CN110257386B - Aptamer for resisting grass carp GCRV I virus as well as construction method and application thereof - Google Patents
Aptamer for resisting grass carp GCRV I virus as well as construction method and application thereof Download PDFInfo
- Publication number
- CN110257386B CN110257386B CN201910599016.1A CN201910599016A CN110257386B CN 110257386 B CN110257386 B CN 110257386B CN 201910599016 A CN201910599016 A CN 201910599016A CN 110257386 B CN110257386 B CN 110257386B
- Authority
- CN
- China
- Prior art keywords
- grass carp
- aptamer
- virus
- gcrv
- gcrvi
- 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- 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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function
Abstract
An aptamer for resisting grass carp GCRVI virus, which comprises the following nucleotide sequences: TCCGTCGGTTCGCCTTGCGAG TGCACGCCGCCAGCCGTTACATAAAGTCTGCCCACT is added. The aptamer provided by the invention has the advantages of small molecular weight, short preparation period, good reproducibility, convenience for in-vitro chemical synthesis and marking, and stable sequence and easiness in transportation and storage. The aptamer has high specificity and affinity to grass carp reovirus I, and has no immunogenicity.
Description
Technical Field
The invention belongs to the technical field of aquatic pathogenic bacteria detection, and particularly relates to a nucleic acid aptamer for resisting grass carp GCRVI virus, and a construction method and application thereof.
Background
Guangxi is a big province of aquaculture in China, and main aquaculture species comprise grass carp, channel catfish, grouper, grass carp, prawn, oyster, pinctada martensii and various edible plants. According to statistics, by the end of 2017, the total value of fishery economy in Guangxi exceeds 621 hundred million yuan, and the total amount of aquaculture exceeds 320.76 ten thousand tons. However, the continuous expansion of aquaculture scale and the continuous increase of aquaculture density lead to the deterioration of aquaculture environment, and various aquaculture epidemic pathogens frequently outbreak. Suspected hemorrhage of Grass carp outbreaks cultured in pond and net cage of Guangxi Nanning, river pond, Liuzhou and the like in 2018 proves that Grass carp reovirus I (Grass carp reovirus, GCRVI) is a main pathogenic pathogen by analyzing and identifying pathogenic microorganisms in diseased Grass carps through Polymerase Chain Reaction (PCR) detection technology. Grass carp hemorrhage is the most common viral disease in Grass carp breeding, and the pathogenic pathogen is Grass Carp Reovirus (GCRV), also known as Grass carp hemorrhagic disease virus (GCHV), belonging to the reoviridae and aquatic animal reovirus genus. GCRV, a highly pathogenic fish-borne virus, is usually characterized as a burst infection, which leads to massive fish death in a very short time. At present, grass carp viral hemorrhagic disease caused by GCRV virus exists widely in China, and the development of aquaculture industry in China is seriously influenced. Therefore, a novel efficient antiviral drug is researched and developed, and meanwhile, aiming at the detection technology of GCRV virus development and operation convenience, low cost, short time consumption and high accuracy, the method is very important for preventing and controlling the harm of grass carp GCRV virus.
The aptamer is a single-stranded oligonucleotide which is obtained by multiple rounds of screening in vitro and can specifically recognize a target substance by using an Exponential Enrichment ligand phylogenetic technology (SELEX). The aptamer has the advantages of high specificity, high affinity, strong stability, easy chemical synthesis and chemical modification and the like. At present, the aptamer serving as a novel and widely-focused novel detection and treatment tool has wide application prospects in the fields of human medical research, disease diagnosis, virus infection mechanism research and the like.
Disclosure of Invention
The invention aims to provide a nucleic acid aptamer for resisting grass carp GCRV I virus, and a construction method and application thereof, so as to realize high-sensitivity and specific detection of grass carp reovirus I.
According to one aspect of the invention, there is provided an aptamer against grass carp GCRV ii virus: comprises the nucleotide sequence of SEQ ID NO. 1.
Preferably, it comprises the nucleotide sequence of SEQ ID NO. 2.
Preferably, at least one nucleotide in its nucleotide sequence is phosphorylated, thiolated, methylated, aminated, or isotopically esterified.
Preferably, the nucleotide sequence is combined with a marker, and the marker is selected from one or more of fluorescent substances, luminescent materials, biotin or enzymes.
Preferably, the fluorescent substance is one or more selected from hydroxyl fluorescein, fluorescein isothiocyanate or carboxyl tetramethyl rhodamine.
According to another aspect of the present invention, there is provided a method for constructing the aptamer against GCRVI virus of grass carp, which adopts SELEX technology, comprising the following steps: (1) establishing a single-stranded DNA random Library50, wherein the nucleotide sequence of the single-stranded DNA random Library50 is as follows:
5' -GTCTGAAGTAGACGCAGGAG (50N) AGTCACACCTGAGTAAGCGT; (2) screening a single-stranded DNA random Library50 by using grass carp reovirus I infected cells to obtain a specific DNA Library of the grass carp reovirus I; (3) the specific DNA library is used as a template, and PCR amplification is carried out with a primer with a nucleotide sequence of SEQ ID NO.3 and a primer with a nucleotide sequence of SEQ ID NO. 4.
Preferably, the amplification procedure for PCR amplification is: 5 minutes at 94 ℃, 1 minute at 94 ℃, 30 seconds at 56 ℃, 1 minute at 72 ℃ and 25 cycles; 5 minutes at 72 ℃.
According to another aspect of the invention, the application of the aptamer resisting grass carp GCRVI virus in detecting grass carp reovirus I is provided.
According to another aspect of the invention, a fluorescence quantitative PCR rapid detection kit for grass carp GCRVI is provided: comprises a fluorescent molecular detection probe, the aptamer for resisting grass carp GCRVI virus is used as the fluorescent molecular detection probe, and the marker is hydroxyfluorescein.
The aptamer for resisting grass carp GCRV I virus provided by the invention has the advantages of small molecular weight, short preparation period, good reproducibility, convenience for in-vitro chemical synthesis, convenience for marking, stable sequence and easiness for transportation and storage. The aptamer has high specificity and affinity to grass carp GCRVI virus, and has no immunogenicity. In addition, the nucleotide sequence constituting the aptamer can be easily substituted or modified. The partially substituted or modified aptamer can keep the molecular structure, the physicochemical property and the function which are basically the same as or similar to those of the original aptamer, and can also effectively realize the detection of the grass carp GCRVI virus. The aptamer provided by the invention is labeled by using hydroxyfluorescein, so that the aptamer is prepared into a fluorescent probe, and the fluorescent probe is combined with a real-time fluorescent quantitative PCR detection technology, so that the real-time qualitative and quantitative detection of the grass carp GCRVI virus with high specificity and high sensitivity can be realized.
Drawings
FIG. 1 is a diagram showing the prediction of the secondary structure of an aptamer against grass carp GCRVI virus having the nucleotide sequence of SEQ ID NO. 2;
FIG. 2 shows the FAM fluorescence value test results of the samples measured by the flow cytometer in example 2;
FIG. 3 shows the observation results of the laser scanning confocal microscope in example 2: (a) a light mirror image of a control sample, (b) a fluorescence image of a control sample, (c) a light mirror image of a test sample, and (d) a fluorescence image of a test sample.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
EXAMPLE 1 screening and preparation of aptamers for detecting GCRV type I virus in grass carp
S1, construction of random single-stranded DNA (ssDNA) library and synthesis of primers
Constructing a random ssDNA Library50, wherein two ends are fixed sequences, the middle 50 nucleotides are random sequences, and the nucleotide sequences are as follows:
5’-GTCTGAAGTAGACGCAGGAG(50N)AGTCACACCTGAGTAAGCGT。
5' primer (SEQ ID NO. 3): 5 '-FAM-GTCTGAAGTAGACGCAGGAG-3'.
3' primer (SEQ ID NO. 4): 5 '-Biotin-ACGCTTACTCAGGTGTGACT-3'.
Both random ssDNA libraries and primers were synthesized by Shanghai Biotechnology, Inc.
S2.SELEX screening (corresponding to positive screening)
Dissolving 10nmol of the random ssDNA library in 500 mu L PBS, carrying out thermostatic water bath at 92 ℃ for 5min, then quickly inserting into ice, carrying out ice bath for 10min, and incubating the treated random ssDNA library and GCRV I virus infected cells of the grass carp on the ice for 1 h.
After incubation and combination are completed, centrifuging to remove the supernatant, washing GCRV I type virus infected cells of the grass carp with 10mL of PBS, carrying out constant-temperature water bath at 92 ℃ for 10min, and centrifuging at 12000g to collect the supernatant, namely the ssDNA nucleic acid library for specifically identifying the GCRV I type virus infected cells of the grass carp.
S3.PCR amplification
And carrying out PCR amplification on 100 mu L of ssDNA library which is obtained by screening and can identify GCRV I type virus infected cells of the grass carp, a 5 'primer and a 3' primer. The PCR reaction system was as follows (1000. mu.L): 10 XBuffer 100. mu.L, dNTP Mix (2.5mM) 80. mu.L, primer 40. mu.L of SEQ ID NO.3, primer of SEQ ID NO.4, ssDNA library 100. mu.L, rTaq enzyme 12.5. mu.L, ddH2O627.5. mu.L. The PCR amplification procedure was: 5min at 94 ℃, 1min at 94 ℃, 30sec at 56 ℃ and 1min at 72 ℃ after 25 cycles; 5min at 72 ℃. The supernatants obtained after the first round of SELEX screening were all used for PCR amplification to obtain a double stranded nucleic acid (dsDNA) library.
S4. preparation of ssDNA library
Incubating 100 mu L of streptavidin-labeled magnetic beads and a dsDNA library prepared from S3 for 20min at normal temperature, utilizing the affinity action of biotin on the dsDNA and streptavidin on the magnetic beads to bond the dsDNA to the surfaces of the magnetic beads, removing supernatant by utilizing a magnetic separator, washing the magnetic beads by using 2mL of PBS, then adding 200 mu L of NaOH solution (200mM) into an EP tube, reacting for 10min at normal temperature to denature and melt the dsDNA, leaving one chain with the biotin bonded with the streptavidin on the magnetic beads, and recovering the supernatant by utilizing a magnetic separation rack after the reaction is finished; adding the supernatant into a desalting column washed by sterile water, and naturally dripping under the action of gravity. To the filtrate was added 500. mu.L of PBS and the solution containing the ssDNA library was collected for the next round of screening.
S5. iterative screening of ssDNA libraries
The SELEX screening, PCR amplification and ssDNA library preparation shown in S2-S4 were repeated 9 times using the ssDNA library obtained in S4 instead of the random ssDNA library in S2.
S6. negative screening
Dissolving the ssDNA library obtained by the second round of S5 and the subsequent round of screening, incubating the ssDNA library with normal cells of the grass carp for 1h on ice after a constant-temperature water bath at 92 ℃ and an ice bath, and centrifugally collecting a supernatant solution after the incubation is finished; then combining the supernatant solution with the normal cell of the grass carp in an ice bath; after the incubation and binding are completed, the supernatant is collected, and at this time, the collected supernatant is the ssDNA library subjected to negative screening.
In the step, normal grass carp cells are used as a control, and the ssDNA library obtained by screening after S5 is subjected to negative screening so as to improve the screening efficiency of the ssDNA library.
S7.9 round of screening
And (3) performing PCR amplification on the supernatant collected in S6 and preparation of an ssDNA library of S4 by S3, then sequentially repeating the processes of S6, S2, S3 and S4, detecting the change condition of the obtained ssDNA library on the identification capability of the GCRV I type virus infected cells of the grass carp by using a flow cytometer, and repeating 9 rounds of screening, wherein the identification capability of the obtained ssDNA library on the GCRV I type virus infected cells of the grass carp is strongest. After the obtained amplification product is subjected to clone sequencing analysis, the ssDNA aptamer for detecting grass carp GCRV I type virus infected cells is finally obtained, and the nucleotide sequence of the ssDNA aptamer is as follows:
TCCGTCGGTTCGCCTTGCGAGTGCACGCCGCCAGCCGTTACATAAAGTCTGCCCACT(SEQ ID NO.1)，
or the like, or, alternatively,
GTCTGAAGTAGACGCAGGAGTCCGTCGGTTCGCCTTGCGAGTGCACGCCGCCAGCCGTTACATAAAGTCTGCCCACTAGTCACACCTGAGTAAGCGT(SEQ ID NO.2)。
MFOLD software (http:// MFOLD. rna. albany. edu/.
Example 2
2.1 Main Instrument
Attune NxT flow cytometer (seimer feishell technology), FV3000 laser scanning confocal microscope (olympus).
2.2 Experimental procedures
The aptamer of SEQ ID NO.2 prepared in example 1 and the random ssDNA Library50 were labeled with hydroxyfluorescein (FAM), respectively.
Test groups: dissolving 0.1nmol FAM-labeled SEQ ID NO.2 aptamer in 500 mu L PBS, performing constant-temperature water bath at 92 ℃ for 5min, then quickly inserting into ice, performing ice bath for 10min, and incubating the treated SEQ ID NO.2 aptamer and grass carp GCRV type I virus infected cells on ice for 1 h. After incubation binding was complete, the supernatant was removed by centrifugation.
Control group: dissolving 0.1nmol FAM-labeled random ssDNA Library50 in 500 μ L PBS, performing thermostatic water bath at 92 ℃ for 5min, then rapidly inserting into ice, performing ice bath for 10min, and incubating the treated random ssDNA Library50 and grass carp GCRV type I virus infected cells on ice for 1 h. After incubation binding was complete, the supernatant was removed by centrifugation.
Cell precipitates obtained by incubation of the test group and the control group are detected by using a flow cytometer and a laser scanning confocal microscope respectively.
2.3 results of the experiment
The detection result of the flow cytometer is shown in fig. 2, and the fluorescence value corresponding to the test group is significantly higher than that of the control group. The detection result of the confocal laser scanning microscope is shown in fig. 3, and the control group hardly has an obvious fluorescence effect, relatively speaking, the fluorescence effect corresponding to the experimental group is obvious, and the cell sample emits strong green light.
The test results of the flow cytometry and the laser scanning confocal microscope show that compared with the random ssDNA Library50, the FAM-labeled SEQ ID NO.2 aptamer has higher affinity and specificity to grass carp GCRV I type virus infected cells.
Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Guangxi academy of sciences
<120> aptamer for resisting grass carp GCRVI virus as well as construction method and application thereof
<130> 190701
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 57
<212> DNA
<213> Artificial sequence
<400> 1
tccgtcggtt cgccttgcga gtgcacgccg ccagccgtta cataaagtct gcccact 57
<210> 2
<211> 97
<212> DNA
<213> Artificial sequence
<400> 2
gtctgaagta gacgcaggag tccgtcggtt cgccttgcga gtgcacgccg ccagccgtta 60
cataaagtct gcccactagt cacacctgag taagcgt 97
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
acgcttactc aggtgtgact 20
Claims (6)
1. An aptamer for resisting grass carp GCRVI virus, which is characterized in that: the nucleotide sequence is SEQ ID NO. 2.
2. The aptamer against grass carp GCRV ii virus according to claim 1, wherein: the nucleotide sequence of the fluorescent material is combined with a marker, and the marker is selected from one or more than one of fluorescent material, luminescent material, biotin or enzyme.
3. The aptamer against grass carp GCRV ii virus according to claim 2, wherein: the fluorescent substance is selected from one or more of hydroxyl fluorescein, fluorescein isothiocyanate or carboxyl tetramethyl rhodamine.
4. Use of the aptamer against grass carp GCRV ii virus according to claim 1 for detecting grass carp reovirus type i, characterized in that: the use is a non-disease diagnostic use.
5. The use of an aptamer against grass carp GCRV ii virus according to claim 2 for detecting grass carp reovirus type i, wherein: the use is a non-disease diagnostic use.
6. A fluorescence quantitative PCR rapid detection kit for resisting grass carp GCRVI virus is characterized in that: the method comprises the steps of using the aptamer of the grass carp GCRVI virus resisting virus as claimed in claim 2 as a fluorescent molecular detection probe, wherein the marker is hydroxyfluorescein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910599016.1A CN110257386B (en) | 2019-07-04 | 2019-07-04 | Aptamer for resisting grass carp GCRV I virus as well as construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910599016.1A CN110257386B (en) | 2019-07-04 | 2019-07-04 | Aptamer for resisting grass carp GCRV I virus as well as construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110257386A CN110257386A (en) | 2019-09-20 |
| CN110257386B true CN110257386B (en) | 2022-06-21 |
Family
ID=67924314
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910599016.1A Active CN110257386B (en) | 2019-07-04 | 2019-07-04 | Aptamer for resisting grass carp GCRV I virus as well as construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110257386B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034220A (en) * | 2017-05-16 | 2017-08-11 | 中国水产科学研究院珠江水产研究所 | Aptamer and its derivative, the screening technique and application of GCRV |
| CN108866063A (en) * | 2018-06-28 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | A kind of aptamer and its preparation method and application of PEG modification |
| CN109207480A (en) * | 2018-09-26 | 2019-01-15 | 广西科学院 | Aptamer and its application of the specificity for egg-shaped pompano nervous necrosis virus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040005543A1 (en) * | 2002-01-18 | 2004-01-08 | Abraham Grossman | Compositions and methods for binding agglomeration proteins |
-
2019
- 2019-07-04 CN CN201910599016.1A patent/CN110257386B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034220A (en) * | 2017-05-16 | 2017-08-11 | 中国水产科学研究院珠江水产研究所 | Aptamer and its derivative, the screening technique and application of GCRV |
| CN108866063A (en) * | 2018-06-28 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | A kind of aptamer and its preparation method and application of PEG modification |
| CN109207480A (en) * | 2018-09-26 | 2019-01-15 | 广西科学院 | Aptamer and its application of the specificity for egg-shaped pompano nervous necrosis virus |
Non-Patent Citations (4)
| Title |
|---|
| "Probing and characterizing the high specific sequences of ssDNA aptamer against SGIV-infected cells";Li OF等;《VIRUS RESEARCH》;20180215;第246卷;46-54 * |
| "RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimidines";Tomohiro Shimada等;《MOLECULAR MICROBIOLOGY》;20070919;第66卷(第3期);744-757 * |
| "Uncultured Lachnospiraceae bacterium clone 2113 16S ribosomal RNA gene, partial sequence";Chehoud C等;《ENA Database》;20130930;Accession NO：KF502972.1 * |
| "基于新型核酸适配体-荧光分子检测探针的石斑鱼虹彩病毒病快速诊断";李鹏飞等;《广西科学》;20180228;第25卷(第1期);63-67 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110257386A (en) | 2019-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111073892B (en) | Nucleic acid aptamer for identifying garrupa iridovirus infected cells, construction method and application thereof | |
| CN111073891B (en) | Aptamer for detecting grouper iridovirus as well as construction method and application thereof | |
| CN110643611B (en) | Aptamer, construction method thereof and application thereof in detection of grouper iridovirus | |
| CN109161547B (en) | Nucleic acid aptamer and application thereof in detection of pathogenic vibrio alginolyticus | |
| CN113215167A (en) | Aptamer and application thereof in detection of cells infected by iridovirus of micropterus salmoides | |
| CN109136229B (en) | Aptamer for specifically recognizing trachinotus ovatus-derived nervous necrosis virus and application thereof | |
| CN109439801A (en) | A kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detection method | |
| CN109207480B (en) | Aptamer specific for trachinotus ovatus nervous necrosis virus and application thereof | |
| CN113462693B (en) | Application of ssDNA aptamer in identifying largemouth black bass iridovirus infected cells | |
| CN111363749B (en) | Nucleic acid aptamer for detecting Chinese softshell turtle iridovirus as well as construction method and application thereof | |
| CN110257386B (en) | Aptamer for resisting grass carp GCRV I virus as well as construction method and application thereof | |
| CN110257385B (en) | Aptamer for resisting grass carp reovirus I, and construction method and application thereof | |
| CN110257384B (en) | Aptamer and construction method and application thereof | |
| CN111363748B (en) | Aptamer, construction method thereof and application thereof in detection of Chinese softshell turtle rainbow virus | |
| CN110257387B (en) | Aptamer for identifying grass carp hemorrhagic disease virus as well as construction method and application thereof | |
| CN111454956B (en) | Nucleic acid aptamer for Chinese soft-shelled turtle iridovirus as well as construction method and application thereof | |
| CN109161548B (en) | Aptamer and application thereof in detection of trachinotus ovatus-derived nervous necrosis virus | |
| CN115992143A (en) | High-flux drug screening method for resisting nervous necrosis virus | |
| CN111118014A (en) | Anti-iridovirus aptamer and construction method and application thereof | |
| CN106103714A (en) | In vivo infection microorganism, parasitic microbe, the noncoding RNA of symbiotic microorganism and qualification thereof and application | |
| CN113462694B (en) | Nucleic acid aptamer for megaphone iridovirus infected cells and application thereof | |
| CN113278621B (en) | ssDNA aptamer and application thereof in identification and detection of vibrio harveyi | |
| CN109161546B (en) | Aptamer and application thereof in detection of trachinotus ovatus source pathogenic vibrio alginolyticus | |
| CN116879549A (en) | Application of aptamer in recognition of garrupa iridovirus and aptamer | |
| CN116891851A (en) | Aptamer and fluorescent molecule detection probe for detecting garrupa iridovirus |
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 |