CN111690648A - Sequence and application of nucleic acid aptamer TDHA for specifically recognizing vibrio parahemolyticus TDH - Google Patents
Sequence and application of nucleic acid aptamer TDHA for specifically recognizing vibrio parahemolyticus TDH Download PDFInfo
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Abstract
The invention relates to a sequence of aptamer TDHA for specifically recognizing vibrio parahemolyticus TDH and application thereof, wherein the sequence of the aptamer TDHA is as follows: 5'-GAGCGGCACGAACCAGTAAAGTCTTCCCGACCGC-3', respectively; the nucleic acid aptamer TDHA can recognize vibrio parahemolyticus TDH with high affinity and high specificity; the nucleic acid aptamer TDHA is used as an identification element of the vibrio parahaemolyticus TDH, the detection method of the vibrio parahaemolyticus TDH and the preparation detection reagent are established, the method can be used for the application fields of monitoring the pollution of the vibrio parahaemolyticus in the environment, diagnosing and evaluating the disease condition of the vibrio parahaemolyticus infection in clinic, analyzing the biological function of the vibrio parahaemolyticus TDH in basic research and the like, and the method has the advantages of simplicity, convenience, rapidness, good characteristics and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a sequence of a nucleic acid aptamer TDHA for specifically recognizing TDH and application thereof in detecting TDH of vibrio parahaemolyticus.
Background
Vibrio Parahaemolyticus (Vibrio Parahaemolyticus) is a gram-negative bacillus, and is in various shapes such as arc, rod and thread, and has no spore. Eating food containing the bacteria can cause food poisoning, also called halophilic bacteria food poisoning. Food poisoning caused by vibrio parahaemolyticus is transmitted by improperly cooked seafood or salted products. Common are jellyfish, sea fish, sea shrimp and various shellfish. Food poisoning can also occur after the bacteria are contaminated by uncooked food containers or chopping boards. The disease can occur all the year round, the incubation period is 5-72 hours, the average time is 24 hours, the clinical manifestations can be from self-limiting diarrhea to moderate cholera-like diseases, and the symptoms comprise diarrhea, abdominal pain, vomiting, low fever and the like. Most of the excrement is water sample, and a few of the excrement is blood water sample, so the excrement can be recovered quickly, the immunity after diseases is not strong, and the infection can be repeated.
Hemolysin is the leading cause of the pathogenesis of vibrio parahaemolyticus and includes Thermostable Direct Hemolysin (TDH), thermostable direct Hemolysin-related Hemolysin (TRH) and Thermolabile Hemolysin (TLH). Epidemiological investigation research shows that TDH is the most main pathogenic factor of vibrio parahaemolyticus, and almost more than 95% of clinically separated vibrio parahaemolyticus can generate hemolytic rings on a special blood plate, which is called Kanagawa Phenomenon (KP). At present, most of the detection research aiming at the vibrio parahaemolyticus TDH at home and abroad adopts molecular biology technology, wherein multiple PCR, fluorescent real-time quantitative PCR and the like are applied more. Although direct detection of TDH of Vibrio parahaemolyticus is more convenient and faster, few immunological techniques have been applied to this aspect due to the problem of antibody specificity.
An Aptamer (Aptamer) is an oligonucleotide sequence (DNA or RNA). Generally, oligonucleotide fragments are obtained from random libraries of nucleic acid molecules using in vitro screening techniques, the exponential enrichment of ligands by phylogenetic evolution, SELEX. The combination of the aptamer and the target has the characteristics of high affinity, high specificity and the like. As nucleic acid molecules, the aptamer can be directly synthesized and subjected to various chemical modifications, and has the advantages of low cost, quick preparation and the like. Aptamers have been used for rapid and accurate detection of various targets as recognition elements for the targets. A sensor detection method capable of detecting the thallus of the vibrio parahaemolyticus has been established by taking an aptamer as an identification element of the vibrio parahaemolyticus (J Agric Food chem.2019, 67: 2313-2320). If the advantages of the aptamer can be utilized to establish a rapid and accurate vibrio parahemolyticus TDH detection method, the method has wide application and basic research value.
The invention provides a nucleic acid aptamer TDHA for specifically recognizing vibrio parahemolyticus TDH. TDHA is obtained by taking vibrio parahemolyticus TDH expressed by escherichia coli as a target, manually screening in vitro by a SELEX technology and optimizing a sequence. TDHA specifically recognizes TDH, and does not bind to hemolysin of other Vibrio parahaemolyticus. At present, no report is found for establishing a vibrio parahaemolyticus TDH detection kit by taking a specific aptamer as an identification element. Disclosure of Invention
The invention aims to provide a sequence of a nucleic acid aptamer TDHA for specifically identifying vibrio parahemolyticus TDH and application thereof.
The invention is realized by the following technical scheme:
firstly, synthesizing a sequence of aptamer TDHA by Shanghai's chemical company, modifying and marking (such as biotin or fluorescent group) the 5' end or 3' end of the TDHA, and then carrying out application research: TDHA is used as an identification element to respectively establish a colorimetric biosensor, a gel migration retardation experiment and an enzyme-linked aptamer adsorption method which can be used for detecting the vibrio parahaemolyticus TDH.
The nucleic acid aptamer TDHA of the vibrio parahaemolyticus TDH is applied to establishing a vibrio parahaemolyticus TDH detection method and preparing a detection kit.
Compared with the prior art, the invention has the advantages that:
1. compared with protein antibodies, the single-chain oligonucleotide is more stable; the aptamer can be directly synthesized and labeled in vitro, so that a labeled secondary antibody is not needed, and the operation is simpler and quicker.
2. The synthesis cost of the aptamer TDHA is lower than that of antibody preparation, the period is short, and the reproducibility is good.
3. The nucleic acid aptamer TDHA can be combined with vibrio parahemolyticus TDH with high affinity and high specificity, and has no recognition function on other vibrio parahemolyticus hemolysin. Therefore, the TDHA sequence has wide application value and wide market prospect in the fields of environmental monitoring, clinical medicine, research on the biological function of the vibrio parahaemolyticus TDH and the like.
Drawings
FIG. 1 is a schematic diagram of the detection of a colorimetric biosensor for detecting Vibrio parahaemolyticus TDH, using a nucleic acid aptamer TDHA as an identification element.
FIG. 2 shows the specific detection of Vibrio parahaemolyticus TDH using a colorimetric biosensor using aptamer TDHA as a recognition element.
FIG. 3 shows the specific detection of Vibrio parahaemolyticus TDH by gel migration inhibition assay using aptamer TDHA as recognition element.
FIG. 4 is a detection diagram of an enzyme-linked aptamer adsorption method for detecting TDH of Vibrio parahaemolyticus using aptamer TDHA as a recognition element.
FIG. 5 shows the specific detection of TDH of Vibrio parahaemolyticus by an enzyme-linked aptamer adsorption method using aptamer TDHA as a recognition element.
FIG. 6 is a graph showing the dissociation constants of the aptamer TDHA binding to Vibrio parahaemolyticus TDH in the fluorescence binding rate experiment. The dissociation constant (Kd) was 3.78 nM. In FIG. 6, the abscissa represents the DNA concentration (nM) and the ordinate represents the fluorescence binding rate.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification:
a nucleic acid aptamer TDHA specifically combined with vibrio parahaemolyticus TDH is obtained by taking vibrio parahaemolyticus TDH expressed by escherichia coli as a target, manually screening in vitro by a SELEX technology and optimizing a sequence. The length of TDHA is 34 bases, and the sequence is as follows: 5'-GAGCGGCACGAACCAGTAAAGTCTTCCCGACCGC-3' (SEQ ID NO: 1).
The nucleic acid aptamer TDHA of the vibrio parahaemolyticus TDH is analyzed by mfold platform at 25 ℃ and 100mM Na+，1mM Mg2+Under the conditions of (a), the spatial structure thereof is as follows:
the aptamer TDHA of the vibrio parahaemolyticus TDH is subjected to chemical modification including but not limited to FITC, amino, biotin, digoxin and the like on the 5 'end or the 3' end of the aptamer TDHA.
The nucleic acid aptamer TDHA of the vibrio parahaemolyticus TDH is subjected to chemical modification including but not limited to FITC, amino, biotin, digoxin and the like on a product obtained by carrying out truncation or extension or partial base replacement on the nucleic acid aptamer TDHA and carrying out structural modification.
The present invention will be further described below by establishing a method for detecting Vibrio parahaemolyticus TDH with the nucleic acid aptamer TDHA as an identification element of Vibrio parahaemolyticus TDH.
The invention is realized by the following technical scheme:
example one: colorimetric biosensor for detecting vibrio parahaemolyticus TDH (time domain of binding protein) by using nucleic acid aptamer TDHA (TDHA) as identification element
(1) Synthesis of aptamer TDHA (synthesized by Shanghai Biotech) with the sequence: 5'-GAGCGGCACGAACCAGTAAAGTCTTCCCGACCGC-3' are provided.
(2) A concentration of aptamer TDHA was dissolved in a suitable volume of buffer (50 mM Tris-HCl, 100mM NaCl, 1mM MgCl)25mM KCl, pH 7.4) and then heat activated. The method of the thermal activation treatment is as follows: after denaturation at 95 ℃ for 5min, the mixture was immediately placed in an ice-water bath for 10min and then at room temperature for 10 min.
(3) TDHA at a concentration of 2. mu.M in a volume of 50. mu.L was mixed with Vibrio parahemolyticus hemolysin TDH (1. mu.M), TDH + TLH + TRH (1. mu.M, respectively), and added to a 96-well plate, and left to stand at room temperature for 30 min. The TDH, TLH and TRH are cloned, expressed and purified by escherichia coli, and the purity is more than 95%. The above system was set up with 3 replicates and a blank.
(4) And (4) adding 50 mu L of gold nanoparticle (AuNPs) solution with the diameter of 15nm into the 96-well plate in the step (3), uniformly mixing, and standing at room temperature for 5 min.
(5) And (4) adding 50 mu L of 300 mM sodium chloride solution into the 96-well plate in the step (4), mixing uniformly, and standing at room temperature for 15 min.
(6) The absorbance of the 96-well plate reaction system at 520nm was measured using a spectrophotometer.
As shown in FIG. 1, the principle of the colorimetric biosensor for detecting Vibrio parahaemolyticus TDH is as follows: when the detector has vibrio parahemolyticus TDH, the TDH in the reaction system is combined with the nucleic acid aptamer TDHA to make the AuNPs in a free state, and the AuNPs are aggregated after a high-concentration sodium chloride solution is added. When no TDH exists in the detection object, the TDHA in the reaction system is non-specifically combined with the AuNPs, so that the AuNPs are in a combined state, and the AuNPs do not aggregate after a high-concentration sodium chloride solution is added. The aggregation degree of AuNPs can be detected by detecting the absorbance value reaction of the reaction system at 520 nm.
As shown in FIG. 2, the absorbance values at 520nm were significantly decreased in the TDH group and the TDH + TLH + TRH group, while the absorbance values in the TLH + TRH group were not decreased, as compared with the blank control group, demonstrating that TDHA was used as a recognition element and TDH can be specifically detected by a colorimetric biosensor.
Example two: detecting the TDH of the vibrio parahaemolyticus by using a gel migration retardation experiment by taking a nucleic acid aptamer TDHA as an identification element
(1) An aptamer TDHA was synthesized, and the 5' end thereof was labeled with a fluorescent group FITC (synthesized by shanghai bio chemical corporation) having the sequence: 5'-GAGCGGCACGAACCAGTAAAGTCTTCCCGACCGC-3' are provided.
(2) A concentration of FITC-labeled aptamer TDHA was dissolved in an appropriate volume of buffer (50 mM Tris-HCl, 100mM NaCl, 1mM MgCl)25mM KCl, pH 7.4) and then heat activated. The method of the thermal activation treatment is as follows: after denaturation at 95 ℃ for 5min, the mixture was immediately placed in an ice-water bath for 10min and then at room temperature for 10 min.
(3) The FITC-labeled TDHA after the heat activation treatment was incubated with two hemolysin mixtures of TDH-containing and TDH-free Vibrio parahaemolyticus, respectively, "+ TDH + TLH + TRH" and "-TDH + TLH + TRH", in a dark box at room temperature for 1 h. The TDH, TLH and TRH are cloned, expressed and purified by escherichia coli, and the purity is more than 95%.
(4) The co-incubation system of the aptamer TDHA and hemolysin mixture is added with 10 Xloading buffer solution, and separated by electrophoresis of 12% PAGE gel.
(5) Observed in a fluorescent gel imaging system and photographed.
As shown in FIG. 3, after PAGE separation, FITC labeled aptamer TDHA can be combined with TDH-containing vibrio parahemolyticus hemolysin mixture "+ TDH + TLH + TRH", that is, TDHA nucleic acid electrophoresis band has migration retardation; and the DNA does not combine with a vibrio parahemolyticus hemolysin mixture without TDH, namely TDH + TLH + TRH, namely a TDHA nucleic acid electrophoresis strip does not generate migration retardation, so that the TDHA marked by a fluorescent group (FITC) is used as an identification element, and the vibrio parahemolyticus TDH can be specifically detected by utilizing a gel migration retardation experiment.
Example three: detecting the TDH of the vibrio parahaemolyticus by using a biotin-labeled aptamer TDHA as an identification element and adopting an enzyme-linked aptamer adsorption method
(1) Synthesis of aptamer TDHA, biotin (synthesized by Shanghai Biochemical Co., Ltd.) was labeled at its 5' end with the sequence: 5'-GAGCGGCACGAACCAGTAAAGTCTTCCCGACCGC-3' are provided.
(2) TDH, TLH and TRH expressed by Escherichia coli are respectively dissolved in carbonate buffer solution with pH of 9.7, added into the enzyme-linked strip according to the amount of 100 mu L/hole, and packaged in a wet box at 4 ℃ overnight.
(3) The coating solution was discarded, and 100. mu.L of a maleic acid blocking solution containing 1% casein was added to each well and blocked at room temperature for 1 hour.
(4) Different concentrations of biotinylated TDHA were dissolved in appropriate volumes of buffer (50 mM Tris-HCl, 100mM NaCl, 1mM MgCl)25mM KCl, pH 7.4) and then heat activated. The method of the thermal activation treatment is as follows: after denaturation at 95 ℃ for 5min, the mixture was immediately placed in an ice-water bath for 10min and then at room temperature for 10 min.
(5) Adding the biotin-labeled TDHA subjected to heat activation treatment into an enzyme-linked strip, fully mixing the mixture at 100 mu L/hole, and incubating the mixture for 2h at 37 ℃. Duplicate wells and blanks were set up in 3 wells.
(6) Discarding the liquid in the hole, washing each hole with 300 μ L of washing solution, repeating the washing for 3 times, and completely spin-drying the liquid in the hole after the last washing.
(7) Adding 100 μ L of HRP enzyme-labeled streptavidin diluted at a ratio of 1:100 into each well, incubating at room temperature for 40min, discarding the liquid in the wells, and washing the plate for 5 times in the same manner as above;
(8) adding 100 mu L of TMB chromogenic substrate into each hole, carrying out dark color development at 37 ℃, adding 10 mu L of stop solution when obvious color changes, and detecting the absorption value of the reaction system at 450nm by an enzyme-linked analyzer.
As shown in FIG. 4, the principle of the enzyme-linked aptamer adsorption method for detecting the TDH of Vibrio parahaemolyticus is as follows: when the TDH of the vibrio parahemolyticus exists in the coating detection object, the TDHA marked by the biotin added into the reaction system is combined with the TDH coated in the enzyme-linked strip, and further the streptavidin marked by the HRP is combined with the biotin and catalyzes the TMB substrate to develop color.
As shown in FIG. 5, compared with the blank control group, the absorption value of TDH group at 450nm was significantly increased, while TLH and TRH were not significantly increased, which proves that TDHA labeled with biotin was used as a recognition element, and TDH of Vibrio parahaemolyticus was specifically detected by the enzyme-linked aptamer adsorption method.
Example four: determination of dissociation constant (KD value) of binding between aptamer TDHA and Vibrio parahaemolyticus TDH
(1) Coupling of vibrio parahaemolyticus TDH and carboxyl magnetic beads: the carboxyl magnetic beads and the coupling reagent thereof are purchased from BangsLaboratories company in the United states. TDH and magnetic bead coupling operation with reference to the manufacturer's instructions, coupling TDH magnetic beads (TDH magnetic beads) dispersed in PBS buffer, 4 degrees C storage.
(2) And respectively mixing FITC labeled aptamer TDHA solutions with different concentrations with TDH magnetic beads, and incubating for 1h at room temperature in a dark box.
(3) The magnetic beads obtained in step (2) were washed 3 times with 0.1% PBST, and the aptamer TDHA bound to the magnetic beads was eluted by boiling 200. mu.L of a selection buffer at 100 ℃ for 5 min.
(4) The fluorescence binding rate of the aptamer TDHA solution with different concentrations and the TDH magnetic beads is obtained and calculated through experiments, the fluorescence binding rate is calculated as (initial fluorescence intensity-elution fluorescence intensity)/initial fluorescence intensity multiplied by 100%, and the calculated value is used for preliminarily representing the binding rate of the aptamer TDHA and the target molecules.
(5) And (3) utilizing the calculated value of the fluorescence binding rate to draw a saturated binding curve of the aptamer TDHA binding TDH, and calculating the dissociation constant of the aptamer TDHA binding TDH through nonlinear regression analysis.
As shown in FIG. 6, the saturated binding curve of aptamer TDHA was calculated to have a dissociation constant of 3.78nM, which indicates that aptamer TDHA binds to Vibrio parahaemolyticus TDH strongly with a dissociation constant on the nanomolar scale.
In a word, the nucleic acid aptamer TDHA can specifically identify the vibrio parahemolyticus TDH, and the nucleic acid aptamer TDHA is used as an identification element, so that the vibrio parahemolyticus TDH can be detected by a colorimetric biosensor, a gel migration blocking experiment and an enzyme-linked aptamer adsorption method. Therefore, the nucleic acid aptamer TDHA has wide application potential and value in the aspects of developing various vibrio parahemolyticus TDH detection methods.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that a person skilled in the art may make several changes, improvements and modifications without departing from the spirit of the present invention, and these changes, improvements and modifications should be construed as the protection scope of the present invention.
SEQUENCE LISTING
<110> winter medical technology Limited of Baoai in Changle region, Fuzhou city
<120> sequence and application of aptamer TDHA for specifically recognizing vibrio parahaemolyticus TDH
<160>1
<210>1
<211>34
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>1
gagcggcacg aaccagtaaa gtcttcccga ccgc 34
Claims (5)
1. A nucleic acid aptamer TDHA for specifically recognizing vibrio parahaemolyticus TDH, which is characterized in that: the sequence is shown in SEQ ID NO. 1; and 100mM Na at 25 deg.C+，1mM Mg2+Under the condition of (2), the spatial structure is as follows:
2. the nucleic acid aptamer TDHA of Vibrio parahaemolyticus TDH according to claim 1, characterized in that: and carrying out chemical modification on the 5 'end or the 3' end of the aptamer TDHA, such as fluorescent group, amino group, biotin, digoxigenin and the like.
3. The aptamer TDHA according to claim 1, which is chemically synthesized in vitro or prepared by PCR or other molecular biological methods.
4. Use of the aptamer TDHA of Vibrio parahaemolyticus TDH according to claim 1 or 2 for establishing a method for detecting Vibrio parahaemolyticus TDH.
5. Use of the aptamer TDHA of Vibrio parahaemolyticus TDH according to claim 1 or 2 in the preparation of a reagent for detecting Vibrio parahaemolyticus TDH.
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| CN113899731A (en) * | 2021-08-17 | 2022-01-07 | 广东省科学院测试分析研究所(中国广州分析测试中心) | One-step detection method for vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113899731A (en) * | 2021-08-17 | 2022-01-07 | 广东省科学院测试分析研究所(中国广州分析测试中心) | One-step detection method for vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters |
| CN113899731B (en) * | 2021-08-17 | 2022-06-14 | 广东省科学院测试分析研究所(中国广州分析测试中心) | One-step detection method for vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters |
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