CN111363749B - Nucleic acid aptamer for detecting Chinese softshell turtle iridovirus as well as construction method and application thereof - Google Patents
Nucleic acid aptamer for detecting Chinese softshell turtle iridovirus as well as construction method and application thereof Download PDFInfo
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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 with the title of "an aptamer for detecting trionyx sinensis iridovirus, its construction method and use" filed by the national intellectual property office on year 2020, month 02 and month 07, having application number 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 a nucleic acid aptamer for detecting Chinese softshell turtle iridovirus as well as a construction method and application thereof.
Background
The soft-shelled turtle is rich in nutrition and high in medicinal value, as a large and important aquaculture species in China, the annual output of the soft-shelled turtle in China currently reaches over 30 ten thousand tons, the direct industrial output value exceeds billions 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 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 defects of complex operation, long time consumption, expensive instrument and reagent and the like exist, and the requirement of rapid and accurate detection and diagnosis on site cannot be met. Therefore, the technology and the functional product for rapidly detecting the Chinese softshell turtle STIV virus, which are convenient to develop and operate, low in cost, short in time consumption, high in accuracy and capable of being used on the site of a farm, are vital to discover and determine the pathogen as soon as possible, and further purposefully establish 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, the DNA sequence shown as SEQ ID NO.2 or the derivative thereof is included.
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:
5' -GACGCTTACTCAGGTGACTCG (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 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: the test kit comprises (a) a light mirror image of a corresponding sample of an experiment II group, (b) a fluorescence image of a corresponding sample of the experiment II group, (c) a light mirror image of a corresponding sample of a control group, and (d) a fluorescence image of a corresponding sample of the control group.
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 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' -GACGCTTACTCAGGTGACTCG (50N) CGAAGGACGCAGATGAAGTCTC, wherein, two 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-GACGCTTACTCAGGTGACTCG-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-GAGACTTCATCTGCGTCTCTCTCTG-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 1h;
and after the S2.2 incubation combination is completed, centrifuging to remove the supernatant, washing the STIV-infected fat head carp cells with 10mL of PBS, performing centrifugation in a 92 ℃ constant-temperature water bath for 10min and 12000g to collect the supernatant, namely the second ssDNA library for specifically identifying the STIV-infected cells.
S3.PCR amplification
A second ssDNA library obtained by 100. Mu.L screening was subjected to PCR amplification with the forward primer and the reverse primer. The PCR reaction was as follows (1000. Mu.L): 10 XBuffer 100. Mu.L, dNTP Mix (2.5 mM) 80. Mu.L, upstream primer 40. Mu.L, downstream primer 40. Mu.L, second ssDNA library 100. Mu.L, rTaq enzyme 12.5. Mu.L, ddH 2 O627.5. Mu.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 the round of screening were all used for subsequent PCR amplification to yield an amplified dsDNA library.
S4. Preparation of third ssDNA library
Incubating 100 mu L of magnetic beads marked by streptavidin with dsDNA library for 20min at normal temperature, binding the dsDNA library to the surface of the magnetic beads by utilizing the affinity action of biotin on the dsDNA library and the streptavidin on the magnetic beads, removing supernatant by utilizing a magnetic separator, washing the magnetic beads by using 2mL PBS, adding 200 mu L of NaOH solution (200 mM) into an EP tube, reacting for 10min at normal temperature, denaturing and melting the dsDNA library, binding one chain with the biotin and the streptavidin on the magnetic beads, taking single-stranded DNA bound with the magnetic beads as a third ssDNA library, and recovering the supernatant by utilizing a magnetic separation frame; after the desalting column was washed with 10mL of sterile water, the supernatant was added to the desalting column and allowed to drip naturally by gravity. 500 μ L PBS was added and the collected solution was used for the next round of screening.
S5, repeatedly screening
And (3) replacing the first ssDNA library with the third ssDNA library obtained in the step (S4), and repeating the positive screening process, the PCR amplification and the single-stranded DNA library preparation process shown in the step (S2-S4) for 10 times.
S6. Negative screening
And in the second round of S5 and the second round of subsequent screening, normal fat head 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. 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 (4) collecting the supernatant containing the ssDNA library in the step S6, performing PCR amplification on the step S3 and preparation on the ssDNA library in the step S4, sequentially repeating the steps 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 times of screening, wherein the identification capacity of the obtained ssDNA library on the STIV infected cells is the 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)。
the secondary structure of SEQ ID NO.2 was predicted on line using 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 (seemer 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 Cyprinus carpio 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 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 Cyprinus carpio 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. Mu.L of PBS, carrying out thermostatic water bath at 92 ℃ for 5min, then quickly inserting into ice, carrying out 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 and removing a 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. And observing cell precipitates obtained after incubation of the test aptamer of the experiment group II and the test aptamer of 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 bind to the surface of STIV-infected fat head carp cells and has high specific recognition capability on the STIV-infected fat head carp 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, guangxi Zhuang nationality autonomous region Water science research institute
<120> nucleic acid aptamer for detecting Chinese softshell turtle iridovirus as well as construction method and application thereof
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> Artificial sequence
<400> 1
acacccaaat tccgtcagtc gtgctcgtaa ttcacaacac cgctggccat 50
<210> 2
<211> 95
<212> DNA
<213> Artificial sequence
<400> 2
gacgcttact caggtgtgac tcgacaccca aattccgtca gtcgtgctcg taattcacaa 60
caccgctggc catcgaagga cgcagatgaa gtctc 95
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
gacgcttact caggtgtgac tcg 23
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence
<400> 4
gagacttcat ctgcgtcctt cg 22
Claims (4)
1. An aptamer for detecting an iridovirus of a trionyx sinensis, comprising: the DNA sequence is SEQ ID NO.2.
2. An aptamer for detecting an iridovirus of a trionyx sinensis, comprising: the DNA sequence of claim 1 is linked with functional group, and the functional group is selected from biotin label, luminescent label and enzyme label.
3. Use of the nucleic acid aptamer for detecting Chinese softshell turtle iridovirus according to claim 1 in preparation of a product for detecting Chinese softshell turtle iridovirus.
4. Use according to claim 3, characterized in that: the product is a fluorescent molecular probe, and the aptamer for detecting the Chinese softshell turtle iridovirus is connected with a luminescent marker.
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| Selection and characterization of novel DNA aptamers specifically recognized by Singapore grouper iridovirus-infected fish cells;Li Pengfei, Zhou L, et al.;《J Gen Virol》;20150825;第96卷(第11期);3348-3359 * |
| 基于核酸适配体Q5的石斑鱼虹彩病毒快速检测技术;余庆 等;《广西科学院学报》;20190815;第35卷(第3期);193-199+212 * |
| 核酸适配体在病原生物学中的应用研究进展;贺宏斌;《热带病与寄生虫学》;20190910;第17卷(第3期);176-180 * |
| 用于检测石斑鱼虹彩病毒(SGIV)感染的DNA核酸适配体筛选;李鹏飞,周伶俐,俞也频,等;《第十二届海洋药物学术年会论文集》;20190404;147-160 * |
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