CN110257387B - Aptamer for identifying grass carp hemorrhagic disease virus as well as construction method and application thereof - Google Patents
Aptamer for identifying grass carp hemorrhagic disease virus as well as construction method and application thereof Download PDFInfo
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Abstract
An aptamer comprising the nucleotide sequence: TGAACCCACCTCAGGGCATCTTACATTTCTTCTAAGTTGTTACCATGTTT. 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 identifying grass carp hemorrhagic disease virus, and a screening 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 fishery economic value in Guangxi already exceeds 621 hundred million yuan, and the total aquaculture amount 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 virus disease in Grass carp breeding, and the pathogenic pathogen of the Grass carp hemorrhage is Grass Carp Reovirus (GCRV), also called Grass Carp Hemorrhage Virus (GCHV), belonging to the family of reoviridae and the genus of aquatic animal reovirus. 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, a detection technology which is convenient to develop and operate, low in cost, short in time consumption and high in accuracy is used for the GCRV virus, so that the technology is very important for preventing and controlling the harm of the GCRV virus of the grass carp.
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 identifying grass carp hemorrhagic disease viruses, 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 present invention, there is provided an aptamer recognizing grass carp hemorrhagic disease 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 of the kit is combined with a marker, and the marker is selected from one or more of fluorescent substances, luminescent materials, biotin and enzymes.
Preferably, the fluorescent substance is one or more selected from the group consisting of hydroxyfluorescein, fluorescein isothiocyanate and carboxytetramethylrhodamine.
According to another aspect of the present invention, there is provided a method for constructing the aptamer for identifying grass carp hemorrhagic disease virus, 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 nucleic acid aptamer for recognizing grass carp hemorrhagic disease virus in detecting grass carp reovirus I is provided.
According to another aspect of the invention, a kit for rapidly detecting grass carp hemorrhagic disease virus by fluorescent quantitative PCR is provided: comprises a fluorescent molecular detection probe, the aptamer for identifying the grass carp hemorrhagic disease virus is used as the fluorescent molecular detection probe, and the marker is hydroxyl fluorescein.
The aptamer for identifying the grass carp hemorrhagic disease 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 reovirus I, 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 reovirus I. The aptamer provided by the invention is labeled by hydroxyl fluorescein, 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 reovirus I with high specificity and high sensitivity can be realized.
Drawings
FIG. 1 is a diagram showing the prediction of the secondary structure of a nucleic acid aptamer for recognizing grass carp hemorrhagic disease virus, the nucleotide sequence of which is 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 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 those skilled in the art better understand the technical solutions of the present invention, 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 of the embodiments.
Example 1 screening and preparation of aptamers for identifying grass carp hemorrhagic disease Virus
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, performing thermostatic water bath at 92 ℃ for 5min, then quickly inserting into ice, performing ice bath for 10min, and incubating the treated random ssDNA library and grass carp GCRV I type virus infected cells on ice for 1h.
After incubation and combination are completed, centrifuging to remove the supernatant, washing the GCRV I type virus infected cells of the grass carp by using 10mL of PBS, performing centrifugation in a constant-temperature water bath at 92 ℃ for 10min and 12000g to collect the supernatant, and obtaining 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 (2.5 mM) 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, ddH 2 O627.5. Mu.L. The PCR amplification procedure was: 94 ℃ for 5min,94 ℃ for 1min,56 ℃ for 30sec,72 ℃ for 1min, and circulating for 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 on a magnetic separator, washing the magnetic beads by using 2mL of PBS, then adding 200 mu L of NaOH solution (200 mM) into an EP tube, reacting for 10min at normal temperature to denature the dsDNA, leaving one chain with the biotin bonded with the streptavidin on the magnetic beads, and recovering the supernatant by using a magnetic separation frame 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 random ssDNA library in S2 was replaced with the ssDNA library obtained in S4, and the SELEX screening, PCR amplification and ssDNA library preparation process shown in S2-S4 were repeated 9 times.
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 thermostatic water bath at 92 ℃ and an ice bath, and centrifugally collecting a supernatant solution after the incubation is finished; then the supernatant solution is combined with the normal cell of the grass carp in an ice bath; after the incubation binding is 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 the S6 and preparation of an ssDNA library of the S4, repeating the processes of the S6, the S2, the S3 and the S4 in sequence, detecting the change condition of the identification capacity of the obtained ssDNA library on the GCRV I type virus infected grass carp cells by using a flow cytometer, and repeating the 9-round screening, wherein the identification capacity of the obtained ssDNA library on the GCRV I type virus infected grass carp cells is the 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:
TGAACCCACCTCAGGGCATCTTACATTTCTTCTAAGTTGTTACCATGTTT
(SEQ ID NO.1)，
or the like, or, alternatively,
GTCTGAAGTAGACGCAGGAGTGAACCCACCTCAGGGCATCTTACATTTCTTCTAAGTTGTTACCATGTTTAGTCACACCTGAGTAAGCGT(SEQ ID NO.2)。
the secondary structure of the aptamer with the nucleotide sequence of SEQ ID NO.2 is predicted on line by using MFOLD software (http:// MFOLD. Rna. Albany. Edu/.
Example 2
2.1 Main Instrument
Attune NxT flow cytometer (seemer 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 1h. After incubation binding was complete, the supernatant was removed by centrifugation.
Control group: 0.1nmol FAM-labeled random ssDNA Library50 was dissolved in 500. Mu.L PBS, incubated in a 92 ℃ constant temperature water bath for 5min, then rapidly inserted into ice, the treated random ssDNA Library50 was incubated with grass carp GCRV type I virus infected cells on ice for 1h in 10min ice bath. 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 will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.
SEQUENCE LISTING
<110> Guangxi academy of sciences
<120> aptamer for identifying grass carp hemorrhagic disease virus, and construction method and application thereof
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> Artificial sequence
<400> 1
tgaacccacc tcagggcatc ttacatttct tctaagttgt taccatgttt 50
<210> 2
<211> 90
<212> DNA
<213> Artificial sequence
<400> 2
gtctgaagta gacgcaggag tgaacccacc tcagggcatc ttacatttct tctaagttgt 60
taccatgttt agtcacacct gagtaagcgt 90
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
<210> 4
<211> 20
<212> DNA
<213> GTCTGAAGTAGACGCAGGAG
<400> 4
acgcttactc aggtgtgact 20
Claims (4)
1. A nucleic acid aptamer for recognizing grass carp hemorrhagic disease virus, which is characterized in that:
the nucleotide sequence is SEQ ID NO.2.
2. The aptamer for recognizing grass carp hemorrhagic disease 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 for recognizing grass carp hemorrhagic disease 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. A fluorescence quantitative PCR rapid detection kit for grass carp hemorrhagic disease virus is characterized in that:
the method comprises a fluorescent molecular detection probe, the aptamer for recognizing the grass carp hemorrhagic disease virus as claimed in claim 2 is used as the fluorescent molecular detection probe, and the marker is hydroxyfluorescein.
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