CN110157824B - Kit for detecting high-toxicity acinetobacter baumannii and use method thereof - Google Patents
Kit for detecting high-toxicity acinetobacter baumannii and use method thereof Download PDFInfo
- Publication number
- CN110157824B CN110157824B CN201910446577.8A CN201910446577A CN110157824B CN 110157824 B CN110157824 B CN 110157824B CN 201910446577 A CN201910446577 A CN 201910446577A CN 110157824 B CN110157824 B CN 110157824B
- Authority
- CN
- China
- Prior art keywords
- acinetobacter baumannii
- toxicity
- kit
- solution
- specific sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000588626 Acinetobacter baumannii Species 0.000 title claims abstract description 122
- 231100000086 high toxicity Toxicity 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title abstract description 23
- 239000003550 marker Substances 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 71
- 238000006243 chemical reaction Methods 0.000 claims description 66
- 238000011144 upstream manufacturing Methods 0.000 claims description 38
- 238000012408 PCR amplification Methods 0.000 claims description 31
- 239000013642 negative control Substances 0.000 claims description 31
- 239000013641 positive control Substances 0.000 claims description 31
- 238000002474 experimental method Methods 0.000 claims description 16
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 238000012070 whole genome sequencing analysis Methods 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 108010006785 Taq Polymerase Proteins 0.000 claims description 6
- 239000005549 deoxyribonucleoside Substances 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 239000001226 triphosphate Substances 0.000 claims description 6
- 235000011178 triphosphate Nutrition 0.000 claims description 6
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 6
- 231100000225 lethality Toxicity 0.000 claims description 4
- 231100001160 nonlethal Toxicity 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 20
- 231100000419 toxicity Toxicity 0.000 abstract description 7
- 230000001988 toxicity Effects 0.000 abstract description 7
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 6
- 230000003321 amplification Effects 0.000 description 29
- 238000003199 nucleic acid amplification method Methods 0.000 description 29
- 239000000047 product Substances 0.000 description 18
- 239000000090 biomarker Substances 0.000 description 7
- 230000001018 virulence Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000005842 biochemical reaction Methods 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010029719 Nonspecific reaction Diseases 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000000546 chi-square test Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 208000009470 Ventilator-Associated Pneumonia Diseases 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for detecting high-toxicity acinetobacter baumannii and a use method thereof, belonging to the technical field of biological detection. Taking a section of specific sequence in the acinetobacter baumannii as a marker, wherein the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii, and the section of gene sequence comprising the 19 open reading frames is specific to the high-toxicity acinetobacter baumannii; and then, a primer is designed by using a marker specific sequence, and finally, the qualitative detection of the high-toxicity acinetobacter baumannii is completed based on polymerase chain reaction, so that the problems that the acinetobacter baumannii detection method in the prior art can only identify the existence of the acinetobacter baumannii, can not accurately and qualitatively distinguish various strains with different toxicity and the like are solved, and the application values of the marker, the kit and the using method for detecting the high-toxicity acinetobacter baumannii are ensured.
Description
Technical Field
The invention relates to a kit for detecting high-toxicity acinetobacter baumannii and a use method thereof, belonging to the technical field of biological detection.
Background
Acinetobacter baumannii is a non-fermented gram-negative bacillus, widely exists in the nature and belongs to conditional pathogenic bacteria. The bacterium is an important pathogenic bacterium of hospital infection, mainly causes respiratory tract infection, and also can cause bacteremia, urinary system infection, secondary meningitis, operation site infection, ventilator-associated pneumonia and the like. Due to the increasing trend of the drug resistance rate of acinetobacter baumannii to common antibiotics (especially carbapenem antibiotics) year by year, the acinetobacter baumannii attracts the close attention of clinicians and microbiologists. Patients infected by acinetobacter baumannii are mostly old, critically ill, weak in body resistance and patients using broad-spectrum antibiotics for a long time.
The difference in virulence exists among various strains of the acinetobacter baumannii, the infection of the strains with high virulence often leads to high mortality of patients, and the existence of the acinetobacter baumannii can be clinically identified only by biochemical reaction or mass spectrometry technology at present, but the difference in virulence among the various strains cannot be effectively distinguished, so the reference value for clinicians is very limited.
The patent document with the publication number of CN105803041A and the name of acinetobacter baumannii capsule staining kit and detection method is disclosed by the national intellectual property office in 2016, 07, 27, and the patent document comprises the following publications: the detection method comprises the following steps: the Acinetobacter baumannii, the Congo red dye solution and the adhesive are mixed and then pushed into a sheet, and the Acinetobacter baumannii capsule is dyed by a Congo red dyeing method. The patent document provides an acinetobacter baumannii capsule staining kit and a detection method, which can be used for quickly evaluating the toxicity of acinetobacter baumannii, are based on a staining method and belong to morphological detection.
Disclosure of Invention
The inventor finds in research that the genome of the high-virulence acinetobacter baumannii contains a gene sequence comprising 19 open reading frames, the gene sequence contains a unique specific sequence, and a primer is designed by taking the specific sequence as a target (namely a target polynucleotide), so that based on the research result, the specific sequence is taken as a biomarker and is applied to a kit for detecting the high-virulence acinetobacter baumannii.
The invention aims to overcome the defects of the prior art and provides a kit for detecting high-toxicity acinetobacter baumannii and a using method thereof. In the technical scheme, the kit takes the specific sequence as a marker, and then completes the qualitative detection of the high-toxicity acinetobacter baumannii based on polymerase chain reaction, so as to solve the problems that the acinetobacter baumannii detection method in the prior art can only identify the existence of the acinetobacter baumannii, can not accurately and qualitatively distinguish various strains with different toxicity, and the like.
In order to achieve the technical purpose, the following technical scheme is proposed:
the application of a section of specific sequence in acinetobacter baumannii as a marker in the preparation of a kit for detecting high-toxicity acinetobacter baumannii is disclosed, wherein the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii; if the specific sequence is detected to be positive, the acinetobacter baumannii is judged to be high-toxicity acinetobacter baumannii; otherwise, if the specific sequence is detected to be negative, the acinetobacter baumannii is judged to be low-toxicity acinetobacter baumannii;
the gene sequence comprising 19 open reading frames is shown as SEQ ID NO. 1;
the specific sequence is shown as SEQ ID NO. 2.
A kit for detecting high-toxicity acinetobacter baumannii comprises PCR reaction liquid, a primer for amplifying a specific sequence SEQ ID NO.2, a positive control and a negative control.
Further, the PCR reaction solution comprises Taq polymerase, tris hydrochloride solution, magnesium chloride solution, potassium chloride solution, triton solution, formamide solution and deoxyribonucleoside triphosphate.
Further, the primer for amplifying the specific sequence SEQ ID NO.2 comprises an upstream primer and a downstream primer, wherein,
the sequence of the upstream primer is as follows: 5'-TTGAGAAGCTAAATTATGGCTCG-3';
the sequence of the downstream primer is: 5'-GATAGCACAGAAATCCATAAAGGAA-3'.
Further, the positive control is clinical acinetobacter baumannii strain nucleic acid which contains a specific sequence SEQ ID NO.2 and has high lethality in animal experiments, and is determined by whole genome sequencing.
Further, the negative control is clinical acinetobacter baumannii strain nucleic acid which does not contain a specific sequence SEQ ID NO.2 and is non-lethal in animal experiments through whole genome sequencing.
A use method of a kit for detecting high-toxicity Acinetobacter baumannii is based on polymerase chain reaction and comprises the following steps:
s1: sampling
Inoculating the acinetobacter baumannii to be detected into a culture medium, culturing, and taking a culture solution as an acinetobacter baumannii sample to be detected;
s2: preparation of PCR amplification System
Taking at least four PCR tubes, and respectively numbering the PCR tubes as a positive control tube, a negative control tube, an experimental tube and a blank control tube; the PCR amplification system is 20 mu L, wherein 15-18 uL of PCR reaction solution, 0.5-1.5 uL of upstream primer, 0.5-1.5 uL of downstream primer and 1-2 uL of positive control are added into the positive control tube;
adding 15-18 uL PCR reaction solution, 0.5-1.5 uL upstream primer, 0.5-1.5 uL downstream primer and 1-2 uL negative control into the negative control tube;
adding 15-18 uL of PCR reaction solution, 0.5-1.5 uL of upstream primer, 0.5-1.5 uL of downstream primer and 1-2 uL of the sample of the acinetobacter baumannii to be detected obtained in the step S1 into an experimental tube;
adding 15-18 uL PCR reaction solution, 0.5-1.5 uL upstream primer, 0.5-1.5 uL downstream primer and 1-2 uL sterilized water into a blank control tube;
s3: amplification reaction
Centrifuging all PCR tubes subjected to PCR amplification system preparation in the step S2 for 30S under the condition that the rotating speed is 3000rpm, and then placing the tubes in a PCR instrument for amplification, wherein the conditions of amplification reaction are as follows:
reacting for 3min at 94 ℃; then 35 cycles are carried out, wherein the reaction is carried out for 30s at 94 ℃, for 30s at 57-63 ℃ and for 30s at 72 ℃ in sequence; finally, reacting for 5min at 72 ℃, and then keeping at 4 ℃;
s4: judgment and result
Respectively carrying out electrophoresis on products subjected to the amplification reaction in the step S3, and observing whether a strip appears at a position of 91 bp;
if the product after the amplification reaction of the positive control tube correspondingly has a strip, and the product after the amplification reaction of the negative control tube and the product after the amplification reaction of the blank control tube do not correspondingly have strips, judging that the experiment is successful, and then judging the result of the experimental sample;
if the product after the amplification reaction of the positive control tube does not have a band correspondingly, or the product after the amplification reaction of the negative control tube has a band correspondingly, or the product after the amplification reaction of the blank control tube has a band correspondingly, the experiment is judged to be unsuccessful, and the experiment needs to be repeated until the experiment is successful, and the result of the experimental sample is judged to be read.
Further, in the acinetobacter baumannii sample to be detected, the OD600nm is 0.6-0.8. Meanwhile, in the step S1, the acinetobacter baumannii sample to be detected in the acinetobacter baumannii culture solution is identified by biochemical reaction or mass spectrum, and the identification technology of the biochemical reaction or the mass spectrum is the existing mature technology; in step S1, the inoculation, culture, bacteria concentration detection, etc. are well known techniques, such as: refer to the "laboratory Manual of clinical bacteriology, in rock, et al.
Furthermore, in a 20 muL PCR amplification system, the concentration of Taq polymerase is 5U/uL, the concentration of tris hydrochloride solution is 200mmol/L, the concentration of magnesium chloride solution is 20mmol/L, the concentration of potassium chloride solution is 500mmol/L, the volume ratio of triton solution is 0.2%, the volume ratio of formamide solution is 10%, and the concentration of deoxyribonucleoside triphosphate is 10mmol/L.
Further, in a 20 muL PCR amplification system, the concentrations of the upstream primer and the downstream primer are both 0.5umol/L.
The PCR instrument is the existing mature technology, such as: dongsheng Longong ETC811.
The electrophoresis is a well-established technique in the prior art, such as: agarose gel electrophoresis.
In the technical scheme, when a PCR amplification system is prepared, the amounts of the PCR reaction solution correspondingly added into the positive control tube, the negative control tube, the experiment tube and the blank control tube are consistent, the amounts of the upstream primer correspondingly added are consistent, the amounts of the downstream primer correspondingly added are consistent, and the amounts of the acinetobacter baumannii sample to be detected obtained by the step S1, which is correspondingly added with the positive control and the negative control, and the sterilized water are consistent.
By adopting the technical scheme, the beneficial technical effects brought are as follows:
1) The kit of the invention takes a specific sequence in a gene sequence comprising 19 open reading frames as a marker, and then completes the qualitative detection of the high-toxicity acinetobacter baumannii based on polymerase chain reaction, so as to solve the problems that the acinetobacter baumannii detection method in the prior art can only identify the existence of the acinetobacter baumannii, and can not accurately and qualitatively distinguish various strains with different toxicity, and the like;
2) The invention has reasonable design principle, easy result judgment and high accuracy. The inventor selects a gene segment comprising 19 open reading frames from the genome of the high-toxicity acinetobacter baumannii by applying a comparative genomics method, and the existence of the gene segment is related to the toxicity of the acinetobacter baumannii, so a plurality of specific primers are designed based on a section of special sequence in the gene segment, and primers for target polynucleotide amplification with excellent amplification effect are selected by optimization, namely, the primers are finally limited to an upstream primer and a downstream primer with smaller nonspecific reaction, so as to ensure that the reaction of a PCR amplification system is smaller nonspecific reaction; finally, qualitatively detecting the high-toxicity acinetobacter baumannii by using polymerase chain reaction, and having higher specificity;
3) In the using process of the kit, a PCR amplification system is prepared based on the detection kit, then amplification reaction is carried out, and small non-specific reaction is ensured under the conditions of specific temperature, specific time, specific cycle times and the like. Meanwhile, in the preparation of a PCR amplification system, the culture solution (sample) of Acinetobacter baumannii is directly added into an experimental tube, so that the detection process is simple and rapid, only 1-2 h is needed, the qualitative detection of the high-toxicity Acinetobacter baumannii can be completed, the time consumption is short, and the applicability is strong;
4) In the using method of the kit, the PCR reaction solution, the upstream primer, the downstream primer and the Acinetobacter baumannii culture solution are added in sequence, and the solutions of all the substances have larger volume difference and are added in the sequence, so that the convenience and operability of the experiment are ensured on the one hand; on the other hand, the solution with smaller volume is not easy to be added into an empty PCR tube, thereby causing false negative, and therefore, the detection accuracy of the high-toxicity Acinetobacter baumannii is indirectly improved.
Drawings
FIG. 1 is a flow chart of a method of using the kit of the present invention;
FIG. 2 shows the result of agarose gel electrophoresis of the amplified reaction product after the PCR reaction, wherein lane M: nucleic Acid (DNA) molecular weight standards (Marker); lane 1: the PCR amplification product of the negative control tube; lane 2: PCR amplification products of the positive control tube; lane 3: PCR amplification products of blank control tubes; lanes 4-9: PCR amplification products of the test tubes (samples);
FIG. 3 is a comparison of lethality of positive and negative samples of high toxicity Acinetobacter baumannii biomarkers in C57BL/6 mice.
Detailed Description
In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the one clinical isolate corresponds to one case, such as: the 62 clinical isolates corresponded to 62 patients infected with Acinetobacter baumannii. And, the specific number of samples is set at the maximum capacity.
Example 1
The application of a section of specific sequence in acinetobacter baumannii as a marker in the preparation of a kit for detecting high-toxicity acinetobacter baumannii is disclosed, wherein the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii; if the specific sequence is detected to be positive, judging that the acinetobacter baumannii is high-toxicity acinetobacter baumannii; otherwise, if the specific sequence is detected to be negative, the acinetobacter baumannii is judged to be low-toxicity acinetobacter baumannii;
the gene sequence comprising 19 open reading frames is shown as SEQ ID NO. 1;
the specific sequence is shown as SEQ ID NO. 2.
Example 2
A kit for detecting high-toxicity acinetobacter baumannii comprises PCR reaction liquid, a primer for amplifying a specific sequence SEQ ID NO.2, a positive control and a negative control.
Example 3
On the basis of the embodiment 2, further:
the PCR reaction solution comprises Taq polymerase, tris hydrochloride solution, magnesium chloride solution, potassium chloride solution, triton solution, formamide solution and deoxyribonucleoside triphosphate.
Example 4
On the basis of example 3, further:
the primer for amplifying the specific sequence SEQ ID NO.2 comprises an upstream primer and a downstream primer, wherein,
the sequence of the upstream primer is as follows: 5'-TTGAGAAGCTAAATTATGGCTCG-3';
the sequence of the downstream primer is: 5'-GATAGCACAGAAATCCATAAAGGAA-3'.
Example 5
On the basis of example 4, further:
the positive control is clinical acinetobacter baumannii strain nucleic acid which contains a specific sequence SEQ ID NO.2 and has high lethality in animal experiments and is determined by whole genome sequencing.
The negative control is clinical acinetobacter baumannii strain nucleic acid which does not contain a specific sequence SEQ ID NO.2 and is non-lethal in animal experiments through whole genome sequencing.
Example 6
Based on embodiments 1-5, a method for using a kit for detecting Acinetobacter baumannii with high toxicity is provided, which specifically comprises the following steps:
s1: sampling
Inoculating the acinetobacter baumannii to be detected into a culture medium, culturing until OD600nm is 0.8, and taking a culture solution as an acinetobacter baumannii sample to be detected;
s2: preparation of PCR amplification System
Taking four PCR tubes, and numbering the four PCR tubes as a positive control tube, a negative control tube, an experimental tube and a blank control tube respectively; the PCR amplification system is 20 mu L, wherein 17uL of PCR reaction solution, 1uL of upstream primer, 1uL of downstream primer and 1uL of positive control are added into the positive control tube;
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL negative control into a negative control tube;
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL Acinetobacter baumannii sample to be detected obtained in the step S1 into an experimental tube;
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL sterilized water into a blank control tube;
in the four 20 muL PCR amplification systems, the concentration of Taq polymerase is 5U/uL, the concentration of tris hydrochloride solution is 200mmol/L, the concentration of magnesium chloride solution is 20mmol/L, the concentration of potassium chloride solution is 500mmol/L, the volume ratio of triton solution is 0.2%, the volume ratio of formamide solution is 10%, and the concentration of deoxyribonucleoside triphosphate is 10mmol/L; the concentrations of the upstream primer and the downstream primer are both 0.5umol/L.
S3: amplification reaction
Centrifuging all PCR tubes subjected to PCR amplification system preparation in the step S2 for 30S under the condition that the rotating speed is 3000rpm, and then placing the tubes in a PCR instrument for amplification, wherein the conditions of amplification reaction are as follows:
reacting for 3min at 94 ℃; then 35 cycles, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 60 ℃ and reacting for 30s at 72 ℃ in sequence; finally, reacting for 5min at 72 ℃, and then keeping at 4 ℃;
s4: judgment and results
Respectively carrying out electrophoresis on products subjected to the amplification reaction in the step S3, and observing whether a strip appears at a position of 91 bp;
if the product after the amplification reaction of the positive control tube has a corresponding strip, and the product after the amplification reaction of the negative control tube and the product after the amplification reaction of the blank control tube have no corresponding strip, judging that the experiment is successful, and then judging the result of the experimental sample;
if the product after the amplification reaction of the positive control tube does not have a band correspondingly, or the product after the amplification reaction of the negative control tube has a band correspondingly, or the product after the amplification reaction of the blank control tube has a band correspondingly, the experiment is judged to be unsuccessful, and the experiment needs to be repeated until the experiment is successful, so that the result of the experimental sample is judged.
In the step S1, the Acinetobacter baumannii sample to be detected in the Acinetobacter baumannii culture solution is identified by biochemical reaction or mass spectrum, and the identification technology of the biochemical reaction or the mass spectrum is the existing mature technology; in step S1, the inoculation, culture, bacteria concentration detection, etc. are well known techniques, such as: refer to the "laboratory Manual of clinical bacteriology, in rock, et al.
Example 7
On the basis of embodiment 6, the present embodiment is different in that:
when the PCR amplification system in the step S2 is prepared, six PCR tubes are taken and respectively numbered as a positive control tube (one), a negative control tube (one), an experimental tube (three) and a blank control tube (one); the PCR amplification system is 20 mu L, wherein,
adding 15uL PCR reaction solution, 1.5uL upstream primer, 1.5uL downstream primer and 2uL positive control into the positive control tube;
adding 15uL PCR reaction solution, 1.5uL upstream primer, 1.5uL downstream primer and 2uL negative control into the negative control tube;
adding 15uL of PCR reaction solution, 1.5uL of upstream primer, 1.5uL of downstream primer and 2uL of the sample of the acinetobacter baumannii to be detected obtained in the step S1 into an experimental tube;
adding 15uL of PCR reaction solution, 1.5uL of upstream primer, 1.5uL of downstream primer and 2uL of sterilized water into a blank control tube;
in the acinetobacter baumannii sample to be detected obtained in the step S1, the OD600nm is 0.6;
in the step S3, the amplification reaction is carried out for 3min at the temperature of 94 ℃; then 35 cycles, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 57 ℃ and reacting for 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.
Example 8
On the basis of examples 6 to 7, the present example differs in that:
when the PCR amplification system in the step S2 is prepared, eight PCR tubes are taken and respectively numbered as a positive control tube (one), a negative control tube (one), an experimental tube (five) and a blank control tube (one); the PCR amplification system is 20 mu L, wherein,
adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL positive control into the positive control tube;
adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL negative control into the negative control tube;
adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL of the acinetobacter baumannii sample to be detected obtained in the step S1 into an experimental tube;
adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL sterilized water into a blank control tube;
in the Acinetobacter baumannii sample to be detected obtained in the step S1, the OD600nm is 0.7;
in the step S3, the amplification reaction is carried out for 3min at the temperature of 94 ℃; then 35 cycles, wherein the reaction is carried out for 30s at 94 ℃, 30s at 63 ℃ and 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.
Example 9
On the basis of examples 6 to 8, the present example differs in that:
during the preparation of the PCR amplification system in the step S2, thirteen PCR tubes are taken and respectively numbered as a positive control tube (two), a negative control tube (two), an experimental tube (seven) and a blank control tube (two); the PCR amplification system is 20 mu L, wherein,
adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL positive control into the positive control tube;
adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL negative control into a negative control tube;
adding 17uL of PCR reaction solution, 0.8uL of upstream primer, 0.8uL of downstream primer and 1.4uL of the Acinetobacter baumannii sample to be detected obtained in the step S1 into an experimental tube;
adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL sterilized water into a blank control tube;
in the Acinetobacter baumannii sample to be detected obtained in the step S1, the OD600nm is 0.8;
in the step S3, the amplification reaction is carried out for 3min at the temperature of 94 ℃; then 35 cycles, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 61 ℃ and reacting for 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.
Example 10
And (3) verifying the relevance of the biomarker and the high-toxicity acinetobacter baumannii.
1. The whole genome sequences of the acinetobacter baumannii with different virulence are compared by using Spine software, a gene sequence (shown as SEQ ID NO. 1) which is positioned on a chromosome and is specific to the acinetobacter baumannii with high virulence and comprises 19 open reading frames is screened, and a section of specific sequence in the gene sequence is taken as a marker;
2. comparing the whole genome sequence of 62 clinical isolates with the marker sequence of the high-toxicity Acinetobacter baumannii by using Parsnp software in a Harvest software package to judge whether the marker exists or not;
3. principle of design
To obtain: the mortality rate of the high-toxicity acinetobacter baumannii marker positive sample is obviously higher than that of the negative sample (as shown in the following table 1), and the result has statistical significance (chi-square test, p < 0.01).
A specific sequence (shown as SEQ ID NO. 2) in a section of high-toxicity acinetobacter baumannii gene sequence is screened out by adopting a comparative genomics method, and a specific primer is designed according to the sequence. On the basis, a kit for detecting high-toxicity acinetobacter baumannii and a using method thereof are provided (shown in figure 1).
4. The detection kit is used based on polymerase chain reaction, and comprises the following steps:
1) Sampling
Inoculating the acinetobacter baumannii to be detected in a culture medium until the OD600nm of the culture is 0.7, and taking a culture solution as an acinetobacter baumannii sample to be detected;
2) Preparation of PCR amplification System
Taking nine PCR tubes, and numbering as a positive control tube (one), a negative control tube (one), an experimental tube (six) and a blank control tube (one) respectively; the PCR amplification system is 20 mu L, wherein,
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL positive control into a positive control tube;
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL negative control into a negative control tube;
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL Acinetobacter baumannii sample to be detected obtained in the step 1) into an experimental tube;
adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL sterilized water into a blank control tube;
3) Amplification reaction
Centrifuging each PCR tube prepared by the PCR amplification system in the step 2) for 30s under the condition that the rotating speed is 3000rpm, and then placing the tube in a PCR instrument (Dongsheng LongETC 811) for amplification, wherein the conditions of the amplification reaction are shown in the following table 2:
the sequence of the upstream primer is as follows: 5'-TTGAGAAGCTAAATTATGGCTCG-3', the downstream primer sequence is: 5'-GATAGCACAGAAATCCATAAAGGAA-3'; in a 20 mu L PCR amplification system, the concentrations of the upstream primer and the downstream primer are both 0.5umol/L.
5. Judgment and result analysis
After the PCR amplification reaction, each PCR reaction product was subjected to conventional agarose gel electrophoresis, and a band of 91bp was found to be positive (as shown in FIG. 2). In 62 Acinetobacter baumannii clinical isolates which are verified by whole genome sequencing, the sample results containing the high-toxicity Acinetobacter baumannii markers are positive; the results of samples without the high-virulence acinetobacter baumannii marker are all negative.
6. Verification of correlation between high-virulence acinetobacter baumannii marker detection positive and virulence
And (3) verifying the toxicity of the high-toxicity acinetobacter baumannii marker positive sample by using an acinetobacter baumannii pneumonia infection model.
Specifically, 3 Acinetobacter baumannii biomarker positive samples and 4 Acinetobacter baumannii biomarker negative samples are randomly selected to be 2.5 multiplied by 10 8 The survival rate of C57BL/6 mice infected with CFU via trachea for 6-8 weeks is observed after 7 days, and the results are shown in FIG. 3 and show that: infecting for 2 days by the positive sample of the biomarker, the survival rate of the mouse is 0, and the death rate is 100%; channel biomarker yinThe sex samples are infected for 7 days, the survival rate of the mice is 50 percent, the death rate is 50 percent, and meanwhile, the death rate has significant difference (p) through log-rank test<0.0001). And the obtained high-toxicity acinetobacter baumannii marker positive strain presents high mortality rate after infecting mice, which indicates that the strain is a high-toxicity strain.
Furthermore, the clinical isolated strain of Acinetobacter baumannii from Anhui proves the application value of the marker, the kit and the using method for qualitatively detecting the high-toxicity Acinetobacter baumannii. The results show that: the mortality rate of the high-toxicity acinetobacter baumannii marker positive sample is obviously higher than that of the high-toxicity acinetobacter baumannii marker negative sample (as shown in table 3), and the results have statistical significance (chi-square test, p is less than 0.01).
In a first aspect, the present invention shows a gene sequence (shown in SEQ ID NO. 1) comprising 19 open reading frames, which is located on a chromosome and is specific to Acinetobacter baumannii;
the second aspect shows that a specific sequence (shown as SEQ ID NO. 2) in the gene sequence (shown as SEQ ID NO. 1) is a marker, and a specific primer is designed based on the specific sequence;
the third aspect shows a kit for detecting high-toxicity acinetobacter baumannii and a using method thereof, under the normal condition, the toxicity of the acinetobacter baumannii is weaker, a clinician cannot accurately judge whether a patient uses antibiotics, and the kit can quickly and accurately detect the high-toxicity acinetobacter baumannii, so that the technical scheme has important reference significance for making a treatment scheme of the acinetobacter baumannii infected patient.
The above is only a general embodiment of the present invention, and it should be noted that, for those skilled in the art, several other methods and improvements can be developed without departing from high virulence acinetobacter baumannii marker sequences, specific primers, etc., which are within the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen citizen hospital
<120> kit for detecting high-toxicity acinetobacter baumannii and use method thereof
<130> 2019.4.3
<141> 2019-04-03
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18813
<212> DNA
<213> Acinetobacter baumannii
<400> 1
atgtctacag atattcaatt aaatttaatt ggcagaacac aagagctttt tgttgatgat 60
attcaacaat ggaataataa gttaatcgaa attgtttcct catcgaagtt tttagtttta 120
ggtggggcgg ggtctattgg ccaagcagtt gttaaagaaa tttttaaacg taatcctaag 180
aaactacacg ttgttgatat ttcggaaaat aatctaacag aagttgttcg tgatattcgt 240
agctcgtttg gctatatcga tggtgatttc caaacatttg cactagatat tggctctgct 300
gagtatgatg catatattca gaatgatggt gattttgact acgttttaaa tctttctgct 360
ttgaagcatg tacgtagtga aaaagatcca tttacattaa tgcgaatgat tcatgtcaat 420
attttgaata ctgaaaaaac gattcagcag tctattgaaa aaggcgtgaa gaagtatttc 480
tgtgtttcaa cagataaagc tgctaaccct gtaaatatga tgggtggctc aaaacgtatt 540
atggaaatgt tccttatgcg tcaaagtcag catattgata tttccacggc tcgttttgcg 600
aatgttgcat tttcagatgg ttctttatta catggtttta atcaacgtat tcagaaaaac 660
cagccaattg ctgcgccaaa tgatattcgc cgttatttcg ttactccaaa agagtcaggt 720
gagttgtgcc tcatgtcttg tttactaggt gagaatagag atattttctt ccctaagctt 780
agcgagcact tacacctcat tacttttgct gatattgcag tcaaatatct taataacctt 840
ggatatgagc cgtatttgtg tgagtctgag gatgaagcgc gtagcttaat tcagactttg 900
ccaaaacaag gaaaatggcc ttgtttattt gctggaagca caactacggg tgagaaagat 960
tttgaagagt tctttaccga taatgaaact ttagatatgc aacgttttaa taatttaggt 1020
gttattaaaa acgaactaaa tgttgaagaa gacaagttgc atgtatttga agaaaaaatt 1080
catgcaatgc tttcaaataa gcagtggaat aaagaagaaa ttgttgatct atttaactat 1140
atgatgccta actttggaca caaagaaaca ggtttatatt tagatgggaa aatgtaatgg 1200
atacttctaa atttaatcag tttgtacgag atatttatca taccaataat tttattccat 1260
tacatgaacc tcgttttctt ggtaatgaaa agaaatatgt attagatacg attgatagta 1320
cttttgtctc tagtgttggt gcatatgtaa atgacttcga agcaaaagtt caacaattta 1380
caggttgtac taaagccatt gcgactgtta atggaacagc tgcattacat attgctcttt 1440
tactagctgg tgtaaagcgc gacgatattg taatcacgca agcgctgact tttgtcgcaa 1500
cctgtaatgc cttgtcttat attggtgcgg aaccattatt tattgatgtt tcattgaaga 1560
cactcggttt gtgcccaaag gcattagacg cttatttgca agaaaatgct tttattgatg 1620
accaaggtgt atgtaaacac aaagcaacta ataaacgtat ttctgcaatc gttccaatgc 1680
atacatttgg tcatcctgtt gaaattgatg agcttcaaca ggtcgtaacc aagtggaata 1740
ttgcattagt tgaagacgct gctgaaagtt taggttctta ctacaaaggt aaacataccg 1800
gaacttttgg tctagtttca acacttagct ttaacggaaa taaagtcatc actacaggcg 1860
gcggcggtat ggtcttgtgt caagatgagg aacttggcat aagagcgaag cacattacga 1920
caactgcaaa aataccgcat ccttatgagt tttatcatga tgaaaatggt tttaactacc 1980
gtttaccaaa tttgaatgct gccttaggtt gtgcacaaat ggaaggcttg gaaggtttcc 2040
ttgctaaaaa gcgtgattta gctcagcaat atcaagattt ctttaaagat agtgatattt 2100
catttgttgt ggaacctgaa gactgccgat ctaactattg gttaaatgcc attgtttgca 2160
aagataaagc ccaacgagat ttgattttgg atgaaaccaa ttcaaataag gtgatgactc 2220
gcccaatctg gactttaatg accagattgc caatgtacca aactgcatta caaggcgatt 2280
taactaactc attatggttg gaagaaagag ttgtaaatat tccaagttca gttcctctgg 2340
agtaataaga atgaaaaaaa tagccgtttt tacaggaact cgagctgaat atggtttgct 2400
gtattggtta atgcgagata ttcagcaaga tccagagcta gaattacaaa ttctagctac 2460
ggctatgcat tattcatctg agcacggtga aacttggaaa actattgttc aagatggttt 2520
cgaaattacc gaatcggtag aaatgctact gtcatctgat acatcgtccg ctgttgtgaa 2580
atcaatgggt gtgggcttac ttggatttgc agatgcattg aaacgtatgc aacctgattt 2640
attagttgtt ttgggtgatc gtttcgaagc tttggcagtg acgcaggctg ctttaatcat 2700
gcatattcct gttgcacatt tgcacggtgg tgaaatcact gaaggtgcat atgacgaatc 2760
aattcgtcat gcgatcacca aaatgtctaa tatccatttt gctgcagcag aagaatacaa 2820
aaaacgaatt attcagttag gcgagcaacc agaaaaagta tttaatgtcg gcgcactagg 2880
cttagatcat atccagcgta caacatttaa aaatattgca gaattgagtg aattatatga 2940
ttttgatttt tcaaaaccat attttctcat tacttatcat cctgaaacta atctactaga 3000
agaaaatgtt gcacctttat ttgatgcact taagcaaata aaagatgtca attttgtctt 3060
tagttatcca aatgcagata atggtaatac taacattgtg aaagcaatgc ttgatttaaa 3120
agcacagttg ccagatcgtg tattgcttgt aaagagtttt ggtattcaaa actatttaag 3180
tgttttgaaa aatgctttag caatggtggg taattcttca agtggtctat cagaagctcc 3240
agcattacag gtaccaacag taaatattgg tgatcgtcaa aaaggacgct tacgttgtga 3300
gtctatactt gatgttagat tagacgaaag tgaaatttta gaagcattgc aaaaagcaat 3360
taatttccca gacgatgaat tgagtaaagt tgttccacca ttagggttag gcaatacttc 3420
tcagaaaata attgaactta ttaaaacgac ggattttaaa aagaaggcac cattttatga 3480
tctttaatca agacgaaatt tatgtcattg cagaaattgg tgtaaatcat aatggctctg 3540
tagagttagc aaaagagctt attttaaaag ccaaagaatg cggtgcgaat gctgtcaaat 3600
ttcaaacatt taaagcagat agtttattat ctgatcaaac tgaaatggct gcatatcaaa 3660
aagaaaatac tagttctagt caaagccaat tagagcttgt aaaaagttta gagctcactt 3720
atgagcagac agaagaaata cagaaatttt gtgatgagca tcaaattact tttatttcaa 3780
cgccgtttga ctctgatagt ttgaagttct tggtgaatga gatcgatgta ccttacttga 3840
aagtatcatc tgctgatatt tcaaatttac cgtttctcta tgaaattgca tgttcaaaaa 3900
agcatgtcat tttatctaca ggaacagcta gcctaggtga tattgaacaa gcattatcag 3960
tatttgcttt tgttatcgat aagggaacgg aagttcgacc atctcagcag ttatttagag 4020
aagcttattc taaaatttca gtacgtaaac agttgaaaga acaggtttct atcttacact 4080
gtgtaactca atatccggca ttatttgaag aaagtaatct taaagccatt tcaaccttaa 4140
aaaatgtatt tgatttggca acagggtttt ctgatcatac gcttgatgaa tacgcggcgg 4200
ttattggtgt aagtcttggt gctcgcattt tcgaaaagca tattacatta gataaaacga 4260
tggctggccc agaccatgca gcatcaatgg aaccagatga atttaagcat tatgtcgaaa 4320
ttttgcataa aacatatgca gctttgggcg atggtattaa atttatgcta gagcaagaga 4380
gcgataacta ttacctagta cgtcgtagta ttgtggctaa aacagatatt gcggaagggg 4440
agctgttaac ctctgacaat gtcacaacaa aacgagcagg tcgcgtttgt ttagaaccaa 4500
ataagtattg ggatgttgtt ggtacaaaag caaaacgtag ctttaaaaag aacgatttta 4560
ttgaaattta aatgaaaaaa ctggcgattt ttggttcagg tggctttgca aaagagctag 4620
tagatttagc aatagatcaa ggctaccgcc atatattttt tttagaaaga aatgccaaag 4680
atggtgacac cctacttggc ttccccattt tgccagaatc agcaatatcg acattaacgg 4740
atacggtata tgcgataggt gttgctgatc caaagattcg taaaaaaata tatgaagctt 4800
atcccgattt accttatccc aatttgattc attctcaatc atcacttggc tatggcattc 4860
gagagttgct agaacatagt aaaggtgtgg taattgcggc tggagcgaga attacaaatt 4920
catgtcgttt tggtcatttt attatagtaa gttttaacag cacaattggg cacgactgta 4980
ttctagaaaa ctacgtttct gtcatgccag gtgttaatgt gtctggttgt attcatttga 5040
agcaaggcac ttacattgga acaaatgctg ctattttgcc gggcaaaagt ccagagcaat 5100
tgaaatattt aggtgaaaac tccgttatag gcgcgggtgc tgtggttgtg aaacatacgg 5160
aaccgaataa ggtctatatt ggatcacctg ctaaggaatt aagtcgtgac tagagatgtt 5220
aataatataa ttttgcataa aaatgattca attttgaaag cattagagct acttgattta 5280
tatgctttaa gaattgtttt ggtcgttgat gatcacaatc aattgatcgg aagtattaca 5340
gacggtgata ttcgtagagg gcttttaaaa ggccaagatg tgcatgcagc tgttgaaaca 5400
atcatgcata ctaaccctta tagtattgaa gaaggtagct taaataaccg ccagattttt 5460
gagatcatgc gtgaaaaaag ttatctggca ttaccagtaa ttaaaaataa tcagctagtt 5520
aatatcatta cgcttgatga tttgattcgt aaaaaacgta aagaaaatcc agtttttatt 5580
atggctggag gatttggaac acgtttacgt cctttaacag atacatgccc aaaaccaatg 5640
ttacctgttg gtggtaagcc tttactagaa accattatct caagctttaa aaatcaaggc 5700
ttttataaat tttatatttc tactcactat ttaccagaag tgattaatga gcattttggt 5760
aatggtgaga agtttgacgt acagattcaa tatgtacatg aaactgatcc gcttggaact 5820
ggcggtgcat tgagtttatt gccagcatca gatattaaat tgccattcat tgtgattaat 5880
ggtgatgtac taacaaacat gaattttgaa aaattattgg actttcatga aaagcgcgaa 5940
gctattgcaa cgatgtgcgt gagagaattc caatatcaaa ttccttatgg cgtagttaac 6000
tctgaagata atgtaataca aagcatgact gaaaaaccaa gttatttctt tgatattaat 6060
acaggtattt atgtcatttc ccctgaattg ttggcacaag ttaatgcaca gtttattggt 6120
atgccgacta tcttagaaca gcaaatggaa aagaaaaata aagtcttaag ctatccttta 6180
catgaatatt ggctcgacat cggtcatatg gaagactata accgtgcgca aaaagatatt 6240
catatgttgg gtctagaata atatgaaaaa tattgctgtt attggtttag gtaatattgc 6300
tacccgacat cgtcgtaacc ttaaaatgct ttttcctgaa gcaaaattaa ttgtgatgtc 6360
tgctagtggc cgtattccta aagagccagt gactgatagc gatcatatag cagaaagtgt 6420
tgatgaaatt attcagttgc aagttcagtt tgcaataatt gcctcaccag ctccttttca 6480
tgcccaacat gcactaccac ttattaaagc aggtatccct gtattaatag aaaaaccagt 6540
atcagttacc cagacagatg ctcaagcttt aattgatgca gaagcccagt ataagacacc 6600
tgtagcagta ggttactgtt tgcgctattt accttcagcg cagcaagtac gacaaatgat 6660
acaagagggt gtcattggaa acttatataa tgcatttatt gaaattggtc agtatttacc 6720
agattggcga cctaccaagg attatcgcga aacagtatct gctaaaactg aattgggggg 6780
aggagtccta ttagagctga gtcacgagtt agattatgct caatggattt taggttcgct 6840
tactcctaaa tatgtcgttt tacgtgcttc agaagagctc ggtttagaag tcgaggataa 6900
tgccgatctt ttaatgacaa cagcgaaagg agctgttgta aatatccact tagatttttt 6960
gcagcgtaaa gcacatcgta aatgtcgttt tattggcagt gaaggatgta ttgagtggga 7020
tttaattcga aatgaagttg tgcttattaa ggctaaagag caacaagaaa tttatagcgc 7080
ccctgagtgg gataaaaatc aaatgtattt agaaatggtt accgatttta ttaaaaaaat 7140
aaacggacag ccaaaccaat ctataagtct gcaagaagct gaaagaacag taggcctgat 7200
tgtagagatg aaagaatttg tgacgaaaga atgagactgc ttatatgaga aattttgcat 7260
ttatttttgc tagaggcggc tctaaaggtt tacctggaaa aaatattaaa ccattggcag 7320
gaaaaccgtt attacaatat tcaattgata ccgctttagc atcggatttg attgaacaag 7380
tttttgtttc aacagatgat caggctattg cacaagtcgc tatagaaggt ggagcaattc 7440
ttatagagcg acctgctgaa ctagcaactg atcaaagtcc agaatggttg tcttggcgtc 7500
atgcagttga gtgggcaact gagcactatg gctcatttga tgggtttgtt agtttacctg 7560
caactagtcc attacgtagc caagaggatg tagaggctgc gattttaaaa agacaagcag 7620
agacagctga tatttgtatt gcagttacac ccgcaagtcg cagtccttat ttcaatatgg 7680
ttaagtacaa tgaggctggt tttgttgagc ttgtcaatca gcctgaaggt gaagtatcac 7740
gccgtcaaga tgctcccaaa gtttttgata ttacaacagt agtttatgcg acaacgcctg 7800
agtttgtatt aaatagctat ggtttatttt cagggaaagt tgcgagtatt gaagtaccaa 7860
aagcaagagc tgtagatatt gatgatattt acgattttcg tttagctgaa gctattatta 7920
aaggtgaata attgatgcaa aatttattac agaataaaac ctttttagta gctggtgccg 7980
gtgggttatt aggcacgcgt ttagtggcgg ctatattaga acaaggtgca aaagttattg 8040
cagctgatat aaaccttgaa tctatggcaa ctcgattaag tagtgtagga gttaatacta 8100
caaacgaaaa gttatcgttg gtttccttag atgtaacaaa tgaaacaagt gtaaagttat 8160
ttttttcaac agatcttaaa attgatgggg ctgttaatac aacataccca cggaataaga 8220
cttatggttc gcattttttt gatgtgactc ttgaaagctt taatgagaac ttatcactac 8280
atttaggtag tatgtttttg tttactcagc aatgtgccaa atattttacg caataccaga 8340
ctcctttttc gcttgtaaat atctcatcta tttatggagt agtggctcca aaatttgata 8400
tttataacaa cacgaaaatg accatgcctg ttgagtatgc tgcaattaaa tcagcaacac 8460
tccatttaaa caaatacaca gttgcttatg tcaacgacag ccgattccgc attaattgtg 8520
taagtccagg tggtatcttt gatcatcaac ctgaaccatt tttagaagca tataagagcc 8580
atacccatgg agctggtatg ttagatgtta cagaaatcat aggttcagtt ttattcttac 8640
tttctgaaca atcaagatat gtaacaggtc aaaatattat tgtcgatgat ggcttttcaa 8700
tataacttaa ttatgagtaa tatatatata ggtgattggt aatatgaata ttattaataa 8760
tatagttgta aaattttttt taacaatagt tttttatata atattcctat taactttaga 8820
tatttttcct aataaacacc tttttgctat tttttcattt agtgtttatt tgttaaatag 8880
accaacattt attttatcac ctcggaatat gatatttata tattatttct tgtggtttgg 8940
tttggcacct atatttgcgg atagatataa ttatttagat tattctgata atatagttta 9000
taaatcttat gtttatttgt cttcaagttt tataactatg atattagttt ctatgtgtat 9060
agaaactaaa tacgaaaaaa agttttcttt tataaaagtt gattatagta atgtaagaat 9120
cgatggtaaa aaactaactt ttacttttat atttagtttt cttttttttc ttttgtattt 9180
atttaatact ggaggattta gttactggat agcaaatatt gatcgagctt ttttgactag 9240
gcaaggggca ggagtatact atttaggctt ttcattgttt ttccccgtat ttatattttt 9300
tactagattt aagtataaaa atttaatttt attaagtaca ttaagtttat tagtgatagc 9360
tttaagtcca tttataggta gtaaacaaaa aattatttat tgttttttat tactgttttc 9420
ttccttaatt ttgagaagaa agttgacact aagtaattct ttaatgattg gcttgcccac 9480
aatcttttta tttgtattag ggaattattt tagaaatgtt tcatggatga cattaagcga 9540
tgttgcttca tattcattta attattttga tactttagat tctttattgc tatttttata 9600
taacagacat tacccatttg aaattatgtc agtaggactc cctttaaata aaattgttaa 9660
tttatataca ggtggcgatg aattttttga tgtaagtgca tattatacta atatatactt 9720
tcctgatgct tggaatatac gagcaacagt acaatttcct gtggaagtag atttattttt 9780
aagttttggt ttttgggtgg gattaattcc tttaggaatt tttgttggaa tatattcatt 9840
aattttcatt aaaatgttat ctagtgaaaa aattgtatat ggttttatat ggtttaattt 9900
atttttctat ttgttaagtc acatgcgtgg tggattattt atttggacgg atttatattt 9960
atatccttat cttattttag tatattttct ttttaaagag gttaaagtta atgtgcctaa 10020
aaaataagaa atatgaaatt ataaaaaaag ctattgaaaa tgaaatttca gtagataatt 10080
atctttttga gaagctaaat tatggctcga aaaagctatc tattttcaaa atgctacgat 10140
tcaaagataa aattccttta tggatttctg tgctatcttc tattttaata ccaatttatt 10200
ttgctattga attttttgtt ttttttggaa ggagtagaaa tacatcaaat cttgaaaatg 10260
atcaatattt tttatctttt tctaactctc caaaagtaat tgcaatttat gaagaatatg 10320
cttctgatat agagtttttg caatgtatta ataataaagt ttattgtttt gttggctatt 10380
taagtcctct agttatcctg aagagtttta ttcattcaat tatactttac tttaaaatga 10440
tattttttgt taatattaaa tattatttac acttaacagc aatttttgaa ttaattttat 10500
tttatgaatt tgttattgta ttaaataaaa gaaatataaa taaaatatgt gtggtaaatc 10560
attatgatag gtggttaact gttttgtcaa atattggtaa ttttgatatt gagataatac 10620
agcatggaat tttaacagaa aattatgttg ttaagcataa aataccaaat atttcttttt 10680
taaaagtttt tgataaatct caattgagta tttttctaaa aaatatatta gagggtatac 10740
ctaataaaat tgattatatg aaaattagtt tagatatcaa cttttctgat aaatgtgaag 10800
tattgataat tagtaatcca ttttttataa atcaggaact gttattatat aaattcttga 10860
aagataaagg gattaaagtt ttttttagac ctcatccact ttatattaat gaagaaataa 10920
aaagtgtagt tgaaaatgat gagttatgtt taggaaaaag atttccatat cctaacgttt 10980
gcttatgtaa tgagtctact ttgggtaaag aatatgaagc aatgggtttt aaagtattat 11040
ggtggaatca aaatacagat tttcataaga tatggaattt agttaataat gcgtaatttt 11100
attttatatg gtttagcttc tgcttttaat agaggtggag tttttcttat actcccttta 11160
ctagctgctt tactttcagt tgtagattac ggtaaatttt ctctctatat gacagtagtt 11220
caacttttag tgccgctaat aagtctaaat atttctttat tggtgggtag ggaaatttac 11280
gaaaaaccta ttgtagttaa aagctttgtt gtactattta atagattttc tatttttaca 11340
ataatagttt tttttacttt aaattttttt attagaaata ttattttatt gattttaatt 11400
tatacctttt tggaagctat ttttttggtg aattctacat atattcgctt taagtatggt 11460
gcagatcaat ttttctatgt ttgttttttt aaaattttta ttctttcttt tttattagga 11520
agtttcttta tctatgataa gcagataata gtatcaataa aaaatcttat catgatcttt 11580
tctttttcta atttatcaat tgtatattat cttgtaaaga cctctttttc tttcagtttt 11640
aagagaatgt ttgtaattat aagcaactat aagagtatgc tacttttttc tattgggtta 11700
ttacctcata cactttctca atgggtaagt tcaagttcag accgtttttt tgtcaaactt 11760
tatacaaatg atatagaact agggtactac tcattttcat actctctggc tgctatattt 11820
atgattgtaa atagttcatt agctctcgga atacctcaat tatgtgttaa aaattttatt 11880
tattttaatt ccaaaaaatt ttactcaata ttcttttcac tagtgtcaat cttatgggtt 11940
ctatttttaa ttatttatat tgggatatta tattattttt cagacaagta tgatgttaaa 12000
tatatattta aaattggata tgtaattcta gtagggatgt attttttatc gttctattat 12060
tattattcgt cttttctttt ttatgataga gcatctaaaa agttatctta tattactttt 12120
tttgtggcag ttttaaatgt ttctctagta gtgcttctta ctcctttgtt tggtatacta 12180
ggtactgcct tcagtacatt aatatcctat ttaatatatg ttttaatgtc ctataaacat 12240
gcaagtgtta attatacggt aactaatttg tatgctcctt tatctttcat agtaattatt 12300
tcagtatcta tatttgtttt tttatacttc tttaaaatag gtggtgtgag ttatgtttaa 12360
aaataaggtt ttattaatta ctggtggtac aggatctttt ggaaatgctg tattaaaacg 12420
atttatagat agtgatttta aagaaatccg tatctttagt cgtgatgaaa aaaagcaaga 12480
tgatatgcgc aagaaatatc aatcagctaa gcttaaattt tatatagggg atgttcgaga 12540
ttttaattct gttttaaatg taactagagg tgttgattat atttttcatg ctgcagcact 12600
taagcaggtt ccttcttgcg aattttatcc aatggaagct gttaaaacaa atgtgttggg 12660
gacagaaaat ttattagaag cagccattca aaacaatgtt acaagggtag tttgcttaag 12720
cacagataaa gctgtatatc ctattaatgc aatgggcatt tctaaagcca tgatggaaaa 12780
agttatggtc gcaaagtcac gtaacctaga aggcttagat acagttattt gtggcactcg 12840
ttatggcaat gtgatggctt cccgtggttc tgtgattcca ttgtttgttg atcaaatacg 12900
tattggtaaa tctattacaa taacagatcc gaatatgaca cgttttatga tgactttgga 12960
agatgcggta gacttagttc tctatgcttt cgagcatggt gaaaacgggg atatttttgt 13020
acaaaaagca cctgcggcta ctattgatac tttagctaga gctattactc aattattaga 13080
taaacctgaa catccaattc atattattgg tactcgccat ggagaaaaag cttatgaggc 13140
tttattaagt cgtgaagaaa aagcatgtgc cgaagatctc ggtgattatt tccgtgtacc 13200
agcagaccaa cgcgacttga actacgaaaa gtatgtagaa gatggtgatt taaaaattac 13260
tgagttcgaa gattacaact cacataacac cactcgtcta gatgtagagg gtatgaaaca 13320
attattgctt aaactagatt ttgttcgcgc attaacaaga ggcgaatata tttcaccaga 13380
ggcgtaagca tgaaagtttt agttaccggt tctaatggtt ttattgcaaa aaactttatt 13440
cagtttttat cagagaaggc tgaagttgag attttaaaaa ctcatcgtga aacaacagat 13500
caagagttag agcaatcagt tcttgcttcg gactggattg tgcatttagc gggtgtgaat 13560
cgccctttaa atgaaagtga atttgcggaa ggcaatacca ctttaacaga aaagatcagt 13620
caaattttgc agcaagcaaa taagaaaaca cctgttatct tatcgtcttc tattcaggtc 13680
gagcgtgata acccctatgg taaaagtaag ctaggtggag agcaggcact ggtcactctt 13740
catcaagcag aaggtaatcc ggtttatatt tgccgtttag ccaacgtttt tggtaaatgg 13800
tcacgcccta attataactc tgcagttgca actttttgcc ataatgttgc aaatgatttg 13860
cccttacaaa ttcatgatga aaatgctgtg attcgtcttg tttatgtcga tgatgtagtt 13920
gaaacattct ggaatattct gaatgggcaa caagttgaac aaattttcca agtcgaacct 13980
gaatatcaaa ttaccgtagg tgatttggct aaagttttaa atggctttaa agcatcaaga 14040
gatacactta ttaccgaccg tgtgggaaca ggtcttactc gtgcacttta ttcaacttat 14100
ttgagttttt taaagcctga gcaattcgat tacagtgttc caaaatatgg tgatgctcgc 14160
ggtgtattcg ttgaaatgct gaaaacccca gatgcagggc aattttctta ctttactgct 14220
catccaggaa ttacccgtgg tggtcattat caccatacaa aaactgaaaa gttccttgtt 14280
attaagggta aggcattatt taagtttaag catgttgtaa caggtgaatt ttatgagctt 14340
gaaacacaag gcgatgaacc acgaattgta gaaacggtgc ctggttggac acatgatatt 14400
actaacatcg gtgatgatga gatggtggta atgctatggg caaatgaaat ttttgatcgt 14460
gataagccag atacttatgc gatgccaata acaaattaag agtatagatt tgtgaaaaag 14520
ttaaagttaa tgacagtggt gggcacacgt ccagaaatta tccgtttgtc ttgtgtaatg 14580
gctgcgtgtg atgaatattt tgaccatatt attgttcata ctggtcaaaa ctatgactat 14640
gaattgaatg aaattttctt tactgatttg ggtatccgta aacctgacca tttcttaaat 14700
gcagctggtg cgacaggtgc agaaacgatt ggtaatgtaa ttattgcggt tgataagatt 14760
ttagatgaag tgcaacctga agctttactt gttttagggg acacaaacag ttgtatggca 14820
gtattacctg caaaacgtcg taaaattcca actttccaca tggaagctgg aaaccgttgt 14880
tttgatatgc gagttcctga agaaattaac cgccgaattg tcgatcatac ggctgatatt 14940
aatttaactt acagtacgat tgcccgcgat tatttattgg cagaaggttt accagctgac 15000
ttagtaatca aaacgggtag tccaatgttt gaggtacttc accattacaa agcaaaaata 15060
gaagcttctg atgttcttga gcgtttaaat ttaaaagagc atgaatactt tattgttagt 15120
gctcatcgtg aagaaaacat taactcagat cagaacttct tagatttggt tgaaatgtta 15180
aatgcagttg cagagcgata tcagtaccct gtgattgtat cgacacatcc tcgtacgcgt 15240
aaacgtattg aagagttaaa tattgatttt catcctttga ttcaattatt aaaaccactc 15300
ggttttagtg actacaacaa gttacagctt tcagcaaaag cggcattgtc tgatagtgga 15360
acgattaacg aagaatcatc aatcttgaat ttccctgcac ttaatttgcg tcaagcacat 15420
gagcgtccag aaggaatgga agaagcggct gtaatgatgg taggtttgaa agccgaacgc 15480
attttacaag gtttggcaat tttagaaggt caaactagag gcgaaaatcg tcttttgcgt 15540
ttagttgaag actacagtat gccaaatgtg agtgaaaaag ttgtgcgtat cattatgagt 15600
tacacagatt atgtaaatcg agtgatttgg aagaagtatt gaggttaagg cagtgaagaa 15660
gaaggtctat ttaatcacgg aatattatca tccatatcaa aatacgacag gttacttgtt 15720
aggaaaattg tatgacacct tgaatgtaca aagtgatata gaccttgttc ttattgcaaa 15780
agaggataca aactgtcctc aacatgcaaa tgcacacttt attaaagcta aaaagccaaa 15840
taaggcaagt ttgtttaaaa gatttttata tgaactaatt atcgctttta gttttttaat 15900
gaagactttg caagtagtga aaaaggatag tatcgttttt acaggtacga ctcctatttt 15960
actgcttttt gttttattta ttattaaaaa gtttctaaat tttagatgga tattattagt 16020
ccatgatgta tttcccgaaa atttagcagc tgctaaaatt ttaaaaaaag atcatttttt 16080
atttaaaatc ttaaaaagct tatttgatag gatttatgca agtgctgatg aggtgattgt 16140
cattggcaaa gatatgaaag agttagtaca tcaaaaaacg catcaaagta acaatattac 16200
tgtagtgcag aattggattg actcatctga tattggtaca gagcctaaaa ctaataaccg 16260
aattttacaa gaattgaact ggttggatag tgaaactacg atttttcaat tcttcggtaa 16320
tattggtcgt gtacaaggcg tagaaaatat tctgcatgct attcagaaaa tgaaatatgc 16380
acatttggca aaattcattt ttattggtga tggtgcatat gttgctcgtc ttaaagagca 16440
aatacagtct ttgggatctc aaaatattct ttattatgga tctttagatc agaaagaaaa 16500
aacaactggt ttaaatgcgt gtgatgttgc attgattact ttggctgatg ggatgttagg 16560
tttaggggtt cctagtaaat cttatttttc tatggcggca gataagcctt tgctagcaat 16620
tatggatagg gagtcagaag tagcagatat gattaatact catcatatcg gttgggtagt 16680
cccacctaat aatgaagagg ctttggcaaa acaacttgat gaaattgtgt tagcaaaata 16740
ctgctatgca ttcaattcat caagagaagt attaaatgaa ttttattcag aaaaagtagc 16800
aatgagtaaa attctaaata ttattagaaa atttacttaa gaatttaatt tttaaaatga 16860
atatcttgat tacaggaagt agtggttttg ttggttcata cttatgtgaa tattttacga 16920
caatgccaga atatagtgtt ctggctcaaa ctcgtaaaaa tacacaaaag ttaagtaatg 16980
atataaagac tataactttt gatataaatg atgatctaac ttctttggac ttgagctcat 17040
tagatgtcat tgtgcattgt gctgggcgtg ctcatattat gaaggaagtt gaaagtgatc 17100
cactatctat ttatagaaaa gtaaatgtag aaggaacact gaatttagct aaaaaagcag 17160
ctaaagctgg tgtaaaatga tttatattta tgagtagtat taaagtaaat ggtgaagtga 17220
gtgatcaagt accttttact cctcatgata atattataaa tattaaagat ccttatggat 17280
tatcaaaata tgaagctgaa caatctctat taaatttagc gcaaaactcg atgatggagg 17340
tggtcattat tcgcccagct ttaatctatg ggccaaatgt taaggcaaat tttaaaagta 17400
tgatcaatct ggcggctaaa ggaattcctt tgcctgttgg atgcctcgat aataaaagaa 17460
gtatggtcag tatttataat ttaagcgatt tcattaatat ttgtttatca catcctttag 17520
caaaaaatca ggctttttta attagtgatc aagacgatat aacagttaaa gaattattta 17580
cgaagttagc taaagttcag ggtagaaatc ttattgctgt gcctatacct aaagtattaa 17640
ttaatttttt aactaaactt cttaataaga gtgctgtcgc ttcaagactt tgttctgtat 17700
tgatcgttga cacctcaaag aatatagaac tattaggatg gagagctcct tttagctttg 17760
atgagagctt aaaattgatg ttcaaaaagt aataggtttg atatgaatgt tattatatct 17820
ttcatcattc tttttcttat ttcttttgta ttaactttta gtgttcgaaa atatgcttta 17880
agaaaaaata ttattgattt acctaacttt agaagttcac atatgaagcc cacaccacgt 17940
ggtggtggag tagcaatagt gataagtgtt ctaatggctt tattatatag ctatatatct 18000
cagtttataa gtttgaatga ttttttcatt atagggggga gttgtttgct tattgctctc 18060
cttggttttt gtgatgatca tgctcatatc aattctatgt tgcgcttaag tattcatttt 18120
attactgcaa catttatcgt atttagttta aatggctttg cccaagtctc tgtttttaat 18180
aatttaaact taggtttttt taccaacatt cttgctattc tttttctggt ttggatgctt 18240
aatttatata actttatgga tggtattaat ggtattgcaa gtatcgaagc aatatcagtc 18300
ggactaagtc tagctttatt atacagttgg tatgacatca ataattttat attacttgca 18360
atcacttcta ttgtctgtgg ttttctcgta tggaattttc caaaagctaa aatctttatg 18420
ggagatgtag gtagtagttt tttaggtttt ttattcgcta tattagcttt atatgcttta 18480
aaaatagact ttaagttatt tctagcttgg atcatttgtc taggggtttt tatagtagat 18540
gcaaccttta caattataag gagaatacta agaggggaga aaatatatca agctcatcga 18600
agccacgctt atcaatacgc tgctaggtat tttgatagtc atacacccgt aactttagca 18660
gttttaatca ttaatcttat atggctctta ccttgtgcat tttttgtatt aaataaagca 18720
atttctccat tagtcggttt aataatcgca tattttcctt taataatttt agctatttat 18780
tttaatgcag gacagttaga acataaaact taa 18813
<210> 2
<211> 1086
<212> DNA
<213> Acinetobacter baumannii
<400> 2
atgtgcctaa aaaataagaa atatgaaatt ataaaaaaag ctattgaaaa tgaaatttca 60
gtagataatt atctttttga gaagctaaat tatggctcga aaaagctatc tattttcaaa 120
atgctacgat tcaaagataa aattccttta tggatttctg tgctatcttc tattttaata 180
ccaatttatt ttgctattga attttttgtt ttttttggaa ggagtagaaa tacatcaaat 240
cttgaaaatg atcaatattt tttatctttt tctaactctc caaaagtaat tgcaatttat 300
gaagaatatg cttctgatat agagtttttg caatgtatta ataataaagt ttattgtttt 360
gttggctatt taagtcctct agttatcctg aagagtttta ttcattcaat tatactttac 420
tttaaaatga tattttttgt taatattaaa tattatttac acttaacagc aatttttgaa 480
ttaattttat tttatgaatt tgttattgta ttaaataaaa gaaatataaa taaaatatgt 540
gtggtaaatc attatgatag gtggttaact gttttgtcaa atattggtaa ttttgatatt 600
gagataatac agcatggaat tttaacagaa aattatgttg ttaagcataa aataccaaat 660
atttcttttt taaaagtttt tgataaatct caattgagta tttttctaaa aaatatatta 720
gagggtatac ctaataaaat tgattatatg aaaattagtt tagatatcaa cttttctgat 780
aaatgtgaag tattgataat tagtaatcca ttttttataa atcaggaact gttattatat 840
aaattcttga aagataaagg gattaaagtt ttttttagac ctcatccact ttatattaat 900
gaagaaataa aaagtgtagt tgaaaatgat gagttatgtt taggaaaaag atttccatat 960
cctaacgttt gcttatgtaa tgagtctact ttgggtaaag aatatgaagc aatgggtttt 1020
aaagtattat ggtggaatca aaatacagat tttcataaga tatggaattt agttaataat 1080
gcgtaa 1086
Claims (6)
1. The application of a section of specific sequence in acinetobacter baumannii as a marker in the preparation of a kit for detecting high-toxicity acinetobacter baumannii is provided, wherein the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii;
the gene sequence comprising 19 open reading frames is shown as SEQ ID NO. 1;
the specific sequence is shown as SEQ ID NO. 2.
2. The kit for detecting high-toxicity acinetobacter baumannii as claimed in claim 1, wherein a PCR amplification system in the kit comprises a PCR reaction solution, a primer for amplifying a specific sequence SEQ ID NO.2, a positive control and a negative control;
the primer for amplifying the specific sequence SEQ ID NO.2 comprises an upstream primer and a downstream primer, wherein,
the sequence of the upstream primer is as follows: 5'-TTGAGAAGCTAAATTATGGCTCG-3' of the formula,
the downstream primer sequence is as follows: 5'-GATAGCACAGAAATCCATAAAGGAA-3'.
3. The kit for detecting Acinetobacter baumannii with high toxicity according to claim 2, wherein the PCR reaction solution comprises Taq polymerase, tris (hydroxymethyl) aminomethane hydrochloride solution, magnesium chloride solution, potassium chloride solution, triton solution, formamide solution and deoxyribonucleoside triphosphate.
4. The kit for detecting Acinetobacter baumannii with high toxicity according to claim 3, wherein the PCR amplification system is 20 μ L, wherein the concentration of Taq polymerase is 5U/uL, the concentration of tris hydrochloride solution is 200mmol/L, the concentration of magnesium chloride solution is 20mmol/L, the concentration of potassium chloride solution is 500mmol/L, the volume ratio of triton solution is 0.2%, the volume ratio of formamide solution is 10%, and the concentration of deoxyribonucleoside triphosphate is 10mmol/L.
5. The kit for detecting Acinetobacter baumannii with high toxicity according to claim 2, wherein the PCR amplification system is 20 μ L, and the concentrations of the upstream primer and the downstream primer are both 0.5umol/L.
6. The kit for detecting high-toxicity acinetobacter baumannii according to claim 2, wherein the positive control is clinical acinetobacter baumannii strain nucleic acid which contains a specific sequence SEQ ID NO.2 and has high lethality in animal experiments and is determined by whole genome sequencing; the negative control is clinical acinetobacter baumannii strain nucleic acid which does not contain a specific sequence SEQ ID NO.2 and is non-lethal in animal experiments, and is determined by whole genome sequencing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910446577.8A CN110157824B (en) | 2019-05-27 | 2019-05-27 | Kit for detecting high-toxicity acinetobacter baumannii and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910446577.8A CN110157824B (en) | 2019-05-27 | 2019-05-27 | Kit for detecting high-toxicity acinetobacter baumannii and use method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110157824A CN110157824A (en) | 2019-08-23 |
| CN110157824B true CN110157824B (en) | 2022-11-11 |
Family
ID=67629329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910446577.8A Active CN110157824B (en) | 2019-05-27 | 2019-05-27 | Kit for detecting high-toxicity acinetobacter baumannii and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110157824B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876509A (en) * | 2020-09-10 | 2020-11-03 | 广州医科大学附属第一医院（广州呼吸中心） | PCR method for detecting high-toxicity acinetobacter baumannii |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119140A (en) * | 2017-06-16 | 2017-09-01 | 北京百康芯生物科技有限公司 | Respiratory tract micro-fluidic chip Fast Detection Technique and kit |
-
2019
- 2019-05-27 CN CN201910446577.8A patent/CN110157824B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119140A (en) * | 2017-06-16 | 2017-09-01 | 北京百康芯生物科技有限公司 | Respiratory tract micro-fluidic chip Fast Detection Technique and kit |
Non-Patent Citations (3)
| Title |
|---|
| An Emerging Clone (ST457) of Acinetobacter baumannii Clonal Complex 92 With Enhanced Virulence and Increasing Endemicity in South China;Kai Zhou等;《Clinical Infectious Diseases》;20181231;第67卷(第2期);摘要 * |
| Complete genome sequence of hypervirulent and outbreak-associated Acinetobacter baumannii strain LAC-4:epidemiology, resistance genetic determinants and potential virulence factors;Hong-Yu Ou等;《SCIENTIFIC REPORTS》;20150302;第5卷;摘要、表3、第6页右栏第1段和第10页左栏第1段 * |
| 广泛耐药鲍曼不动杆菌ST238克隆株毒力及适应性特征;何小庆等;《中国感染与化疗杂志》;20161120(第06期);第779-795页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110157824A (en) | 2019-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tzanakaki et al. | Simultaneous single-tube PCR assay for the detection of Neisseria meningitidis, Haemophilus influenzae type b and Streptococcus pneumoniae | |
| Kee et al. | Detection of leptospiral DNA by PCR | |
| CN105018489A (en) | Kit for recognizing Brucella wild strain and vaccine strains A19 and S2 | |
| CN105002173A (en) | Kit for identifying Brucella S2 vaccine strain and wild strain | |
| CN102947467A (en) | Assays and kits for serotyping pseudomonas aeruginosa and oligonucleotide sequences useful in such methods and kits | |
| CN111020041B (en) | 16 different serotype salmonella specific new molecular targets and rapid detection method thereof | |
| CN110669852A (en) | Kit for detecting high-toxicity non-mucus Klebsiella pneumoniae | |
| US20110287965A1 (en) | Methods and compositions to detect clostridium difficile | |
| KR20190002362A (en) | Specific primer set for discriminating pathogenic bacteria and uses thereof | |
| EP1795614B1 (en) | Diagnostic method and products useful therein | |
| CN110157824B (en) | Kit for detecting high-toxicity acinetobacter baumannii and use method thereof | |
| CN110079623B (en) | Method for detecting toxicity of acinetobacter baumannii | |
| CN102424862B (en) | Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila | |
| WO2006043368A1 (en) | Method of detecting streptococcus pneumoniae, primer set for the detection and kit for the detection | |
| CN103160587B (en) | Genetic typing chip of 10 common pathogenic legionella and detection kit | |
| CN109811073B (en) | Primer for rapidly detecting streptococcus agalactiae and streptococcus iniae at early stage by double PCR (polymerase chain reaction) and application of primer | |
| CA2775273A1 (en) | Composition, method and kit for detecting bacteria by means of sequencing | |
| RU2435853C1 (en) | TEST SYSTEM FOR QUANTITATIVE DETERINATION OF Streptococcus agalactiae IN BIOLOGICAL MATERIAL | |
| CN104328206B (en) | The LAMP detection method and its primer special of clostridium difficile binary toxin and test kit | |
| JP2004519225A (en) | Method for detecting pathogenic bacteria | |
| CN112961926B (en) | Primer, kit and method for simultaneously detecting listeria monocytogenes CC87 and listeria monocytogenes type 88 strains | |
| WO2005030027A2 (en) | Salmonella detection identification | |
| EP1445313A1 (en) | METHOD OF DETECTING AND QUANTIFYING HEMOLYSIN-PRODUCING BACTERIA BY OVERWHELMINGLY DETECTING AND QUANTIFYING THERMOSTABLE HEMOLYSIN-RELATED GENES (tdh-RELATED HEMOLYSIN GENES) OF FOOD POISONING BACTERIA | |
| KR20090122554A (en) | Primer set and probe for detecting salmonella typhimurium | |
| US7101668B2 (en) | Molecular markers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20190823 Assignee: Guangzhou Saimo Biotechnology Co.,Ltd. Assignor: SHENZHEN PEOPLE'S Hospital Contract record no.: X2024980005517 Denomination of invention: A reagent kit for detecting high virulence Acinetobacter baumannii and its usage method Granted publication date: 20221111 License type: Common License Record date: 20240510 |