CN112816682A - Triple helix DNA molecular switch probe and application thereof in OTA colorimetric rapid detection - Google Patents
Triple helix DNA molecular switch probe and application thereof in OTA colorimetric rapid detection Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a triple-helix DNA molecular switch probe and application thereof in OTA colorimetric rapid detection. The triple-helix DNA molecular switch probe is mainly formed by hybridizing and assembling a DNA chain GP27 which is rich in G basic group and forms a parallel G-quadruplex structure and can improve the peroxidase activity of G-quadruplex-hemin and a DNA chain AP9 containing ochratoxin A (OTA) aptamer sequence, and the triple-helix DNA molecular switch probe formed by hybridizing and assembling is applied to OTA colorimetric rapid detection. The triple-helix DNA molecular switch probe can be used for sensitive and rapid colorimetric detection of OTA, improves the detection sensitivity, avoids using DNA amplification or nano material to amplify a detection signal, does not need to label a used DNA chain, does not need steps of separation, cleaning and the like in the detection process, greatly simplifies the detection step while maintaining the detection sensitivity, and has the advantages of simple and rapid detection, low cost and good application prospect.
Description
Technical Field
The invention belongs to the technical field of rapid detection of mycotoxins, and particularly relates to design and assembly of a triple helix DNA molecular switch probe and application of the triple helix DNA molecular switch probe in ochratoxin A (OTA) colorimetric rapid detection.
Background
Ochratoxin a (ota) is a secondary metabolite produced by toxic fungi such as aspergillus and penicillium. Research shows that OTA has renal toxicity and hepatotoxicity, can cause DNA damage, inhibit RNA and protein synthesis and other hazards, has teratogenic, carcinogenic and mutagenic effects, and is listed as a 2B carcinogen by the cancer research institute of the world health organization. Agricultural products are easily polluted by ochratoxin in the processes of harvesting, storing, transporting and processing, and the easily polluted agricultural products (food) comprise grains, beans, nuts, spices, coffee beans, cocoa, wine, beer and the like. In 2013, domestic investigation shows that the OTA detection rate in 77 parts of red wine is as high as 57%, and the maximum value is 5.65 mug/kg. In 2010, a study of the academy of Chinese medical sciences showed that OTA was detected in 30 of 57 Chinese medicinal materials. In 2019, Hajok et al detected 473 food samples available in the Western Pachys market in Poland, and the results showed that 22% of the samples were contaminated with OTA, with the highest OTA content in raisins reaching 34.0. mu.g/kg, which is 3.5 times the standard. The researches show that OTA has high toxicity, wide distribution and high possibility of causing high incidence, so that the establishment of a simple, quick, low-cost and high-sensitivity detection method is one of effective technical means for realizing OTA pollution monitoring and reducing the influence of OTA on the health of people and animals to the maximum extent. At present, the common detection method of OTA is a high performance liquid chromatography-fluorescence/mass spectrometry detection method, and has the characteristics of high sensitivity and good reproducibility. However, these methods rely on expensive analytical testing equipment and skilled technicians, and are not conducive to point-of-care testing (POCT) of large numbers of samples, limiting their application and spread. Antibody-based immunoassays, such as: the enzyme-linked immunosorbent assay and the immune test strip have high detection flux and high speed, are also commonly applied to the detection of OTA, but have low OTA immunogenicity, large difficulty in antibody development, time consumption, high cost and easy generation of false positive. Therefore, it is still very urgent to develop a rapid, sensitive, easy-to-operate and low-cost OTA detection method.
DNA has long been considered as a material for storing and transmitting life genetic information and has been studied in large quantities. However, from a material point of view, DNA can be found to have several unique physical and chemical properties compared to other synthetic polymers, including sequence editability, molecular recognition, binding and catalytic chemical reactions. In addition to the well-known classical double helix structure, DNA can be folded into a variety of structures by intramolecular hydrogen bonding, base stacking, electrostatic interactions, and metal ion coordination, such as: specific three-dimensional structures such as DNA triple helix structures (triple helix), G-quadruplex structures (G-quadruplex), aptamers (aptamer), and the like. The structure of a substance determines its properties. After the DNA forms a specific structure, the DNA can show unique properties and has the functions of identification, combination, catalysis and the like. For example, a G-quadruplex structure formed by a specific G-rich base sequence can be combined with hemin (hemin) to form a DNAse (DNAzyme) having peroxidase-like activity. The peroxidase activity of the DNase can be used for catalyzing and generating and amplifying a detection signal, so that the detection sensitivity is improved. The DNA aptamer can form a stable structure and can specifically recognize and bind to a target object similarly to an antibody. Compared with antibodies, the DNA aptamer has some obvious advantages, such as: good chemical stability, low production cost, small batch-to-batch difference and the like.
In recent years, with the development of various nanomaterials and DNA amplification technologies, researchers use DNase and aptamers and combine the DNA amplification technology and the nanomaterials to amplify detection signals, so that a new method is provided for rapid and sensitive detection of OTA. However, the preparation and modification of the nano-materials and the labeling and amplification of the DNA make the detection operation steps complicated, the cost is increased, and the practicability is reduced.
Disclosure of Invention
In order to solve the problems, the invention designs a new G-quadruplex structure forming sequence to improve the peroxidase activity of DNA enzyme, simultaneously designs an aptamer sequence, assembles to form a triple helix DNA molecular switch probe, and develops the sensitive and rapid colorimetric detection technology of OTA. The technology does not need to use DNA amplification or nano material to amplify detection signals, so the used DNA chain does not need to be marked, the detection process does not need steps of separation, cleaning and the like, the detection steps are greatly simplified while the detection sensitivity is kept, the detection cost is low, and the application prospect is good.
The technical scheme adopted by the invention is as follows:
one, three spiral DNA molecule switch probe
The triple-helix DNA molecular switch probe is mainly formed by hybridizing and assembling a DNA chain GP27 capable of improving the peroxidase activity of G-quadruplex-hemin and a DNA chain AP9 containing ochratoxin A (OTA) aptamer sequence.
The DNA chain GP27 capable of improving the peroxidase activity of the G-quadruplex-hemin is rich in G base, forms a parallel G-quadruplex (G-quadruplex) structure, and has the base sequence: 5'-GGTGGTGGTGGTTGTGGAGGAGGAGGA-3', as shown in SEQ ID No. 1. The 3' end of the DNA chain GP27 is provided with an adenine base A which greatly enhances the peroxidase activity of G-quadrupulex-hemin, so that a detection signal is amplified without using DNA amplification or nano materials for amplifying the detection signal, therefore, the used DNA chain does not need to be marked, the detection process does not have the steps of separation, cleaning and the like, and the detection step is greatly simplified while the detection sensitivity is maintained.
The DNA chain AP9 containing the OTA aptamer sequence has 36 base sequences capable of selectively identifying and combining OTA in the middle, the arm end sequences at the front end and the rear end are all sequences consisting of 6 cytosine C bases and 3 thymine T bases, and the base sequences are as follows: 5'-TCCTCCTCCGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACCT CCTCCT-3', as shown in SEQ ID No. 2.
The triple-helix DNA molecular switch probe has a stem-loop structure and is assembled by respectively matching a TCCTCCTCC base sequence at the front arm end of a DNA chain AP9, a CCTCCTCCT base sequence at the rear arm end of a DNA chain AP9 and a AGGAGGAGG base sequence at the 3' end of a DNA chain GP27 through Watson-Crick and Hoogsteen base pairs; the assembled triple-helix DNA molecular switch probe has a stem-loop structure, a 36 base sequence GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA in the middle of a DNA chain AP9 forms a ring part of the triple-helix DNA molecular switch probe and is used for identifying and combining a target object OTA to be detected, a TCCTCCTCC base sequence at the front arm end of the DNA chain AP9 and a CCTCCTCCT base sequence at the rear arm end of the DNA chain AP9 are respectively paired with a AGGAGGAGG base sequence close to the 3' end of the DNA chain GP27 through Watson-Crick and Hoogsteen bases to form a stem part of the triple-helix DNA molecular switch probe, and the DNA chain GP27 is locked.
Too short or too long an end-of-arm sequence can reduce the sensitivity of the detection. When the sequence of the arm terminal is too short, the triple-helix DNA molecular switch probe is unstable and easy to untie, and releases a DNA chain GP27, so that a higher background signal is generated, and the signal-to-noise ratio is reduced; when the sequence of the arm end is too long, the triple-helix DNA molecular switch probe is too stable, the target object OTA to be detected is difficult to combine with the triple-helix DNA molecular switch probe, the triple-helix DNA molecular switch probe is difficult to open, and a detection signal is difficult to generate or even impossible.
Preparation method of triple-helix DNA molecular switch probe
The method specifically comprises the following steps:
and (3) uniformly mixing the DNA chain GP27 and the DNA chain AP9 in PBS buffer solution with the pH value of 6.2, hybridizing at normal temperature for 30 minutes, assembling to obtain the triple-helix DNA molecular switch probe for detecting the target object OTA to be detected, and storing at normal temperature.
The ratio of the amounts of the substances of the DNA strand AP9 to the DNA strand GP27 was equal to 1.8: 1. In principle, when the concentration ratio of the DNA chain AP9 to the DNA chain GP27 is 1:1, the DNA chain AP9 and the DNA chain GP27 are just completely assembled into the triple helix DNA molecular switch probe, and the DNA chain GP27 is completely locked. However, the triple helix DNA molecular switch probe assembled by the DNA chain AP9 and the DNA chain GP27 is a reversible chemical reaction in nature, and analysis from the chemical reaction equilibrium principle shows that increasing the amount of the DNA chain AP9 is beneficial to promoting the combination and assembly of the DNA chain AP9 and the DNA chain GP27, so that the DNA chain GP27 is more completely combined, and the free DNA chain GP27 is reduced, thereby reducing the background signal, further improving the signal to noise ratio when the probe detects the target object to be detected, and generating higher sensitivity.
Third, OTA rapid detection method based on the triple helix DNA molecular switch probe
The method specifically comprises the following steps:
uniformly mixing a triple-helix DNA molecular switch probe solution and a sample solution containing a target object OTA to be detected in a PBS buffer solution to obtain a reaction solution, and reacting for 30 minutes at 25 ℃; then adding HEPES buffer solution and Hemin (Hemin) into the reaction solution, uniformly mixing to obtain a detection reaction solution, and reacting for 30 minutes at normal temperature; finally, adding 2, 2-azino-bis- (3-ethylbenzodihydro thiazoline-6-sulfonic acid) diammonium salt ABTS into the detection reaction solution2-And H2O2The solution develops color. The higher the OTA content in the sample is, the darker the green color of the detection reaction solution is, and an enzyme-labeling instrument or an ultraviolet-visible spectrophotometer can be used for measuring the absorbance value of the detection reaction solution at 418nm to realize accurate quantification. With the increase of the concentration of the target object OTA to be detected, the color of the detection reaction solution is gradually changed from light green to dark green, so that a colorimetric signal visible to naked eyes is generated to quickly detect the OTA. The detection of the object OTA to be detected is completed within 60 minutes, and the speed is higher than that of the liquid chromatography and the enzyme-linked immunosorbent assay method of the current national standard method.
The absorbance value increase rate of the detection reaction solution at 418nm is positively and linearly related with the concentration of the target object OTA to be detected from 0.01 to 1.5 mg/kg.
1) The rapid detection method of the OTA content in the reaction buffer solution comprises the following steps:
incubating a triple-helix DNA molecular switch probe assembled by a DNA chain AP9 and a DNA chain GP27 with OTA in PBS buffer solution at 25 ℃ for 30 minutes, wherein the OTA is combined with an aptamer sequence in the DNA chain AP9, so that the triple-helix DNA molecular switch probe is opened to release the DNA chain GP 27; then adding HEPES buffer solution and Hemin, forming a G-quadruplex structure by a DNA chain GP27 and forming DNA enzyme with peroxidase-like activity with the Hemin; adding ABTS finally2-And H2O2DNase catalysis H2O2Oxidation of ABTS2-Make the solution lookDarkening, and judging the content of OTA in the sample according to the increase percentage of the absorbance of the solution at 418 nm;
2) the method for rapidly detecting OTA in the actual peanut oil sample comprises the following steps:
a peanut oil sample is purchased from a local supermarket, and no OTA is detected through high performance liquid chromatography, so that 3 grades of OTA with different concentrations are added into the peanut oil sample, and an addition recovery test is carried out to verify the accuracy and stability of the OTA in the actual sample detected by the triple helix DNA molecular switch probe. The added samples were pretreated according to the national standard (GB 5009.96-2016) method, namely: extracting, separating and filtering. And then, detecting by using a triple-helix DNA molecular switch probe, calculating the recovery rate and the relative standard deviation, and comparing with the detection result of the high performance liquid chromatography detection method.
And fourthly, the triple helix DNA molecular switch probe is applied to OTA colorimetric rapid detection.
The invention designs a new G-quadruplex structure forming sequence to enhance the peroxidase activity of DNase, so that the DNase catalyzes H2O2Oxidation of the substrate ABTS2-The efficiency of generating detection signals is higher, the detection sensitivity is improved, the use of nano materials and DNA amplification technology is avoided, the detection operation steps are simplified, the cost is reduced, and the practicability of the technology is improved.
The invention has the beneficial effects that:
1) the present invention designs a G-rich base sequence GP26 which can form a G-quadruplex structure, and by simply adding only one A base at its 3' end (i.e.: GP27) greatly improves the catalytic activity of the dnase formed thereby, thus improving the sensitivity of detection;
2) by ingenious design, the GP27 and the AP9 two DNA chains are assembled through Watson-Crick and Hoogsteen base pairing to form the triple helix DNA molecular switch probe. When detecting OTA, the probe and the OTA are only required to be uniformly mixed in a buffer solution and react for 30 minutes at room temperature to carry out subsequent color reaction, and the detection is finished. The preparation of the detection probe and the transduction of the detection signal do not need DNA amplification or amplification of the detection signal by any nano material, and the steps of separation, cleaning and the like are not needed, so that the cost is low, and the OTA can be rapidly detected by a very simple operation step of 'mixing and detecting';
3) the rapid detection of OTA based on the triple helix DNA molecular switch probe of the invention can be completed within 60 minutes, the speed is far faster than that of a high performance liquid chromatography detection method, the linear range of the detection is 0.01-1.5 mg/kg, the detection limit is 0.004mg/kg, and the detection limit is lower than the maximum residual limit (primary grains: 0.005mg/kg, soluble coffee 0.01 mg/kg);
4) the three-helix DNA molecular switch probe based on the invention can be used for rapidly detecting OTA, and the detection probe only responds to the target object OTA due to the good specificity of the aptamer, so that the detection of the OTA can not be interfered when mycotoxins such as AFB1, AFB2, AFG1 and OTB exist in a sample;
5) the triple-helix DNA molecular switch probe designed and assembled by the invention has expansibility, wherein the aptamer sequence of AP9 is changed into the aptamer sequences of other objects to be tested, and after proper optimization, the triple-helix DNA molecular switch probe capable of specifically responding to different objects to be tested can be assembled.
Summarizing, the triple-helix DNA molecular switch probe can be used for sensitive and rapid colorimetric detection of OTA, improves the detection sensitivity, avoids using DNA amplification or nano materials to amplify detection signals, has no steps of labeling, separating, cleaning and the like in the detection process, greatly simplifies the detection steps while maintaining the detection sensitivity, and has the advantages of simple and rapid detection, low cost and good application prospect.
Drawings
FIG. 1 is a schematic diagram of the assembly of triple helix DNA molecular switch probes and their use for OTA rapid detection;
FIG. 2 is a schematic diagram of base pairing when AP9 and GP27 are assembled to form a triple helix DNA molecular switch probe;
FIG. 3 shows the enhancement of the catalytic activity of DNase and the response of triple helix DNA molecular switch probe to the target object OTA to be detected. 3(A) is the characterization that the A base enhances the catalytic activity of the DNase; 3(B) is A basic group to enhance the catalytic activity of the DNase and accelerate the generation rate of a detection signal; 3(C) verifying the assembly of the triple-helix DNA molecular switch probe and the response of the triple-helix DNA molecular switch probe to OTA by adopting an ultraviolet visible absorption spectrum; 3(D) adopting circular dichroism to characterize and verify the assembly of the triple-helix DNA molecular switch probe and the response of the triple-helix DNA molecular switch probe to OTA;
FIG. 4 is a colorimetric chart of the sample solution for colorimetric detection of OTA. C is positive control, containing only GP27, no triple helix DNA molecular switch probe, no OTA; S1-S7 are samples containing triple helix DNA molecular switch probes and different concentrations of OTA, wherein the concentration of the OTA is gradually increased from 0 to 2 mg/kg;
FIG. 5 shows the response result of absorption spectrum and the specific detection result of OTA colorimetric rapid detection using triple helix DNA molecular switch probe. 5(A) is an ultraviolet visible absorption spectrum response diagram of the OTA detected by the triple-helix DNA molecular switch probe through colorimetry, the color of a sample detection reaction solution is changed from light green to dark green along with the increase of the concentration of the OTA, and the absorbance at 418nm is increased along with the color; 5(B) is that the percentage increase of the absorbance value of the sample detection reaction solution at 418nm increases with the increase of the concentration of OTA; 5(C) is a linear regression equation fitting graph of the absorbance value increase percentage of the sample detection reaction solution at 418nm and the logarithmic value of the concentration of the OTA when the concentration of the OTA is 0.01 to 1.5 mg/kg; and 5(D) is the specificity of the triple helix DNA molecular switch probe for colorimetric rapid detection of OTA.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It should be understood that the preferred embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
One, three spiral DNA molecule switch probe
The triple helix DNA molecular switch probe is mainly formed by hybridizing and assembling a DNA chain GP27 capable of improving the peroxidase activity of G-quadruplex-hemin and a DNA chain AP9 containing ochratoxin A (OTA) aptamer sequence.
A DNA chain GP27 capable of improving the peroxidase activity of G-quadruplex-hemin is rich in G base, forms a parallel G-quadruplex (G-quadruplex) structure, and has the base sequence as follows: 5'-GGTGGTGGTGGTTGTGGAGGAGGAGGA-3', as shown in SEQ ID No. 1.
As shown in FIG. 3, the 3' end of the DNA chain GP27 is provided with an adenine base A, which greatly enhances the peroxidase activity of G-quadruplex-hemin, so that the detection signal is amplified without using DNA amplification or nano material amplification, therefore, the used DNA chain does not need to be marked, the detection process does not have the steps of separation, cleaning and the like, and the detection step is greatly simplified while the detection sensitivity is maintained. FIG. 3(A) is a representation of the A base enhancing DNase catalytic activity; FIG. 3(B) is a diagram showing that the A base enhances the catalytic activity of DNase and accelerates the generation rate of a detection signal; FIG. 3(C) is a graph demonstrating the assembly of triple-helical DNA molecular switch probes and their response to OTA using UV-visible absorption spectroscopy; FIG. 3(D) is a graph of the validation of the assembly of triple-helical DNA molecular switch probes and their response to OTA using circular dichroism;
a DNA chain AP9 containing OTA aptamer sequence has 36 base sequences capable of selectively recognizing and combining OTA in the middle, the arm end sequences of the front end and the rear end are all sequences consisting of 6 cytosine C bases and 3 thymine T bases, and the base sequences are as follows: 5'-TCCTCCTCCGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACCT CCTCCT-3', as shown in SEQ ID No. 2.
As shown in FIG. 2, the triple-helix DNA molecular switch probe has a stem-loop structure, and is assembled by respectively matching the TCCTCCTCC base sequence at the front arm end of a DNA chain AP9, the CCTCCTCCT base sequence at the rear arm end of the DNA chain AP9 and the AGGAGGAGG base sequence at the 3' end of the DNA chain GP27 through Watson-Crick and Hoogsteen base pairs; the assembled triple-helix DNA molecular switch probe has a stem-loop structure, a 36 base sequence GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA in the middle of a DNA chain AP9 forms a ring part of the triple-helix DNA molecular switch probe and is used for identifying and combining a target object OTA to be detected, a TCCTCCTCC base sequence at the front arm end of the DNA chain AP9 and a CCTCCTCCT base sequence at the rear arm end of the DNA chain AP9 are respectively paired with a AGGAGGAGG base sequence close to the 3' end of the DNA chain GP27 through Watson-Crick and Hoogsteen bases to form a stem part of the triple-helix DNA molecular switch probe, and the DNA chain GP27 is locked.
Too short or too long an end-of-arm sequence can reduce the sensitivity of the detection. When the sequence of the arm terminal is too short, the triple-helix DNA molecular switch probe is unstable and easy to untie, and releases a DNA chain GP27, so that a higher background signal is generated, and the signal-to-noise ratio is reduced; when the sequence of the arm end is too long, the triple-helix DNA molecular switch probe is too stable, the target object OTA to be detected is difficult to combine with the triple-helix DNA molecular switch probe, the triple-helix DNA molecular switch probe is difficult to open, and a detection signal is difficult to generate or even impossible.
Preparation method of triple-helix DNA molecular switch probe
The method specifically comprises the following steps:
and (3) uniformly mixing the DNA chain GP27 and the DNA chain AP9 in PBS buffer solution with the pH value of 6.2, hybridizing at normal temperature for 30 minutes, assembling to obtain the triple-helix DNA molecular switch probe for detecting the target object OTA to be detected, and storing at normal temperature.
The ratio of the amounts of substance of DNA strand AP9 to DNA strand GP27 was equal to 1.8: 1. In principle, when the concentration ratio of the DNA chain AP9 to the DNA chain GP27 is 1:1, the DNA chain AP9 and the DNA chain GP27 are just completely assembled into the triple helix DNA molecular switch probe, and the DNA chain GP27 is completely locked. However, the triple helix DNA molecular switch probe assembled by the DNA chain AP9 and the DNA chain GP27 is a reversible chemical reaction in nature, and analysis from the chemical reaction equilibrium principle shows that increasing the amount of the DNA chain AP9 is beneficial to promoting the combination and assembly of the DNA chain AP9 and the DNA chain GP27, so that the DNA chain GP27 is more completely combined, and the free DNA chain GP27 is reduced, thereby reducing the background signal, further improving the signal to noise ratio when the probe detects the target object to be detected, and generating higher sensitivity.
Third, OTA rapid detection method based on the triple helix DNA molecular switch probe
The method specifically comprises the following steps:
uniformly mixing a triple-helix DNA molecular switch probe solution and a sample solution containing a target object OTA to be detected in a PBS buffer solution to obtain a reaction solution, and reacting for 30 minutes at 25 ℃; then adding HEPES buffer solution and Hemin into the reaction solution, uniformly mixing to obtain a detection reaction solution, and reacting for 30 minutes at normal temperature; finally adding ABTS into the detection reaction solution2-And H2O2The solution develops color. The higher the OTA content in the sample is, the darker the green color of the detection reaction solution is, and the detection can be carried out by an enzyme-linked immunosorbent assay or an ultraviolet-visible spectrophotometerAnd detecting the absorbance value of the reaction solution at 418nm to realize accurate quantification. With the increase of the concentration of the target object OTA to be detected, the color of the detection reaction solution is gradually changed from light green to dark green, so that a colorimetric signal visible to naked eyes is generated to quickly detect the OTA. The detection of the object OTA to be detected is completed within 60 minutes, and the speed is higher than that of the liquid chromatography and the enzyme-linked immunosorbent assay method of the current national standard method.
As shown in FIG. 5(A), the absorbance increase rate of the detection reaction solution at 418nm is positively linearly related to the concentration of the target OTA to be detected from 0.01 to 1.5 mg/kg.
1) The rapid detection method of the OTA content in the reaction buffer solution comprises the following steps:
incubating a triple-helix DNA molecular switch probe assembled by a DNA chain AP9 and a DNA chain GP27 with OTA in PBS buffer solution at 25 ℃ for 30 minutes, wherein the OTA is combined with an aptamer sequence in the DNA chain AP9, so that the triple-helix DNA molecular switch probe is opened to release the DNA chain GP 27; then adding HEPES buffer solution and Hemin, forming a G-quadruplex structure by a DNA chain GP27 and forming DNA enzyme with peroxidase-like activity with the Hemin; adding ABTS finally2-And H2O2DNase catalysis H2O2Oxidation of ABTS2-The color of the solution is darkened, and the content of the OTA in the sample is judged according to the increasing percentage of the absorbance of the solution at 418 nm;
2) the method for rapidly detecting OTA in the actual peanut oil sample comprises the following steps:
a peanut oil sample is purchased from a local supermarket, and no OTA is detected through high performance liquid chromatography, so that 3 grades of OTA with different concentrations are added into the peanut oil sample, and an addition recovery test is carried out to verify the accuracy and stability of the OTA in the actual sample detected by the triple helix DNA molecular switch probe. The added samples were pretreated according to the national standard (GB 5009.96-2016) method, namely: extracting, separating and filtering. And then, detecting by using a triple-helix DNA molecular switch probe, calculating the recovery rate and the relative standard deviation, and comparing with the detection result of the high performance liquid chromatography detection method.
And fourthly, the triple helix DNA molecular switch probe is applied to OTA colorimetric rapid detection.
The invention designs a new G-quadruplex structure forming sequence to enhance the peroxidase activity of DNase, so that the DNase catalyzes H2O2Oxidation of the substrate ABTS2-The efficiency of generating detection signals is higher, the detection sensitivity is improved, the use of nano materials and DNA amplification technology is avoided, the detection operation steps are simplified, the cost is reduced, and the practicability of the technology is improved.
The preferred embodiment of the invention is as follows:
example 1
As shown in FIG. 1, a triple-helix DNA molecular switch probe for colorimetric rapid detection of a target object OTA to be detected is assembled by using a DNA chain GP27 and a DNA chain AP 9.
mu.L of 10. mu.M DNA strand GP27 and 7.2. mu.L of 10. mu.M DNA strand AP9 in a 1 XPBS buffer (20mM PBS, 20mM NaCl, 2.5mM MgCl) at pH 6.2 were taken2) And uniformly mixing, hybridizing for 30 minutes at room temperature, and assembling to obtain the triple-helix DNA molecular switch probe of the target object OTA to be detected. The prepared probe solution is stored at room temperature for later use.
Example 2
And rapidly detecting the OTA in the aqueous solution by using a triple helix DNA molecular switch probe.
S1: 100 μ L of the triple helix DNA molecular switch probe solution assembled in example 1 was added with 4 μ L of OTA standard solution (methanol as solvent) of a certain concentration, mixed well, and reacted at 25 ℃ for 30 minutes.
S2: to the sample were added 20. mu.L of 10 XHEPES buffer solution (250mM HEPES, 200mM KCl, 2000mM NaCl, 0.05% Triton X-100, pH 5.3), 4. mu.L of Hemin at a concentration of 10. mu.M, and 32. mu.L of ultrapure water (to make each sample volume 160. mu.L), mixed well, and reacted at room temperature for 30 minutes.
S3: 20 μ L ABTS at 20mM concentration was added to the sample2-And 20. mu.L of 100mM H2O2And (5) mixing the solution and the mixture. Transferring the samples to a 96-well plate, adding H from the sample2O2Timing was started after the solution, and the absorbance value of the sample solution at 418nm was measured by a microplate reader until the 7 th minute of reaction.
S4: steps S1 to S3 were repeated using OTA standard solutions of different concentrations (methanol as solvent), with the final concentration of OTA in the samples being 0.002, 0.01, 0.05, 0.1, 0.3, 0.5, 1, 1.5, 2, 3, 4mg/kg in order, with 3 replicates per concentration.
S5: the concentration of OTA was plotted on the abscissa, and the absorbance value increase Rate%0) /A0) X 100% as ordinate (A, A)0Respectively representing the absorbance values of the reaction solution when the sample contains OTA and does not contain OTA), and establishing a working curve of OTA detection.
FIG. 4 is a colorimetric chart of the sample solution for colorimetric detection of OTA. C is positive control, containing only GP27, no triple helix DNA molecular switch probe, no OTA; S1-S7 are samples containing triple helix DNA molecular switch probes and varying concentrations of OTA, where the concentration of OTA was gradually increased from 0 to 2 mg/kg.
And (3) detection results:
as shown in FIG. 5(B), the absorbance value of the sample solution at 418nm gradually increased as the final concentration of OTA increased from 0.002 to 4mg/kg (from low to high, 0.002, 0.01, 0.05, 0.1, 0.3, 0.5, 1, 1.5, 2, 3, 4 mg/kg).
As shown in FIG. 5(C), the absorbance value increase rate of the sample solution at 418nm was fitted to the concentration of OTA (0.01 to 1.5mg/kg) to obtain a linear regression equation IR of 0.484C +0.131, C representing the concentration of OTA, and a linear correlation coefficient r20.997, and the linear range of detection is 0.01 to 1.5 mg/kg. The detection limit was calculated to be 0.004mg/kg based on 3S/S (S is the standard deviation at the lowest concentration of 0.01mg/kg, and S is the slope of the calibration curve).
Example 3
Detection of OTA in peanut oil
S1: pretreating peanut oil according to national standard method (GB 5009.96-2016), weighing peanut oil 5g, placing into a centrifuge tube, adding sodium chloride 1g and methanol 25mL (V)Methanol：VWater (W)80: 20) shaking for 30 min, centrifuging at 6000rpm for 10 min, collecting 15mL of upper layer extractive solution, adding 30mL of phosphate buffer solution, mixing, filtering with glass fiber filter paper membrane for 2 times, and preparing OTA sample with concentration of 0, 0.01, 0.05, and 0.1mg/kgAnd (3) solution.
S2: and (3) taking 100 mu L of the assembled triple helix DNA molecular switch probe solution in the example 1, adding 4 mu L of the OTA sample solution with a certain concentration into the solution, mixing the solution evenly, and reacting the mixture for 30 minutes at 25 ℃.
S3: to the sample were added 20. mu.L of HEPES at a concentration of 250mM, 4. mu.L of Hemin at a concentration of 10. mu.M, and 32. mu.L of ultrapure water (to make the volume of each sample 160. mu.L), mixed well, and reacted at room temperature for 30 minutes.
S4: 20 μ L ABTS at 20mM concentration was added to the sample2-And 20. mu.L of 100mM H2O2And (5) mixing the solution and the mixture. Transferring the samples to a 96-well plate, adding H from the sample2O2The solution was timed to begin, and the absorbance value of the sample solution at 418nm was measured by a microplate reader by the 7 th minute of reaction.
S5: and (4) calculating the increase rate of the absorbance value of the sample solution, substituting the increase rate into a regression equation of a standard curve, and calculating the concentration, the addition recovery rate and the relative standard deviation of the OTA in each sample solution.
S6: OTA solutions with different concentrations are used for addition and recovery, so that the addition concentration of 3 grades is 0.01, 0.05 and 0.1mg/kg respectively, and 3 samples are made in parallel for each grade of concentration. And repeating the steps S2 to S4, and calculating the detection result.
The results of the addition recovery test are shown in Table 1.
TABLE 1
The above examples are merely the results of the present invention on this example, but the specific implementation of the present invention is not limited to this example. Any alternatives which have similar effects according to the principles and concepts of the invention should be considered as the protection scope of the invention.
The DNA sequence related to the invention is as follows:
SEQ ID No.1：
name: DNA chain GP27 base sequence
The source is as follows: artificial Sequence (Artificial Sequence)
GGTGGTGGTGGTTGTGGAGGAGGAGGA
SEQ ID No.2：
Name: DNA chain AP9 base sequence
The source is as follows: artificial Sequence (Artificial Sequence)
TCCTCCTCCGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACC TCCTCCT。
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> triple-helix DNA molecular switch probe and application thereof in OTA colorimetric rapid detection
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggtggtggtg gttgtggagg aggagga 27
<210> 2
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcctcctccg atcgggtgtg ggtggcgtaa agggagcatc ggacacctcc tcct 54
Claims (8)
1. A triple-helix DNA molecular switch probe is characterized in that the triple-helix DNA molecular switch probe is formed by hybridizing and assembling a DNA chain GP27 capable of improving the peroxidase activity of G-quadruplex-hemin and a DNA chain AP9 containing ochratoxin A (OTA) aptamer sequences.
2. The triple-helical DNA molecular switch probe of claim 1, wherein: the DNA chain GP27 capable of improving the peroxidase activity of the G-quadruplex-hemin is rich in G base, forms a parallel G-quadruplex structure, and has the following base sequence: 5'-GGTGGTGGTGGTTGTGGAGGAGGAGGA-3', as shown in SEQ ID No. 1.
3. The triple-helical DNA molecular switch probe of claim 1, wherein: the DNA chain AP9 containing the OTA aptamer sequence has 36 base sequences capable of selectively identifying and combining OTA in the middle, the arm end sequences at the front end and the rear end are all sequences consisting of 6 cytosine C bases and 3 thymine T bases, and the base sequences are as follows: 5'-TCCTCCTCCGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACCTCCTCCT-3', as shown in SEQ ID No. 2.
4. The triple-helical DNA molecular switch probe of claim 1, wherein: the triple-helix DNA molecular switch probe has a stem-loop structure, a 36 base sequence GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA in the middle of a DNA chain AP9 forms a loop part of the triple-helix DNA molecular switch probe, and a TCCTCCTCC base sequence at the front arm end of the DNA chain AP9 and a CCTCCTCCT base sequence at the rear arm end of the DNA chain AP9 are respectively in base pairing with a AGGAGGAGG base sequence close to the 3' end of the DNA chain GP27 through Watson-Crick and Hoogsteen to form a stem part of the triple-helix DNA molecular switch probe.
5. A method for preparing a triple-helix DNA molecular switch probe according to any one of claims 1 to 4, which comprises the following steps:
and (3) uniformly mixing the DNA chain GP27 and the DNA chain AP9 in PBS buffer solution with the pH value of 6.2, hybridizing at normal temperature for 30 minutes, assembling to obtain the triple-helix DNA molecular switch probe, and storing at normal temperature.
6. The method for preparing a triple-helix DNA molecular switch probe according to claim 5, wherein: the ratio of the amounts of the substances of the DNA strand AP9 to the DNA strand GP27 was equal to 1.8: 1.
7. A method for colorimetric rapid detection of a target object OTA to be detected by using a triple helix DNA molecular switch probe is characterized by comprising the following steps:
in PBS buffer solution, the mixture isUniformly mixing the triple-helix DNA molecular switch probe solution with a sample solution containing the target object OTA to be detected to obtain a reaction solution, and reacting for 30 minutes at 25 ℃; then adding HEPES buffer solution and hemin into the reaction solution, uniformly mixing to obtain a detection reaction solution, and reacting for 30 minutes at normal temperature; finally, 2-azino-bis- (3-ethylbenzodihydro thiazoline-6-sulfonic acid) diammonium salt and H are added into the detection reaction solution2O2The solution develops color.
8. The use of the triple-helical DNA molecular switch probe of claim 1, wherein: the method is applied to OTA colorimetric rapid detection.
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