CN116445490A - Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection - Google Patents

Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection Download PDF

Info

Publication number
CN116445490A
CN116445490A CN202310464012.9A CN202310464012A CN116445490A CN 116445490 A CN116445490 A CN 116445490A CN 202310464012 A CN202310464012 A CN 202310464012A CN 116445490 A CN116445490 A CN 116445490A
Authority
CN
China
Prior art keywords
tetracycline
dna
aptamer
seq
aptamer probe
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.)
Pending
Application number
CN202310464012.9A
Other languages
Chinese (zh)
Inventor
刘猛
李晓倩
常洋洋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian University of Technology
Original Assignee
Dalian University of Technology
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Dalian University of Technology filed Critical Dalian University of Technology
Priority to CN202310464012.9A priority Critical patent/CN116445490A/en
Publication of CN116445490A publication Critical patent/CN116445490A/en
Pending legal-status Critical Current

Links

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
    • C12N15/115—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses an aptamer probe based on a structure conversion signal and application thereof in tetracycline detection, and belongs to the technical field of nucleic acid probes. According to the invention, through an in vitro screening technology, a structure conversion aptamer probe capable of specifically recognizing tetracycline is obtained, when the tetracycline does not exist, the aptamer can keep a stable double (duplex) structure with the complementary antisense DNA (FAM mark and Dabcyl mark), and fluorescence is quenched due to the fact that the distance between two complementary chains is similar; the existence of the tetracycline can induce the conversion of the DNA/DNA dual structure to the DNA/target composite structure, so that the distance between the marker Dabcyl and the FAM is increased, thereby generating a fluorescent signal, and detecting the tetracycline with different concentrations.

Description

Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection
Technical Field
The invention belongs to the technical field of nucleic acid probes, and particularly relates to an aptamer probe based on structural conversion signals and application of the aptamer probe in tetracycline detection.
Background
Functional nucleic acids are mainly classified into two major classes according to different action mechanisms, wherein the first class of functional nucleic acids is called a nucleic acid Aptamer (Aptamer), and the Aptamer can perform a similar recognition function as a protein antibody and can specifically recognize metal ions, small molecules, proteins, cells, tissues and even organs, so that the functional nucleic acids are often used as recognition units for constructing functional nucleic acid probes. Another class of functional nucleic acids is known as deoxyribozymes (DNAzyme), which bind to cofactors and have similar catalytic functions as proteases. An aptamer is a single stranded oligonucleotide that can selectively recognize and bind a target molecule obtained by the in vitro exponential enrichment ligand evolution System (SELEX). Compared with an antibody, the aptamer has the advantages of wide target range, simple acquisition process, simple chemical structure, strong stability and the like, so that the aptamer is a powerful tool for molecular recognition. Researchers obtain nucleic acid aptamer capable of recognizing and combining with metal ions, small molecules, proteins, pathogenic bacteria and the like through in-vitro screening technology. The versatility of targets, high binding affinity and specificity, and simplicity of in vitro selection, make aptamers attractive as molecular tools for bioanalytical applications.
Tetracyclines (TCs) are a broad spectrum of antibiotics produced by actinomycetes, including chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC), and semisynthetic derivatives of methacycline, doxycycline, minocycline, etc., all of which have a tetracyclic skeleton. Among them, tetracyclines are an important group of antibiotics, with a common tetracyclic scaffold that inhibit bacterial protein synthesis by preventing the attachment of aminoacyl-tRNA to ribosomes. While most tetracyclines are currently used only in animals, their residues of immersion in the environment and in foods have caused serious health problems.
In recent years researchers have reported a number of aptamers for detecting tetracyclines, which interact with a variety of RNAs with micromolar affinities, including 30S ribosomal RNAs, tRNA' S, and a number of ribozymes. In 2001, berens et al reported an RNA aptamer recognizing tetracycline, K d About 100nM. DNA aptamers are more stable and easier to modify than RNA aptamers and are therefore more attractive for biosensor development. In 2008, niazi et al reported DNA aptamers that recognize tetracycline. All of these aptamers have been obtained so far by immobilization of target molecules, whereas in vitro screening of libraries immobilized (also known as capture SELEX) can use unmodified free targets. This selection strategy will result in a signal aptamer, which in the selection step will result in a DNA aptamer that has not been modified at all, and then be converted into an effective signaling probe that will synchronize target binding with signal generation. The aptamer probe has potential application prospects in aspects of biosensing, proteomics and drug discovery. Standard aptamers can be easily obtained by in vitro selection techniques. However, for biosensing applications, post-selection modifications must be made in order to convert the relevant aptamer to a signaling probe. Although some conversion strategies have reported, the modification process typically requires lengthy optimization steps to ensure that the affinity and specificity of the original aptamer is not lost, and that the labeled and modified signal aptamer is able to significantly modulate the fluorescent signal after binding to the target molecule.
Disclosure of Invention
In view of the above, the present invention aims to obtain a structure-based signal aptamer probe through an in vitro screening scheme, identify tetracycline, and achieve the purpose of rapid and sensitive detection of tetracycline, and by utilizing a screening strategy of immobilizing a library, evolve and select a signal aptamer with a structure-based switching mechanism to act, when tetracycline is not present, the aptamer maintains a stable double (duplex) structure with complementary antisense DNA (a FAM tag and a Dabcyl tag) due to the fact that two complementary chains are close in distance, and fluorescence is quenched; the existence of tetracycline can induce the conversion of a DNA/DNA dual structure to a DNA/target composite structure, so that the distance between a marker Dabcyl and FAM is increased to generate a fluorescent signal, the detection of tetracycline with different concentrations is realized, the aptamer probe can report the existence of a target molecule through the real-time fluorescent signal, and no more separation and purification steps are needed.
The purpose of the invention is achieved by the following means:
the invention provides an aptamer probe, the nucleotide sequence of the aptamer probe is shown as SEQ ID NO. 7, and the nucleotide sequence shown as SEQ ID NO. 7 and the sequences shown as SEQ ID NO. 8 and SEQ ID NO. 9 or SEQ ID NO. 10 form a structure conversion signal molecule;
wherein, the 5 'end of the sequence shown in SEQ ID NO. 8 is marked with a fluorescent group, and the 3' end of the sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10 is marked with a quenching group.
Further, in the above technical solution, the fluorescent group is FAM.
Further, in the above technical scheme, the quenching group is Dabcyl.
The invention also provides a detection kit comprising the aptamer probe.
Further, in the above technical scheme, the detection kit includes an auxiliary factor, and the auxiliary factor is Mg 2+ 、Na + And K + 。
The invention also provides application of the aptamer probe or the detection kit in biological sensing, which is used for specifically identifying tetracycline antibiotics.
Further, in the above technical scheme, the tetracycline antibiotics include tetracycline and oxytetracycline.
Further, in the above technical scheme, the conditions for specifically recognizing the tetracycline antibiotics of the aptamer probe are as follows: pH is 7-8, at room temperature, mg 2+ 、Na + 、K + As a cofactor.
Furthermore, in the technical scheme, the response concentration of the tetracycline antibiotics is more than or equal to 1 mu M.
Compared with the prior art, the invention has the following effects:
(1) The invention obtains a structure conversion aptamer probe, and the molecular signal of the aptamer probe increases along with the extension of time.
(2) The aptamer probe provided by the invention has the advantages of good stability, high sensitivity and good specificity, and can play an advantage in the aspect of biosensing application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of an in vitro screening procedure for structurally transformed aptamers.
FIG. 2 is a graph of in vitro screening process.
FIG. 3 is a schematic diagram of aptamer structural transformations.
FIG. 4 is a validation graph of in vitro screening results.
FIG. 5 is a sequence diagram of TC5 structural switching aptamers.
Fig. 6 is a feasibility analysis diagram of TC 5.
FIG. 7 is a graph of TC5 sensitivity analysis to tetracycline.
FIG. 8 is a graph of TC5 selectivity for tetracycline.
Detailed Description
Embodiments of the technical scheme of the present invention will be described in detail below. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, which should not be construed as limiting the scope of the present invention.
Examples include construction of DNA libraries, reverse screening, forward screening, PCR amplification, etc., and the names and sequences of the nucleic acids referred to in the examples are shown in Table 1.
TABLE 1 nucleic acid names, sequences and uses
EXAMPLE 1 construction of DNA library modules
An amount of library DNA (SEQ ID NO: 1) was mixed with 1.5-fold molar amounts of P1 (SEQ ID NO: 3), P2 (SEQ ID NO: 4) and 5-fold molar amounts of BDNA (SEQ ID NO: 2), heated at 90℃for 3min, cooled to room temperature for 10min, and base complementary pairing of the DNA in the complementary region of the library was achieved during annealing. Then adding suspension of streptavidin magnetic beads, incubating for 2h at room temperature in a magnetic bead coupling buffer solution, and coupling biotin at the BDNA5' end with streptavidin on the magnetic beads to realize library immobilization in the screening process. After incubation, magnetic separation is carried out by a magnetic frame, and the uncoupling DNA which is free in the solution is eluted, and the uncoupling DNA is respectively eluted by a magnetic bead buffer solution and ddH 2 O and 1 Xreaction buffer were eluted 3 times to complete the construction of the DNA module for in vitro screening.
DNA library construction system:
1) Construction of DNA library modules
TABLE 2 reaction Components of DNA library Assembly
Reagent(s) Volume (mu L)
Library DNA (100. Mu.M) 5
P1(100μM) 7.5
P2(100μM) 7.5
BDNA(100μM) 25
2 Xbuffer 50
Water and its preparation method Supplement to 100
2) Coupling of libraries to magnetic beads
TABLE 3 ligation reaction composition Table
Reagent(s) Volume (mu L)
DNA module 100
Magnetic bead (10 mg/mL) 250
2 Xcoupling buffer 312.5
Water and its preparation method Supplement to 625
Example 2 preparation of target molecules
As stock solutions, 10mM tetracycline, kanamycin and ampicillin solutions were prepared, respectively, and all stock solutions were prepared.
Example 3 in vitro screening
The in vitro screening is carried out by the steps of reverse screening, forward screening, PCR amplification, library component construction and the like, and the high-throughput sequencing is carried out after 13 rounds of screening. The screening flow is shown in fig. 1, and the specific screening steps include:
1. reverse screening
The DNA library assembly constructed in example 1 was resuspended in 200. Mu.L of ultrapure water and reacted in a reaction-containing buffer.
TABLE 4 reverse sieve reaction composition table
Reagent(s) Volume (mu L)
DNA library component 200
2 Xreaction buffer solution 250
Kanamycin solution (10 mM) 10
Ampicillin solution (10 mM) 10
Water and its preparation method Supplement and fill up to 500
The constructed library components are mixed by shaking before the reaction, and then 2 x reaction buffer solution, kanamycin solution, ampicillin solution and water are added, and incubated for 2 hours in a DNA mixer at room temperature.
Wherein 2 Xthe reaction buffer is dissolvedThe liquid comprises the following components: 100mM HEPES,300mM NaCl,100mM KCl,30mM MgCl 2 ，0.02％Tween20，pH 7.5。
The product after reaction is magnetically separated by a magnetic frame, the nonspecific DNA sequence which is free in the solution is eluted, and the specific DNA is still fixed on the magnetic beads.
2. Forward screening
The uneluted DNA/magnetic bead complex obtained in the reverse screening was resuspended in 200. Mu.L of ultrapure water and reacted in a reaction buffer containing tetracycline.
TABLE 5 Positive Screen reaction composition Table
Reagent(s) Volume (mu L)
DNA library component 200
2 Xreaction buffer solution 250
Tetracycline solution (10 mM) 10
Water and its preparation method Supplement and fill up to 500
The recovered DNA/magnetic bead complex was mixed well by shaking before the reaction, then 2 Xreaction buffer solution, tetracycline solution and water were added and incubated for 1h at room temperature in a DNA mixer.
Wherein 2 x reaction buffer composition: 100mM HEPES,300mM NaCl，100mM KCl，30mM MgCl 2 ，0.02％Tween20，pH 7.5。
Magnetically separating the reacted product through a magnetic rack, recovering DNA free in the solution, combining the DNA with tetracycline, eluting the specific DNA, and obtaining a sequence capable of carrying out structural conversion through a cold ethanol precipitation method.
3. PCR amplification
The DNA sequences recovered by forward screening, which are capable of undergoing structural transformation, are used as templates for the mass amplification of the sequence of interest by two-step PCR using the upstream primer FP and the downstream primers P2 and RP. The PCR products were separated and purified by 10% dPAGE. Since the downstream primer RP has a steric modification, sense strand (77 nt) and antisense strand (97 nt) with different lengths are obtained after PCR amplification, and the sense strand is recovered by cutting and the content is measured. Wherein, the sequence of the upstream primer FP is shown as SEQ ID NO. 5, and the sequences of the downstream primer P2 and RP are shown as SEQ ID NO. 4 and SEQ ID NO. 6 respectively.
PCR amplification conditions:
TABLE 6 PCR amplification conditions
Name of the name Temperature (. Degree. C.) Time(s)
Hold 94 60
Denaturation (denaturation) 94 30
Annealing 50 45
Extension 72 45
PCR reaction system:
1)PCR1
TABLE 7 PCR1 reaction conditions
Reagent(s) Volume (mu L)
Library 1
Upstream primer FP (100. Mu.M) 0.5
Downstream primer P2 (100. Mu.M) 0.5
10 XPCR buffer 5
dNTPs(2.5mM) 1
Taq enzyme (5U/. Mu.L) 1
Water and its preparation method Supplement and fill to 50
2)PCR2
TABLE 8 PCR2 reaction conditions
Reagent(s) Volume (mu L)
Library (PCR 1 product) 1
Upstream primer FP (100. Mu.M) 0.5
Downstream primer RP (100. Mu.M) 0.5
10 XPCR buffer 5
dNTPs(2.5mM) 1
Taq enzyme (5U/. Mu.L) 1
Water and its preparation method Supplement and fill to 50
4. Construction of DNA library modules
The library sense strand DNA obtained by PCR was mixed with 1.5-fold molThe amounts of P1 (SEQ ID NO: 3), P2 (SEQ ID NO: 4) and 5 times the molar amount of BDNA (SEQ ID NO: 2) were mixed, heated at 90℃for 3min, cooled to room temperature for 10min, then streptavidin magnetic beads were added, incubated in a coupling buffer for 2h at room temperature, biotin on BDNA was coupled with streptavidin on the magnetic beads, and library immobilization was achieved during screening. After incubation, magnetic separation is carried out by a magnetic frame, and the uncoupling DNA which is free in the solution is eluted, and the uncoupling DNA is respectively coupled with buffer solution and H by magnetic beads 2 Eluting with O and 1 Xreaction buffer solution for 3 times to obtain the DNA component for the next round of screening.
Percent sequence conversion (Clv%) was expressed as conversion activity, i.e., the ratio of free sequence from the beads during the screening to the total amount of sequence put into the reaction, and used as a measure for assessing the progress of the screening to characterize the degree of enrichment of the DNA library after each round of screening, as shown in fig. 2. The percent conversion of the forward screening was significantly increased over the percent conversion of the reverse screening and the percent conversion of the blank control over the multiple rounds of screening, with the percent conversion of the 13 th round of forward screening chain library being approximately 10.0%. Library enrichment was complete and DNA turnover was defined as: the amount of DNA eluted in the forward selection, as compared to the amount of DNA dosed prior to incubation.
And through high-throughput sequencing, selecting the five sequences with the top ranking for experimental verification. After finding out the DNA sequence TC5 (SEQ ID NO: 7) meeting the target requirements and constructing molecular signal components with antisense oligonucleotide chains FDNA and LQDNA, the structural conversion activity of the aptamer is verified (FIG. 2).
Example 4 structural switching aptamer characterization
1. In vitro screening results validation
The top five sequences were chemically synthesized and named TC1, TC2, TC3, TC4, TC5, respectively, and their structure-switching activity after binding to tetracycline was verified (fig. 4). The enzyme-labeled instrument is used for monitoring the real-time fluorescence signal, and TC5 is found to have obvious fluorescence enhancement, and the fluorescence intensity of the rest sequences is kept unchanged. After the addition of tetracycline, the fluorescence intensity of TC5 tends to increase with time, indicating that TC5 can be used as a structural transition aptamer, the sequence structure of which is shown in FIG. 5, and can be used as a signal molecule in the aspect of biological sensing application for detecting antibiotics.
2. Feasibility analysis
In the feasibility analysis, a structure conversion assembly is constructed after truncated QDNA (SQDNA) is mixed with TC5 and FDNA, and is used for real-time fluorescence analysis by an enzyme labeling instrument, the change of fluorescence intensity of the structure conversion along with time is explored, the equilibrium point when the reaction reaches saturation is explored, the reaction time is optimized for the subsequent characterization experiment, the highest value of a response fluorescence signal of TC5 to tetracycline can appear within 5min, and the reaction reaches equilibrium (figure 6).
3. Sensitivity analysis
Under the condition that the concentration of the structural conversion aptamer and the proportion of the structural conversion aptamer and the complementary antisense sequence in the reaction system are kept consistent, the concentration of the tetracycline in the reaction system is changed, and the structural conversion probe can generate signal responses with different intensities to the tetracycline with different concentrations, and the minimum signal response can reach 1 mu M (figure 7).
4. Selective analysis
Response signals of TC5 to different antibiotics are explored (FIG. 8 a), TC5 responds to non-fluorescent signals of Kanamycin (KANA), ampicillin (AMP), chloramphenicol (CPL), polymyxin B (PMB) and the like, and tetracycline has obvious fluorescent signal generation in the presence of tetracycline, so that the structure conversion system has better specificity to the tetracycline. In addition, the signal response characteristics of TC5 to different types of tetracycline antibiotics in the family were also explored (FIG. 8 b), TC5 did not respond to aureomycin (CTC), even showed a trend of fluorescence decrease, the structure was different from that of tetracycline due to the presence of Cl atoms in aureomycin, and the decrease in fluorescence was probably due to Cl; since Oxytetracycline (OTC) differs from Tetracycline (TC) by only one O atom, and has very high structural similarity, TC5 also produces a fluorescent response to OTC.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (9)

1. The aptamer probe is characterized in that the nucleotide sequence of the aptamer probe is shown as SEQ ID NO. 7, and the nucleotide sequence shown as SEQ ID NO. 7 and the sequences shown as SEQ ID NO. 8 and SEQ ID NO. 9 or SEQ ID NO. 10 form a structure conversion signal molecule;
wherein, the 5 'end of the sequence shown in SEQ ID NO. 8 is marked with a fluorescent group, and the 3' end of the sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10 is marked with a quenching group.
2. The aptamer probe of claim 1, wherein the fluorophore is FAM.
3. The aptamer probe of claim 1, wherein the quenching group is Dabcyl.
4. A test kit comprising the aptamer probe of any one of claims 1-3.
5. The kit of claim 4, wherein the kit comprises a cofactor, wherein the cofactor is Mg 2+ 、Na + And K + 。
6. Use of an aptamer probe according to any one of claims 1 to 3 or a detection kit according to claim 4 or 5 for biosensing, for the specific recognition of tetracycline antibiotics.
7. The use according to claim 6, wherein the tetracycline antibiotics comprise tetracycline and oxytetracycline.
8. Root of Chinese characterThe use according to claim 6, wherein the conditions for specific recognition of tetracycline antibiotics are: pH is 7-8, at room temperature, mg 2+ 、Na + 、K + As a cofactor.
9. The use according to claim 6, wherein the tetracycline antibiotic has a response concentration of 1 μm or more.
CN202310464012.9A 2023-04-26 2023-04-26 Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection Pending CN116445490A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310464012.9A CN116445490A (en) 2023-04-26 2023-04-26 Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310464012.9A CN116445490A (en) 2023-04-26 2023-04-26 Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection

Publications (1)

Publication Number Publication Date
CN116445490A true CN116445490A (en) 2023-07-18

Family

ID=87127301

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310464012.9A Pending CN116445490A (en) 2023-04-26 2023-04-26 Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection

Country Status (1)

Country Link
CN (1) CN116445490A (en)

Similar Documents

Publication Publication Date Title
AU2008275917B2 (en) Multiplexed analyses of test samples
US9404919B2 (en) Multiplexed analyses of test samples
Zhang et al. Peptide bond formation by in vitro selected ribozymes
US7855054B2 (en) Multiplexed analyses of test samples
CN106834295B (en) Broad-spectrum nucleic acid aptamer for specifically recognizing bacterial lipopolysaccharide and directional screening method thereof
CN107446929B (en) Aptamer for specifically recognizing ochratoxin A and preparation method thereof
Wang et al. Recent advances in the rapid detection of microRNA with lateral flow assays
JP2022530041A (en) New isothermal single reaction probe set and its uses
CN111122847B (en) Method for rapidly detecting aflatoxin B1 on site based on aptamer
Morse Direct selection of RNA beacon aptamers
EP1476557B1 (en) A rapid and sensitive assay for the detection and quantification of coregulators of nucleic acid binding factors
Zhao et al. Exonuclease I aided enzyme-linked aptamer assay for small-molecule detection
Song et al. Label-free visual detection of nucleic acids in biological samples with single-base mismatch detection capability
US20100035769A1 (en) Biomolecule assay chip
CN116445490A (en) Aptamer probe based on structure conversion signal and application of aptamer probe in tetracycline detection
CN114807147B (en) Nucleic acid aptamer of aflatoxin B1 and application thereof
CN112462046A (en) Method for rapidly detecting antibiotics based on colorimetric aptamer sensor
AU2017202493B2 (en) Multiplexed analyses of test samples
CN114621958B (en) Single-stranded DNA aptamer sequence for specifically recognizing ATP and application thereof
WO2018003809A1 (en) Rna structure library
CN113549676B (en) Novel RNA pull down method based on magnetic beads
Laios et al. Novel hybridization assay configurations based on in vitro expression of DNA reporter molecules
CN114058624B (en) Aptamer, sensor, kit and application for detecting sulfanilamide-5-methoxypyrimidine
CN117487813B (en) Single-stranded DNA aptamer sequence for specifically recognizing azithromycin and application thereof
CN116769783A (en) Aptamer capable of specifically recognizing shiga toxin type II B subunit and application thereof

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