CN111363749A - Aptamer for detecting Chinese softshell turtle iridovirus and construction method and application thereof - Google Patents
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Abstract
The invention provides an aptamer for detecting Chinese softshell turtle iridovirus, a construction method and application thereof, wherein the aptamer for detecting Chinese softshell turtle iridovirus comprises a DNA sequence shown as SEQ ID NO.1 or a derivative thereof. The aptamer provided by the invention has higher affinity and specificity to the Chinese softshell turtle iridovirus, and also has the advantages of no immunogenicity, short preparation period, good reproducibility, small molecular weight, convenience for in-vitro chemical synthesis, convenience for marking, easiness for modifying and replacing different parts of the aptamer, stable sequence, easiness for transporting and storing and the like. When the rapid detection method based on the aptamer is used for detecting the Chinese softshell turtle iridovirus, the operation is simple and rapid, and higher accuracy and sensitivity can be achieved. The aptamer can be used for constructing a molecular probe or a detection kit, and is combined with detection equipment such as an enzyme labeling instrument, a flow cytometer, a fluorescence microscope and the like to realize the accurate detection of the Chinese softshell turtle iridovirus.
Description
The present application claims priority of chinese patent application entitled "an aptamer for detecting iridovirus of trionyx sinensis, a method of constructing the same, and use thereof" filed by the national intellectual property office on year 2020, month 02, and month 07, application No. CN202010082169.1, the entire contents of which are incorporated herein by reference.
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to an aptamer for detecting Chinese softshell turtle iridovirus as well as a construction method and application thereof.
Background
The soft-shelled turtles are rich in nutrition and high in medicinal value, and as large and important aquaculture species in China, the annual output of the soft-shelled turtles in China currently reaches over 30 million tons, the direct output value of the industry exceeds billions of yuan, and the economic value is extremely high. However, with the increase of the breeding density and the enlargement of the breeding scale of the turtles, various diseases are frequently outbreak, and huge economic losses are caused. The Chinese Softshell Turtle Iridovirus (STIV) is obtained by separating from the body of a Chinese softshell turtle suffering from red neck disease, and the STIV is used as a main viral pathogen causing the disease of the Chinese softshell turtle, so the lethality rate is extremely high, and therefore, the research and the development of a detection technology which is convenient to operate, low in cost, short in time consumption and high in accuracy are extremely important for controlling the harm of the STIV of the Chinese softshell turtle. However, the current diagnostic methods for the Chinese softshell turtle STIV virus mainly comprise a molecular biological detection method, an immunological detection method and the like, and comprise a PCR technology, a nested PCR technology, a fluorescent quantitative PCR technology, an ELISA technology and the like. The detection result of the PCR technology is accurate and reliable, but the PCR technology has the defects of complex operation, long time consumption, expensive instrument and reagent and the like, and can not meet the requirement of rapid and accurate detection and diagnosis on site. Therefore, the rapid detection technology and the functional product of the Chinese softshell turtle STIV virus, which are convenient to develop and operate, low in cost, short in time consumption and high in accuracy and can be used on the site of a farm, are important for finding and determining the pathogen as soon as possible and further purposefully making a treatment scheme to control pathogen diffusion and reduce loss.
The aptamer is a single-stranded oligonucleotide capable of specifically recognizing a target substance, which is obtained by in vitro multiple rounds of screening from an artificially synthesized random sequence library 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. Based on the biological characteristics that the aptamer can recognize pathogenic microorganisms or pathological cells with high specificity, the nucleic acid aptamer is widely applied to the development of detection technology and the construction of biosensors, and can realize the accurate detection and diagnosis of pathogeny or disease. Therefore, the aptamer has wide application prospect in various biological fields such as disease diagnosis, virus infection mechanism research and the like.
Disclosure of Invention
The invention aims to provide an aptamer for detecting Chinese softshell turtle iridovirus as well as a construction method and application thereof, so as to improve the detection level of the Chinese softshell turtle iridovirus.
According to one aspect of the present invention, there is provided an aptamer for detecting trionyx sinensis iridovirus: comprises a DNA sequence shown as SEQ ID NO.1 or a derivative thereof.
Preferably, the derivative is a DNA sequence shown in SEQ ID NO.1 in which at least one base is phosphorylated, thiolated, methylated, aminated or isotopically esterified.
Preferably, it comprises the DNA sequence shown in SEQ ID NO.2 or a derivative thereof.
Preferably, its secondary structure is as follows:
preferably, the derivative is a DNA sequence shown in SEQ ID NO.2 in which at least one base is phosphorylated, thiolated, methylated, aminated or isotopically esterified.
Preferably, the DNA sequence is linked with a functional group, and the functional group is selected from a biotin label, a luminescent label and an enzyme label. Optionally, the luminescent marker is selected from one or more of hydroxyfluorescein, fluorescein isothiocyanate or carboxytetramethylrhodamine.
According to another aspect of the invention, the application of the nucleic acid aptamer for detecting the trionyx sinensis rainbow virus in preparation of products for detecting the trionyx sinensis rainbow virus is provided.
Preferably, the product is a fluorescent molecular probe, and the aptamer is connected with a luminescent marker.
According to another aspect of the present invention, there is provided a method for constructing the above aptamer for detecting trionyx sinensis iridovirus, comprising the steps of: step one, providing a first ssDNA library and a pair of PCR primers, wherein the first ssDNA library comprises the following single-stranded DNA sequences:
the 5' -GACGCTTACTCAGGTGTGACTCG (50N) CGAAGGACGCAGATGAAGTCTC, PCR primer comprises an upstream primer and a downstream primer, wherein the upstream primer comprises a DNA sequence shown as SEQ ID NO.3, and the downstream primer comprises a DNA sequence shown as SEQ ID NO. 4; step two, incubating the random ssDNA library and fat head carp cells infected with the Chinese softshell turtle iridovirus, and screening to obtain a second ssDNA library for specifically identifying the Chinese softshell turtle iridovirus; step three, taking the second ssDNA library as a template, and performing PCR amplification by using a PCR primer to obtain a dsDNA library; and step four, incubating the dsDNA library with magnetic beads marked by streptavidin, carrying out magnetic separation on the magnetic beads after incubation, and then purifying and separating a third ssDNA library which is combined on the magnetic beads and specifically recognizes the trionyx sinensis iridovirus to obtain the aptamer for detecting the trionyx sinensis iridovirus. In the single-stranded DNA sequences of the first ssDNA library, the two ends are fixed sequences and the middle "50N" represents a random sequence of 50 nucleotides in length.
Preferably, in step three, PCR amplification is performed according to the following procedure: 5 minutes at 92 ℃, 1 minute at 92 ℃, 30 seconds at 60 ℃ and 1 minute at 72 ℃ through 25 cycles; 5 minutes at 72 ℃.
Compared with the existing protein antibodies, the aptamer obtained by SELEX screening has higher affinity and specificity to the Chinese softshell turtle iridovirus, and has the characteristics that the protein antibodies do not have, such as no immunogenicity, short preparation period, good reproducibility, small molecular weight, convenience for in-vitro chemical synthesis, convenience for marking, easiness for modifying and replacing different parts of the aptamer, stable sequence, easiness for transporting and storing and the like. When the rapid detection method based on the aptamer is used for detecting the Chinese softshell turtle iridovirus, the operation is simple and rapid, and higher accuracy and sensitivity can be achieved. The aptamer can be used for constructing a molecular probe or a detection kit, and is combined with detection equipment such as an enzyme labeling instrument, a flow cytometer, a fluorescence microscope and the like to realize the accurate detection of the Chinese softshell turtle iridovirus. The method has important significance for rapid diagnosis of the Chinese softshell turtle STIV virus, and has good application prospect in the field of detection of the Chinese softshell turtle STIV virus.
Drawings
FIG. 1 is a diagram showing the prediction of the secondary structure of an aptamer having the DNA 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) the light mirror image of the experiment II group corresponding to the sample, (b) the fluorescence image of the experiment II group corresponding to the sample, (c) the light mirror image of the control group corresponding to the sample, and (d) the fluorescence image of the control group corresponding to the 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 detection of STIV
S1, construction of a first ssDNA library and synthesis of primers
A synthetic first ssDNA Library 50 was designed, the nucleotide sequence of which is as follows:
5' -GACGCTTACTCAGGTGTGACTCG (50N) CGAAGGACGCAGATGAAGTCTC, wherein both ends are fixed sequences and the middle 50 nucleotides are random sequences.
The upstream primer comprises a DNA sequence shown as SEQ ID NO.3 and is marked by hydroxyfluorescein (FAM), and the specific sequence is as follows: 5 '-FAM-GACGCTTACTCAGGTGTGACTCG-3'.
The downstream primer comprises a DNA sequence shown as SEQ ID NO.4 and is marked by Biotin (Biotin), and the specific sequence is as follows: 5 '-Biotin-GAGACTTCATCTGCGTCCTTCG-3'.
Both the first ssDNA library and the primers were synthesized by Shanghai Biotechnology Ltd.
S2.SELEX screening to obtain nucleic acid aptamer (positive screening) for specifically recognizing STIV infected fat head carp cell
S2.1, dissolving 10nmol of the first 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 first ssDNA library and STIV-infected fat head carp cells on the ice for 1 h;
s2.2 after incubation and combination are completed, centrifuging to remove the supernatant, washing the STIV-infected fat head carp cells with 10mL of PBS, carrying out constant-temperature water bath at 92 ℃ for 10min, and centrifuging at 12000g to collect the supernatant, namely the second ssDNA library for specifically identifying the STIV-infected cells.
S3.PCR amplification
PCR was performed in a 100. mu.L second ssDNA library screened with the forward primer and the reverse primer in a PCR reaction system (1000. mu.L) of 10 × Buffer 100. mu.L, dNTP Mix (2.5mM) 80. mu.L, forward primer 40. mu.L, reverse primer 40. mu.L, second ssDNA library 100. mu.L, rTaq enzyme 12.5. mu.L, ddH2O627.5 μ L. PCR amplification was performed according to the following procedure: 5 minutes at 92 ℃, 1 minute at 92 ℃, 30 seconds at 60 ℃ and 1 minute at 72 ℃ through 25 cycles; 5 minutes at 72 ℃. The supernatants from the first round of screening were all used for subsequent PCR amplification to obtain an amplified dsDNA library.
S4. preparation of third ssDNA library
Incubating 100 mu L of streptavidin-labeled magnetic beads and a dsDNA library for 20min at normal temperature, utilizing the affinity action of biotin on the dsDNA library and streptavidin on the magnetic beads to bond the dsDNA library 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 the dsDNA library, leaving one strand with biotin bonded with the streptavidin on the magnetic beads, taking single-stranded DNA bonded with the magnetic beads as a third ssDNA library, and recovering the supernatant by utilizing a magnetic separation rack; after the desalting column was washed with 10mL of sterile water, the supernatant was added to the desalting column and allowed to drip naturally under the action of gravity. 500 μ L of PBS was added and the collected solution was used for the next round of screening.
S5, repeatedly screening
The third ssDNA library obtained in S4 was used in place of the first ssDNA library, and the positive selection process, PCR amplification and single-stranded DNA library preparation process shown in S2-S4 were repeated 10 times.
S6. negative screening
In the second round and the subsequent rounds of screening of S5, normal caplet cells are used as a control, and the ssDNA library obtained by screening after S5 is subjected to negative screening to improve the screening efficiency. The specific negative screening process is as follows: and dissolving the ssDNA library obtained by screening, incubating the ssDNA library with normal fat head carp cells for 1h on ice in a thermostatic water bath at the temperature of 92 ℃, and centrifugally collecting supernatant solution after incubation is finished, so as to obtain the ssDNA library subjected to negative screening.
S7.11 round of screening
And (3) collecting the supernatant containing the ssDNA library from the S6, performing PCR amplification of S3 and preparation of the ssDNA library of the S step 4, sequentially repeating the processes of S6, S2, S3 and S4, detecting the change of the identification capacity of the obtained ssDNA library on the STIV infected cells by using a flow cytometer, and repeating 6 rounds of screening, wherein the identification capacity of the obtained ssDNA library on the STIV infected cells is strongest. After the obtained amplification product is subjected to clone sequencing analysis, the aptamer which can be used for detecting the STIV infected cell in the embodiment is finally obtained, and the DNA sequence of the aptamer is as follows:
ACACCCAAATTCCGTCAGTCGTGCTCGTAATTCACAACACCGCTGGCCAT(SEQ IN NO.1),
or the like, or, alternatively,
GACGCTTACTCAGGTGTGACTCGACACCCAAATTCCGTCAGTCGTGCTCGTAATTCACAACACCGCTGGCCATCGAAGGACGCAGATGAAGTCTC(SEQ ID NO.2)。
MFOLD software (http:// MFOLD. rna. albany. edu/. Similarly, aptamers with the DNA sequence of SEQ ID NO.1 also form specific stem-loop and hairpin structures.
Example 2
2.1 Main Instrument
Attune NxT flow cytometer (seimer feishell technology), FV3000 laser scanning confocal microscope (olympus).
2.2 Experimental group setting mode
The aptamers of SEQ ID NO.1 and SEQ ID NO.2 constructed in example 1 were labeled with FAM, respectively.
Experiment i group: 10nmol of the aptamer of SEQ ID NO.1 was dissolved in 500. mu.L of PBS, incubated in a thermostatic water bath at 92 ℃ for 5min, then rapidly inserted into ice, ice-incubated for 10min, the treated first ssDNA library and STIV-infected decapitated carp cells were incubated on ice for 1h, and after completion of incubation binding, the supernatant was removed by centrifugation.
Experiment II group: 10nmol of the aptamer of SEQ ID NO.2 was dissolved in 500. mu.L PBS, incubated in a thermostatic water bath at 92 ℃ for 5min, then rapidly inserted into ice, ice-incubated for 10min, the treated first ssDNA library and STIV-infected decapitated carp cells were incubated on ice for 1h, and after completion of incubation binding, the supernatant was removed by centrifugation.
Control group: dissolving 10nmol of the aptamer of SEQ ID NO.1 in 500 μ L of PBS, performing thermostatic water bath at 92 ℃ for 5min, then rapidly inserting into ice, performing ice bath for 10min, incubating the treated first ssDNA library and normal fat head carp cells on ice for 1h, and after incubation and combination are completed, centrifuging to remove the supernatant.
And respectively detecting the binding effect and specificity of the aptamer tested in the experiment I group, the experiment II group and the control group and the fat head carp cell by using a flow cytometer. Observing cell precipitates obtained after incubation of the reference aptamers of the experiment group II and the control group with the fat head carp cells respectively by using a laser confocal microscope.
2.3 results of the experiment
The detection results of the flow cytometer are shown in fig. 2, and the results prove that the fluorescence value of the surface of the STIV-infected thick head carp cell detected by the flow cytometer in the experiment I group and the experiment II group is obviously increased compared with the fluorescence value of the surface of the normal thick head carp cell detected by the flow cytometer in the control group, namely, the aptamer of SEQ ID No.1 or SEQ ID No.2 has high specific recognition capability for the STIV-infected thick head carp cell.
The detection result of the confocal laser microscope is shown in fig. 3, and the result proves that compared with the normal cells in the control group, the aptamer in the experiment II group is obviously combined on the surface of the STIV infected fat head carp cell, namely the aptamer of SEQ ID No.2 has high specific recognition capability on the STIV infected fat head carp cell. Similarly, the aptamer of SEQ ID NO.2 can be bound on the surface of the STIV infected fat head carp cell, and has high specific recognition capability on the STIV infected fat head carp cell.
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, Guangxi Zhuang nationality autonomous region Water science research institute
<120> aptamer for detecting Chinese softshell turtle iridovirus, and construction method and application thereof
<130>
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<170>PatentIn version 3.5
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acacccaaat tccgtcagtc gtgctcgtaa ttcacaacac cgctggccat 50
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gacgcttact caggtgtgac tcgacaccca aattccgtca gtcgtgctcg taattcacaa 60
caccgctggc catcgaagga cgcagatgaa gtctc 95
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gacgcttact caggtgtgac tcg 23
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gagacttcat ctgcgtcctt cg 22
Claims (10)
1. An aptamer for detecting an iridovirus of a trionyx sinensis, comprising: comprises a DNA sequence shown as SEQ ID NO.1 or a derivative thereof.
2. The aptamer for detecting trionyx sinensis iridovirus according to claim 1, wherein: the derivative is obtained by phosphorylating, sulfhydrylating, methylating, aminating or isotopic alkylating at least one basic group on the DNA sequence shown in SEQ ID NO. 1.
3. The aptamer for detecting trionyx sinensis iridovirus according to claim 1, wherein: comprises a DNA sequence shown as SEQID NO.2 or a derivative thereof.
4. The aptamer for detecting Chinese softshell turtle iridovirus according to claim 3, having the secondary structure:
5. the aptamer for detecting Chinese softshell turtle iridovirus according to claim 3, wherein: the derivative is obtained by phosphorylating, sulfhydrylating, methylating, aminating or isotopic alkylating at least one basic group on the DNA sequence shown in SEQ ID NO. 2.
6. The aptamer for detecting Chinese softshell turtle iridovirus according to any one of claims 1-5, wherein: the DNA sequence is connected with a functional group, and the functional group is selected from a biotin marker, a luminescent marker and an enzyme marker.
7. Use of the nucleic acid aptamer for detecting Chinese softshell turtle iridovirus according to any one of claims 1-5 in preparation of products for detecting Chinese softshell turtle iridovirus.
8. The use of claim 7, wherein: the product is a fluorescent molecular probe, and the aptamer for detecting the Chinese softshell turtle iridovirus is connected with a luminescent marker.
9. A method for constructing the aptamer for detecting Chinese softshell turtle iridovirus according to claim 1 or 3, comprising the steps of:
step one, providing a first ssDNA library and a pair of PCR primers,
the first ssDNA library comprises the following single-stranded DNA sequences: 5' -GACGCTTACTCAGGTGTGACTCG (50N) CGAAGGACGCAGATGAAGTCTC, wherein the PCR primer comprises an upstream primer and a downstream primer, the upstream primer comprises a DNA sequence shown as SEQ ID NO.3, and the downstream primer comprises a DNA sequence shown as SEQ ID NO. 4;
step two, incubating the first ssDNA library and fat head carp cells infected with the Chinese softshell turtle iridovirus, and screening to obtain a second ssDNA library for specifically identifying the Chinese softshell turtle iridovirus;
step three, taking the second ssDNA library as a template, and performing PCR amplification by using the PCR primer to obtain a dsDNA library;
and step four, incubating the dsDNA library with magnetic beads marked by streptavidin, carrying out magnetic separation on the magnetic beads after incubation, and then purifying and separating a third ssDNA library which is combined on the magnetic beads and specifically recognizes the trionyx sinensis iridovirus to obtain the aptamer for detecting the trionyx sinensis iridovirus.
10. The method for constructing an aptamer for detecting trionyx sinensis iridovirus according to claim 9, wherein in the third step, PCR amplification is performed according to the following procedure:
5 minutes at 92 ℃, 1 minute at 92 ℃, 30 seconds at 60 ℃ and 1 minute at 72 ℃ through 25 cycles; 5 minutes at 72 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111454956B (en) * | 2020-02-07 | 2023-10-10 | 广西科技大学 | Nucleic acid aptamer for Chinese soft-shelled turtle iridovirus as well as construction method and application thereof |
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