
Third, template preparation and sequencing
Preparing a sequencing Template from the library obtained in the second step by using an Ion PGM OT 2200 Template Kit (Thermo Fisher company, No.448094), performing on-machine sequencing on the obtained sequencing Template, wherein the emulsification and enrichment of the Template in the sequencing process are completed on an Ion One Touch System (Thermo Fisher company), and the on-machine sequencing is completed on an Ion Proton sequencer (Thermo Fisher company) by using an Ion P1 chip. All steps are performed according to the instructions.
Fourthly, analyzing sequencing results
And converting ram data in a bam format obtained by sequencing into a sam file through Samtools (version 1.6) software, then extracting an effective fasta format sequencing result, filtering low-quality data and short fragments, respectively comparing the obtained data with a drug-resistant gene sequence of the pathogen downloaded by NCBI (national center for Biotechnology information) by using bowtie (version 1.2.0) software, extracting a comparison result, and determining the type of the drug-resistant gene contained in the pathogen to be detected according to the number and the proportion of the genes reads obtained by sequencing.
Samtools command behavior: sam file output by samtools view-h original sequence bam file >; bowtie command behavior: output paths of files in the bowtie-f-a-m 20-v 1-al comparison, output paths of files in the un-compared and output paths of the non reference paths of sequence files needing comparison.
Fifth, the result of drug-resistant gene detection
The results of detection of the drug-resistant gene of each pathogen are shown in table 9.
TABLE 9 detection results of drug-resistant genes of the respective pathogens
(II) evaluation of drug resistance of pathogen to be tested
First, pathogen resistance phenotype detection
The resistance phenotypes of the individual pathogens shown in Table 4 were screened using 13 different classes of antibiotics shown in Table 10. The method comprises the following specific steps: each of the bacteria shown in Table 4 was cultured overnight at 37 ℃ for 8 hours in a shaker, and then diluted to 0.5 McLeod using PBS. And dipping the diluted bacteria liquid with a cotton swab, and uniformly coating the diluted bacteria liquid on the MHA culture medium. Using tweezers to pick up the drug sensitive paper pieces, and sticking the corresponding drug sensitive paper pieces on the MHA culture medium according to the requirements of CLSI. Culturing different bacteria for 18-24 hours according to the CLSI requirement, measuring the diameter of a bacteriostatic circle by using a vernier caliper after the culture is finished, and comparing the diameter with the file of the 2018 CLSI to determine the sensitivity, the mediation and the drug resistance of the pathogen. The specific procedure for drug resistance evaluation is described in the literature "Urbaniak C, Sielfaff A C, Frey K G, et al.Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces [ J ]. Sci Rep,2018,8(1): 814".
TABLE 10 antibiotic classes
| Name of drug | Dosage form | Unit of | Classes of antibiotics |
| Gentamicin | 10 | μg | Aminoglycosides |
| Cefoxitin | 30 | μg | Cephalosporins |
| Oxacillin | 1 | μg | Penicillins |
| Chloromycetin | 30 | μg | Chloramphenicol |
| Tetracycline derivatives | 30 | μg | Tetracyclines |
| Penicillin G | 10 | units | Penicillins |
| Levofloxacin | 5 | μg | Quinolones(fluoroquinolones) |
| Ceftazidime | 30 | μg | Cephalosporins |
| Imipenem | 10 | μg | Carbapenems |
| Piperacillin | 100 | μg | Penicillins |
| Minocycline | 30 | μg | Tetracyclines |
| Vancomycin | 30 | μg | Glycopeptides |
| Ampicillin | 10 | μg | Penicillins |
Second, the detection result
The results of screening each pathogen for the resistance phenotype of 13 different classes of antibiotics are shown in table 11.
TABLE 11 screening of drug resistant phenotypes of the respective pathogens against 13 different classes of antibiotics
The detection result of the drug-resistant phenotype of each pathogen is basically consistent with that of the drug-resistant gene, which shows that the method can accurately and effectively detect whether the bacteria to be detected carry the drug-resistant gene and the type of the carried drug-resistant gene.
Sequence listing
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<120> detection method of bacterial drug resistance gene and special kit thereof
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<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 45
atggaaaatt actttgacag tcccttca 28
<210> 46
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 46
aagctggctc ttcttgcatg ta 22
<210> 47
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 47
cggcatgact gggtaacatc tt 22
<210> 48
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 48
ctgtgcagac caaaaatgtc tgaag 25
<210> 49
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 49
atgaaaaact actttaacag ccctttcaa 29
<210> 50
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 50
gaaagcaggc tcctcttgca ta 22
<210> 51
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 51
gcatgactgg gcatcatcct tc 22
<210> 52
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 52
acttatgcag accaaaaata tttgacga 28
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 53
ctttagttgg cggtactccc tt 22
<210> 54
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 54
actgttgcaa atgtggaaac aagg 24
<210> 55
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 55
gcttcggatt cttcaggctt gt 22
<210> 56
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 56
gacgaagaag ctacccattc ca 22
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 57
cgatcggcga tgagacgttt 20
<210> 58
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 58
gggtaaaata ggtcaccaga acca 24
<210> 59
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 59
gtgacaaaga gagtgcaacg gat 23
<210> 60
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 60
gtactgcaca ttggaaagcg tt 22
<210> 61
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 61
gagtcaccca caacttgagc ta 22
<210> 62
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 62
tcgaggagat cacgaacgat tc 22
<210> 63
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 63
gagcgaatca aggagaaagg aattc 25
<210> 64
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 64
aagtgaatgc gctcttgagt ct 22
<210> 65
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 65
atgaaagttt ctttgattgc tgcgat 26
<210> 66
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 66
cgattgattc aaggttcttc ctacctaata 30
<210> 67
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 67
gactgggaat atgttaaaaa tactacaaag gg 32
<210> 68
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 68
acggattttt atcattcttt atcccttttt gg 32
<210> 69
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 69
tgtattcttt atatttttgt tttgcagcat tgc 33
<210> 70
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 70
gccgtaaatg gagtgtcaat taggtaag 28
<210> 71
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 71
ctaaacatgg tttggtggtt cttgt 25
<210> 72
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 72
caccgaataa tattttcctt tcaggcaa 28
<210> 73
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 73
tgtcactgta tcgccgtcta gt 22
<210> 74
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 74
cctgttgtca gatatttttc cgagatg 27
<210> 75
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 75
ccatccgtta cggcaaaaat gc 22
<210> 76
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 76
cctttagcca atcaacaaac tgct 24
<210> 77
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 77
gctactgatc accacgctgt ta 22
<210> 78
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 78
cccaaaccaa tgatacgcat gataaa 26
<210> 79
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 79
caagccgaga ccaaggatct at 22
<210> 80
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 80
catatcaggc ttggttgctt gtac 24
<210> 81
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 81
gaagtagaaa tgactgaacg tccgataa 28
<210> 82
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 82
atatgctgtt cctgtattgg ccaat 25
<210> 83
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 83
tcagaacgtg gtaaaatttt agaccga 27
<210> 84
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 84
cagagttaat gggaccaaca taacctaat 29
<210> 85
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 85
atcatcataa acttttccag atatagtagc att 33
<210> 86
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 86
gaacgtgtag ttaataaaac acatagggat ga 32
<210> 87
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 87
ccagatttac caataattcg ggcataat 28
<210> 88
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 88
agcaaggtat gcaagatttg gga 23
<210> 89
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 89
agaagcaggt ttggcttcag at 22
<210> 90
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 90
gccgtaccga acgatcagaa tat 23
<210> 91
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 91
atcagcacgc gcacatacag ac 22
<210> 92
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 92
cgatttccgg ctttatcggt ca 22
<210> 93
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 93
gcgatgccaa aaccgtcttt aa 22
<210> 94
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 94
ttacccaact ggcgaaaagc at 22
<210> 95
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 95
ggcggaagtg gacatataat ccaa 24
<210> 96
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 96
ccatttttgc gtgggttatt tcgat 25
<210> 97
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 97
ctacattgaa aactaccttg acctctgt 28
<210> 98
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 98
ggctgtacgc aaactggtat tg 22
<210> 99
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 19
cctgcaaaag ctgatcgaca tc 22
<210> 100
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 100
cataacgctc gttgaaatag gcttt 25
<210> 101
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 101
atgagaaaaa ccaaaaccga agcctt 26
<210> 102
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 102
gtgttcgcat tttaaaaaca ggatgttg 28
<210> 103
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 103
gctgcaaagc aacgacatct ac 22
<210> 104
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 104
ggttttccaa gttgtccatc attatcc 27
<210> 105
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 105
aacggtgata ttgtcactgg tgt 23
<210> 106
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 106
gcttccaacg gtttgatcgt ca 22
<210> 107
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 107
gcaggttgat ctcctgcttg at 22
<210> 108
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 108
atggaattgc ccaatattat gcacc 25
<210> 109
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 109
tacaggatcg acagcggata gt 22
<210> 110
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 110
ctgtccagta ctccgacttc aac 23
<210> 111
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 111
ccataggagg ctaggttcag gt 22
<210> 112
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 112
gccgaactcg aagacatcta tcg 23
<210> 113
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 113
aaagacggtg tacgtctact ttgac 25
<210> 114
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 114
gctgtgcttt accatcttta tcaacgata 29
<210> 115
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 115
ggtactgacc gatcaggatc gt 22
<210> 116
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 116
gcaacggttt tggcgttaac 20
<210> 117
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 117
cgatcaacgg cgttgaaaac at 22
<210> 118
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 118
gccagctcat ccttcacttt cag 23
<210> 119
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 119
aagtacgact cgctgtatat gcg 23
<210> 120
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 120
ccaaactctt cttcggtatg caga 24
<210> 121
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 121
ttgttagccg ttaaaattaa gcccttt 27
<210> 122
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 122
cgttgcacac tttgctttat tgaattg 27
<210> 123
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 123
ttttacttta cgatgcatcc acaaacg 27
<210> 124
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 124
tactttcgag ccatgcttct gttaat 26
<210> 125
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 125
gtcaatggtg tcttcgtgct tt 22
<210> 126
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 126
ggccttccaa ccagaatttg tc 22
<210> 127
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 127
aaaaattttc ctatggcaac cagaatatca 30
<210> 128
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 128
tccatgattt tggtgggaat ggatt 25
<210> 129
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 129
cctcaggtgt gctggttatt caaa 24
<210> 130
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 130
cctgctgtcc aatttcagca ttac 24
<210> 131
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 131
tcttgatctc atgcaaaaag aagtaaaacg 30
<210> 132
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 132
atcaataatt cattacgtat agatgccgg 29
<210> 133
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 133
tattttactt gctatgtggt tgcttctct 29
<210> 134
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 134
gcggcttaga gcattaccat atagattaat 30
<210> 135
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 135
acttcctata ttcagcattt ctattctagt ttc 33
<210> 136
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 136
gcagggacat attctttatt tgctcgt 27
<210> 137
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 137
gagggtaaaa atcttagcac ctatggt 27
<210> 138
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 138
ggcaaaatta acttcttgta ctggtgtaat 30
<210> 139
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 139
gaacacaggt cgataatttt tggttagttg 30
<210> 140
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 140
ttaaatgatt ccaagatttt ctagcgactt 30
<210> 141
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 141
atgaacatta aaacactctt acttataaca agc 33
<210> 142
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 142
atgtcctttt cccattctgg gaatag 26
<210> 143
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 143
agtatttaag tgggacgggc aaaa 24
<210> 144
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 144
tcttctatga ataacatgga ttgcacttca 30
<210> 145
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 145
ctaataaaac gcttccattt agccca 26
<210> 146
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 146
atttctaagt taagggagaa cgctacaata ttt 33
<210> 147
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 147
gatgctgtgt ttgtcacata tgatgg 26
<210> 148
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 148
ccctttcaac caaaattgat caacttcc 28
<210> 149
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 149
ttgcaacgta ttggttatgg caatatg 27
<210> 150
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 150
cccaacttat ctagcacatc taaagacaat 30
<210> 151
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 151
gagtttagtt tgcttaagca taagtattgg g 31
<210> 152
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 152
cggtctaaat gcgtgccata ttttt 25
<210> 153
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 153
cttatatagt gactgctaat ccaaatcaca gt 32
<210> 154
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 154
tcttttccca ttctgggaat agcc 24
<210> 155
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 155
aaccaccaca gaagtattta aatggga 27
<210> 156
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 156
tgcctcttgc tgaggagtaa tttttag 27
<210> 157
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 157
cagccgttct cctatcgcta 20
<210> 158
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 158
gcacgtgttc gcacaattcg 20
<210> 159
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 159
gtggactggg taccgaactt 20
<210> 160
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 160
ggtctcgtag accttctgca 20
<210> 161
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 161
accgaagccg agatcatcac 20
<210> 162
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 162
gtcatcagct cttcgacatc ga 22
<210> 163
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 163
cgacccgcat cgtcatcatc 20
<210> 164
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 164
gcacctcgct gagttcgaag 20
<210> 165
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 165
tcgagcgcat cggcttta 18
<210> 166
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 166
ctcccaggca gtcgataaac tc 22
<210> 167
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 167
ggaagtttaa atacgcttgt cgaca 25
<210> 168
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 168
cgccaagtga tttaccgtgg aa 22
<210> 169
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 169
caagtaggta ccttgcggtt tca 23
<210> 170
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 170
aagatttgac cctctttggt cacaa 25
<210> 171
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 171
tttgaaggca ggattgatgg tattga 26
<210> 172
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 172
aacagctatt caaaccttta caaaagaagg 30
<210> 173
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 173
gtgggtagac aggcgtgtta atta 24
<210> 174
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 174
ccaaccgttt agggcaccat a 21
<210> 175
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 175
atgaacaaac caacgattct gca 23
<210> 176
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 176
ccaggatacg gatggtatca atcc 24
<210> 177
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 177
gtttctatcg aagaccatcg cttctt 26
<210> 178
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 178
agcaaggcta actgaggtaa acttaaatta 30
<210> 179
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 179
gacagcagct caaaactact atggt 25
<210> 180
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 180
cttggtctac atttgtgtag acatcca 27
<210> 181
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 181
ggaagtttcc aaaacctttc tatccca 27
<210> 182
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 182
agcatcagtt ctccaaattt gacagaa 27
<210> 183
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 183
agatttcagg atcagccaaa tagtaatcc 29
<210> 184
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 184
aaatttaaca gccattcgat tccgattc 28
<210> 185
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 185
ggtatttttg atacagattg ataatgtcac g 31
<210> 186
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 186
ggtacagccg aaagctatgt gg 22
<210> 187
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 187
tttagtcaat tttgcggtca ttattggg 28
<210> 188
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 188
gctccaaagg aaacttgctt gagat 25
<210> 189
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 189
tggaagaatc ctatgtaaga gagcaact 28
<210> 190
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 190
taccgtttgt tgggaaactt gttct 25
<210> 191
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 191
atgaaactgc tggtagtgga aga 23
<210> 192
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 192
gaacggcttg accacgtagt 20
<210> 193
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 193
caagtccttc ccgatcctga t 21
<210> 194
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 194
gtgatgccgc atgaggtatt c 21
<210> 195
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 195
cgctgaccgc ctacgaat 18
<210> 196
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 196
gcgctcggtg aacaggtag 19
<210> 197
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 197
tacagcatcg tcaccatgca a 21
<210> 198
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 198
ctgcgcaaca ggcggttaag 20
<210> 199
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 199
cgagctggac cagatcgaat 20
<210> 200
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 200
tgatagccta tctgctggct cat 23
<210> 201
<211> 201
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 201
cctgctggag aacgcctatc 20
<210> 202
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 202
tgtagcgaaa cgtatgcgga a 21
<210> 203
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 203
atgagaatac tgctggccga 20
<210> 204
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 204
ctgcagttcg tcgagatcga 20
<210> 205
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 205
gtgccgacga ctacctga 18
<210> 206
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 206
tcgatgccgt agccgatg 18
<210> 207
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 207
gggtgctcca gctgaacat 19
<210> 208
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 208
ggcgtgcgga tttcatgg 18
<210> 209
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 209
ggcctcatcg acgaactcaa 20
<210> 210
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 210
agctcgatgt ccttgtcgat g 21
<210> 211
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 211
ggctccatca ccgagatcat c 21
<210> 212
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 212
caggttttcg ctgatccaga tct 23
<210> 213
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 213
aaaaagatgg actgcactag tagtattagc 30
<210> 214
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 214
ctgcaatacc aagtaaaaat cgaatagct 29
<210> 215
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 215
cattagctat atttttcgca gaaagtgca 29
<210> 216
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 216
gtccaatcat gcctgcaatt gataaaat 28
<210> 217
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 217
caaaagaaaa gtctcactcg tgggata 27
<210> 218
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 218
acaacctgta accattgtga agctaataa 29
<210> 219
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 219
gataaagttt ttcagcaaga ggatttctca 30
<210> 220
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 220
gttgcaacct gagatataag ccga 24
<210> 221
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 221
gcttctacaa tcaagtcagc cataaga 27
<210> 222
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 222
cagcactatt actcccaagg gtt 23
<210> 223
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 223
atgatgactc tggcgttagt tg 22
<210> 224
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 224
gtattggtga tataggcgct acaaaac 27
<210> 225
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 225
ggcgcaagtt ttatgaatat gatcacc 27
<210> 226
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 226
ataccaagac gttccaggag caa 23
<210> 227
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 227
ccggcaaatc cagcttca 18
<210> 228
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 228
cgcctgaaag cttatgatta cgc 23
<210> 229
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 229
ctgagcaggt ggcggaaaca t 21
<210> 230
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 230
ccatagcaag cagagtcttc atcag 25
<210> 231
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 231
ccctaccgga aggattacat ctc 23
<210> 232
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 232
ccagtaccag tacatcgctg tttc 24
<210> 233
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 233
gcttgagcaa attaaacaaa gcgaaag 27
<210> 234
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 234
cgcaaaaagg cagtcaatcc tg 22
<210> 235
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 235
gccattacca tgagcgataa cag 23
<210> 236
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 236
cggtcttatc ggcgataaac cag 23
<210> 237
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 237
gcagtggatg gtggacgat 19
<210> 238
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 238
cgttgccagt gctcgatca 19
<210> 239
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 239
ttcttaccgg acgaggacaa ga 22
<210> 240
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 240
tccaatcgca gatagaaggc aag 23
<210> 241
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 241
tttttggtga atcgcattct gactg 25
<210> 242
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 242
aaatccgctc cagacagatc ag 22
<210> 243
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 243
atgcttggtg ataacggcaa ttc 23
<210> 244
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 244
ttcgacacga gccaaaagat cgag 24
<210> 245
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 245
tgagaacaat ctgatgttgc tcgaata 27
<210> 246
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 246
tcatgatgca gatcgccatg tag 23
<210> 247
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 247
gattatagcc gatcaaatga tgagcaatg 29
<210> 248
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 248
tctgtcgcac ctgcttgatc 20
<210> 249
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 249
cgcctgtttt tcctgctcat tg 22
<210> 250
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 250
gcaacgatgt gagagagcat tc 22
<210> 251
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 251
cggtgttcgg cattctgaat ct 22
<210> 252
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 252
gcctcagcaa tatcgggata gag 23
<210> 253
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 253
gctacctgaa cgatatccaa ggatttc 27
<210> 254
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 254
ttccgcgaca ccgagaccaa ta 22
<210> 255
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 255
gtcgaacctt caaaagctga agtc 24
<210> 256
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 256
cgtcgcgact gcgaaatttc 20
<210> 257
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 257
acatttccgt gtcgccctta tt 22
<210> 258
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 258
gatgcttttc tgtgactggt gagta 25
<210> 259
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 259
gccgcataca ctattctcag aatg 24
<210> 260
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 260
cctccatcca gtctattaat tgttgcc 27
<210> 261
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 261
gcaaactatt aactggcgaa ctacttac 28
<210> 262
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 262
cacctatctc agcgatctgt ctatttc 27
<210> 263
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 263
cccagtgctg ttgttgtcat taa 23
<210> 264
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 264
ccctacttcg gtatctgtat tatcacgt 28
<210> 265
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 265
gtggttatgt tttggttccg tgaa 24
<210> 266
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 266
ccttcagata taaacgctaa aaaggcaaa 29
<210> 267
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 267
tgcattgcct ccaattccca ta 22
<210> 268
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 268
agctatattc gtgctcgtcg ttt 23
<210> 269
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 269
tttttcgtca acctcaactc aacag 25
<210> 270
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 270
cagcattctc gctatcaaaa taaagctaat 30
<210> 271
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 271
tagctcatgt tgacgcagga aa 22
<210> 272
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 272
atctctcgca gaaattagta ggattgc 27
<210> 273
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 273
ttttagcaga agtatatcgc tcgttatcag 30
<210> 274
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 274
tcatttcctt ctattaccac atcccattg 29
<210> 275
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 275
ccctaatatg tgtgtgatga gctgta 26
<210> 276
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 276
aaagttcaga cttctttctg tatgttgatg 30
<210> 277
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 277
agtgattaac agcgcattag agct 24
<210> 278
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 278
ctgtaggccg tgtacctaaa tgt 23
<210> 279
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 279
atgtgcttta ctaagtcatc gcgat 25
<210> 280
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 280
gacccacttt cacatttaag ttgtttttct 30
<210> 281
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 281
atgaatagaa taaaagttgc aatactgttt gg 32
<210> 282
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 282
ctgaatagca attgtcgttt tccca 25
<210> 283
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 283
tggtgtatgg aaaatgtgcg aaaaac 26
<210> 284
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 284
ttaacaaaaa caggataggt aaacgtagct 30
<210> 285
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 285
catgggctgc ttgtcatgaa ag 22
<210> 286
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 286
ttacgccaaa ggacgaacct ga 22
<210> 287
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 287
gcttacctac cctgtctttg tga 23
<210> 288
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 288
gtaatcatcg cattttctga gccttttt 28
<210> 289
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 289
gtatcttccg catccatcag ga 22
<210> 290
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 290
aatcaggctg tcaatcagtg ca 22
<210> 291
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 291
tctacccgtc caatggtctc at 22
<210> 292
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 292
cacaactaag ttgtcggtcg aatg 24
<210> 293
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 293
cttcggtcca gtagaactct tctatc 26
<210> 294
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 294
gcgcttttac aacattcgtt gtgt 24
<210> 295
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 295
gttattggtc tatttgaccg cgtctat 27
<210> 296
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 296
gctgtatcaa tcaaaagcaa ctcatcac 28
Claims (10)
1. The complete set of single-stranded DNA consists of 296 single-stranded DNAs shown in sequences 1-296 in a sequence table.
2. Use of the set of single-stranded DNA of claim 1 in the preparation of a product for detecting or aiding in the detection of a bacterial drug resistance gene.
3. Use of the set of single-stranded DNA of claim 1 in the preparation of a product for detecting or aiding in the detection of whether a test bacterium carries a drug-resistant gene and/or a drug-resistant gene species.
4. Use of the set of single-stranded DNA of claim 1 in the preparation of a product for detecting or aiding in the detection of a test sample containing a bacterium harboring a drug-resistant gene.
5. Use according to any one of claims 2 to 4, characterized in thatThe method comprises the following steps: the drug-resistant gene is at least one of the following:aac (2’)-lb、aadA1、aadA3、aadB、aadD、adeb、ant（4’-llb）、aph3-la、armA、blaZ、catB3、catB6、 catB8、cmlA5、CTX-M-14、dfrA19、dfrG、E.coli_gyrA、E_coli_parC、IMP-1、KPC-1、mecA、 mecC、mcr-1、mtrC、mtrD、mtrE、mtrR、NDM-1、oprD、oqxA、oqxB、bla OXA-10 、bla OXA-23 、bla OXA-24 、 bla OXA-1 、bla OXA-51 、bla OXA-58 、bla OXA-66 、bla OXA-69 、bla OXA-82 、PAO_gyrA_parC、patA、patB、pbp1a、 pbp2b、pbp2x、phoP、phoQ、pmrA、pmrB、qacA、qnrA1、qnrB4、rpsJ、sat-1、bla SHV-60 、strA、 strB、sul1、bla TEM-12 、tetA、tetD、tetM、tetR、vanA、vanB、VIM-2。
6. use according to any one of claims 2 to 4, characterized in that: the bacteria are at least one of the following: acinetobacter baumanniiAcinetobacter baumanniiEnterococcus faecalisEnterococcus faecalis、Enterococcus faeciumEnterococcus faecium、Klebsiella pneumoniaeKlebsiella pneumoniaeKlebsiella pneumoniae with beta-lactam drug resistanceKlebsiella pneumoniae with beta-lactamase、Vancomycin resistant enterococcivancomycin-resistant Enterococci、Pseudomonas aeruginosaPseudomonas aeruginosaStaphylococcus aureusStaphylococcus aureusStaphylococcus epidermidisStaphylococcus epidermidisStaphylococcus haemolyticusStaphylococcus haemolyticusHuman staphylococcusStaphylococcus hominis、Methicillin-resistant staphylococcus aureusmethicillin-resistant Staphylococcus aureus。
7. A kit comprising a set of single stranded DNA of claim 1; the function of the kit is 1) or 2) or 3) as follows:
1) detecting or assisting in detecting bacterial drug resistance genes;
2) detecting or assisting to detect whether the bacteria to be detected carry the drug-resistant genes and/or carry the types of the drug-resistant genes;
3) and detecting or assisting to detect whether the sample to be detected contains bacteria carrying the drug-resistant gene.
8. A method for detecting or assisting in detecting whether a bacterium to be detected carries a drug-resistant gene and/or carries a type of the drug-resistant gene comprises the following steps:
(1) extracting the genome DNA of bacteria to be detected, and amplifying the genome DNA by using the set of single-stranded DNA of claim 1 to obtain an amplification product;
(2) constructing a sequencing library by using the amplification product based on an Ampliseq technology;
(3) sequencing the sequencing library to obtain a sequencing result; comparing the sequencing result with a drug-resistant gene sequence to determine whether the bacteria to be detected carry the drug-resistant gene and the type of the drug-resistant gene;
the methods are for non-disease diagnostic and therapeutic purposes.
9. The kit of claim 7 or the method of claim 8, characterized in that: the drug-resistant gene is at least one of the following:aac(2’)-lb、aadA1、aadA3、aadB、aadD、adeb、ant（4’-llb）、aph3-la、 armA、blaZ、catB3、catB6、catB8、cmlA5、CTX-M-14、dfrA19、dfrG、E.coli_gyrA、E_coli_ parC、IMP-1、KPC-1、mecA、mecC、mcr-1、mtrC、mtrD、mtrE、mtrR、NDM-1、oprD、oqxA、oqxB、 bla OXA-10 、bla OXA-23 、bla OXA-24 、bla OXA-1 、bla OXA-51 、bla OXA-58 、bla OXA-66 、bla OXA-69 、bla OXA-82 、PAO_ gyrA_parC、patA、patB、pbp1a、pbp2b、pbp2x、phoP、phoQ、pmrA、pmrB、qacA、qnrA1、qnrB4、 rpsJ、sat-1、bla SHV-60 、strA、strB、sul1、bla TEM-12 、tetA、tetD、tetM、tetR、vanA、vanB、VIM- 2。
10. the kit of claim 7 or the method of claim 8, characterized in that: the bacteria are at least one of the following: acinetobacter baumanniiAcinetobacter baumanniiEnterococcus faecalisEnterococcus faecalis、Enterococcus faeciumEnterococcus faecium、Klebsiella pneumoniaeKlebsiella pneumoniaeKlebsiella pneumoniae with beta-lactam drug resistanceKlebsiella pneumoniae with beta-lactamase、Vancomycin resistant enterococcivancomycin-resistant Enterococci、Pseudomonas aeruginosaPseudomonas aeruginosaStaphylococcus aureusStaphylococcus aureusStaphylococcus epidermidisStaphylococcus epidermidisStaphylococcus haemolyticusStaphylococcus haemolyticusHuman staphylococcusStaphylococcus hominis、Methicillin-resistant staphylococcus aureusmethicillin-resistant Staphylococcus aureus。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910119759.4A CN109762915B (en) | 2019-02-18 | 2019-02-18 | Detection method of bacterial drug resistance gene and special kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910119759.4A CN109762915B (en) | 2019-02-18 | 2019-02-18 | Detection method of bacterial drug resistance gene and special kit thereof |
Publications (2)
| Publication Number | Publication Date |
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| CN109762915A CN109762915A (en) | 2019-05-17 |
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