CN112816682B - 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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Abstract
The invention discloses a triple helix DNA molecular switch probe and application thereof in colorimetric rapid detection of OTA. The triple helix DNA molecular switch probe is mainly formed by hybridization and assembly of a DNA strand GP27 which is rich in G bases and forms a parallel G-quadruplex structure and can improve the peroxidase activity of G-quad-hemin and a DNA strand AP9 containing an ochratoxin A (OTA) aptamer sequence, and the triple helix DNA molecular switch probe formed by hybridization and assembly is applied to OTA colorimetric rapid detection. The triple helix DNA molecular switch probe can be used for sensitive and quick colorimetric detection of OTA, improves the detection sensitivity, avoids using DNA amplification or nano material to amplify detection signals, does not need labeling, has no steps of separation, cleaning and the like in the detection process, greatly simplifies the detection steps while maintaining the detection sensitivity, has simple and quick 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 colorimetric rapid detection of ochratoxin A (OTA).
Background
Ochratoxin a (OTA) is a secondary metabolite produced by toxic fungi such as aspergillus and penicillium. Studies show that OTA has nephrotoxicity and hepatotoxicity, can cause DNA damage and inhibit synthesis of RNA and protein, has teratogenic, carcinogenic and mutagenic effects, and is listed as a 2B-type carcinogen by the cancer research of the world health organization. Agricultural products are easily contaminated with ochratoxins during harvesting, storage, transportation and processing, and the agricultural products (foods) which are easily contaminated include cereals, beans, nuts, spices, coffee beans, cocoa, wine, beer and the like. In 2013, in one domestic investigation, the OTA detection rate in 77 parts of red wine is up to 57%, and the highest value is 5.65 mug/kg. In 2010, a study of the national academy of medical science shows that 30 of 57 Chinese medicinal materials detected OTA. In 2019, hajok et al detected 473 food samples available in the Bond Cyriluya market, and the results showed that 22% of the samples were contaminated with OTA, with the highest OTA content in raisins reaching 34.0 μg/kg, exceeding 3.5 times. The researches show that the OTA has high toxicity, wide distribution and easy 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 the monitoring of OTA pollution so as to reduce the influence of OTA on human and animal health to the greatest extent. At present, the conventional detection method of the 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, which are disadvantageous for point-of-care testing (POCT) of large amounts of samples, limiting their application and popularization. Antibody-based immunoassays, such as: the ELISA method and the immune test strip have high detection flux and high speed, are also commonly applied to detection of OTA, but the OTA has low immunogenicity, and the antibody is difficult, time-consuming and expensive to develop and is easy to generate false positive. Therefore, development of a rapid, sensitive, easy to operate, and low-cost OTA detection method is still urgent.
DNA has long been considered as a substance for storing and transmitting vital genetic information and has been studied extensively. However, from a materials perspective, DNA can be found to have a number of unique physical, chemical properties, including sequence-editable, molecular recognition, binding, and catalytic chemical reactions, as compared to other synthetic polymers. In addition to the well-known classical double helix structure, DNA can fold to form various structures by intramolecular hydrogen bonding, base stacking, electrostatic interactions, metal ion complexation, etc., such as: specific three-dimensional structures such as a DNA triple helix (triple helix), a G-quadruplex (G-quadruplex), and an aptamer (aptamer). 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 recognition, combination, catalysis and the like. For example, a G-quadruplex structure formed from a particular G-rich base sequence may combine with hemin (hemin) to form a DNAzyme having peroxidase-like activity. The peroxidase activity of the DNase can be used for catalyzing, generating and amplifying detection signals, and improving the detection sensitivity. While DNA aptamers can form stable structures, and can specifically recognize and bind to a target in a similar manner to antibodies. DNA aptamers offer some distinct advantages over antibodies, such as: good chemical stability, low production cost, small batch-to-batch difference and the like.
In recent years, with the development of various nano materials and DNA amplification technologies, researchers amplify detection signals by utilizing DNase and an aptamer and combining the DNA amplification technology and the nano materials, thereby providing a new method for rapid and sensitive detection of OTA. However, the preparation, modification and labeling and amplification of DNA of the nanomaterial make the detection operation steps complicated and complicated, raise the cost, and reduce the practicality.
Disclosure of Invention
In order to solve the problems, the invention designs a novel G-quadruplex structure forming sequence to improve the peroxidase activity of DNase, designs an aptamer sequence, assembles to form a triple helix DNA molecular switch probe, and develops a sensitive and rapid colorimetric detection technology of OTA. The technology does not need to amplify detection signals by using DNA amplification or nano materials, so that the used DNA chains do 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 maintained, the detection cost is low, and the technology has good application prospect.
The technical scheme adopted by the invention is as follows:
1. three-helix DNA molecular switch probe
The triple-helix DNA molecular switch probe is mainly formed by hybridization and assembly of a DNA strand GP27 capable of improving the peroxidase activity of G-quad-hemin and a DNA strand AP9 containing an ochratoxin A (OTA) aptamer sequence.
The DNA chain GP27 capable of improving the peroxidase activity of the G-quad-hemi is rich in G bases, and forms a parallel G-quad (G-quad) structure, wherein the base sequence is as follows: 5'-GGTGGTGGTGGTTGTGGAGGAGGAGGA-3', SEQ ID No.1. The 3' -end of the DNA strand GP27 is provided with an adenine base A, and the A base greatly enhances the peroxidase activity of G-quad-hemin, so that the detection signal is amplified without using DNA amplification or nano materials to amplify the detection signal, therefore, the used DNA strand does not need labeling, the detection process does not have steps of separation, cleaning and the like, and the detection step is greatly simplified while the detection sensitivity is maintained.
The DNA strand AP9 containing the OTA aptamer sequence has 36 base sequences in the middle, wherein the 36 base sequences can be selectively identified and combined with OTA, the sequences of the arm ends 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', SEQ ID No.2.
The triple-helix DNA molecular switch probe has a stem-loop structure, and is formed by assembling a TCCTCCTCC base sequence at the front arm end of a DNA strand AP9 and a CCTCCTCCT base sequence at the rear arm end of the DNA strand AP9 with a AGGAGGAGG base sequence, close to the 3' end, of a DNA strand GP27 through Watson-Crick and Hoogsteen base pairing; the assembled triple-helix DNA molecular switch probe has a stem-loop structure, 36 base sequences GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA in the middle of a DNA strand AP9 form a loop part of the triple-helix DNA molecular switch probe and are used for identifying and combining an object to be detected OTA, TCCTCCTCC base sequences at the front arm end of the DNA strand AP9 and CCTCCTCCT base sequences at the rear arm end of the DNA strand AP9 are respectively matched with AGGAGGAGG base sequences near the 3' end of a DNA strand GP27 through Watson-Crick and Hoogsteen base pairing to form a stem part of the triple-helix DNA molecular switch probe, and the DNA strand GP27 is locked.
Too short or too long an arm end sequence will reduce the sensitivity of the detection. When the arm end sequence is too short, the triple helix DNA molecular switch probe is unstable and easy to unwind, and the DNA strand GP27 is released, so that a higher background signal is generated, and the signal to noise ratio is reduced; when the arm end sequence is overlong, the triple helix DNA molecular switch probe is too stable, the object to be detected OTA 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 even impossible.
2. Preparation method of triple-helix DNA molecular switch probe
The method specifically comprises the following steps:
mixing the DNA strand GP27 and the DNA strand AP9 in PBS buffer solution with pH of 6.2, hybridizing for 30 min at normal temperature, assembling to obtain the triple helix DNA molecular switch probe for detecting the target object OTA, and preserving at normal temperature.
The ratio of the amounts of the substances of the DNA strand AP9 to the DNA strand GP27 is equal to 1.8:1. In principle, when the concentration ratio of the DNA strand AP9 to the DNA strand GP27 is 1:1, the three-helix DNA molecular switch probe is just assembled, and the DNA strand GP27 is completely locked. However, the assembly of the DNA strand AP9 and the DNA strand GP27 into the triple helix DNA molecular switch probe is a reversible chemical reaction, and analysis from the chemical reaction balance principle point of view, the increase of the quantity of the DNA strand AP9 is beneficial to promoting the combination and assembly of the DNA strand AP9 and the DNA strand GP27, so that the DNA strand GP27 is more completely combined, the free DNA strand GP27 is reduced, thereby reducing the background signal, further improving the signal to noise ratio when the probe detects the object to be detected, and generating higher sensitivity.
3. OTA rapid detection method based on triple-helix DNA molecular switch probe
The method specifically comprises the following steps:
uniformly mixing a triple helix DNA molecular switch probe solution with a sample solution containing an object to be detected OTA in PBS buffer solution to obtain a reaction solution, and reacting at 25 ℃ for 30 minutes; then adding HEPES buffer solution and Hemin (Hemin) into the reaction solution, uniformly mixing to obtain detection reaction solution, and reacting for 30 minutes at normal temperature; finally, adding into the detection reaction solution2, 2-azino-bis- (3-ethylbenzodihydrothiazoline-6-sulphonic acid) diammonium salt ABTS 2- And H 2 O 2 The solution developed. The higher the OTA content in the sample, the darker the green color of the detection reaction solution, and the accurate quantification can be realized by measuring the absorbance value of the detection reaction solution at 418nm by using an enzyme-labeled instrument or an ultraviolet-visible spectrophotometer. Along with the increase of the concentration of the object to be detected OTA, 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 rapidly detect OTA. The detection of the target object OTA to be detected is completed within 60 minutes, and the detection speed is faster than that of the existing national standard method liquid chromatography and enzyme-linked immunosorbent assay.
The absorbance value increasing rate of the detection reaction solution at 418nm is in positive linear correlation with the concentration of the target object OTA to be detected from 0.01 to 1.5mg/kg.
1) The method for rapidly detecting the OTA content in the reaction buffer solution comprises the following steps:
incubating a triple-helix DNA molecular switch probe assembled by a DNA strand AP9 and a DNA strand GP27 with OTA in PBS buffer solution for 30 minutes at 25 ℃, and combining the OTA with an aptamer sequence in the DNA strand AP9 to open the triple-helix DNA molecular switch probe and release the DNA strand GP27; then adding HEPES buffer solution and Hemin, wherein the DNA strand GP27 forms a G-quadruplex structure and forms DNase with peroxidase-like activity with Hemin; finally add ABTS 2- And H 2 O 2 DNase-catalyzed H 2 O 2 Oxidizing ABTS 2- Making the color of the solution become deep, 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 the OTA in the peanut oil of the actual sample comprises the following steps:
peanut oil samples are purchased from a local supermarket, and no OTA is detected by high performance liquid chromatography, so 3 grades of OTA with different concentrations are added into the peanut oil samples, and an addition recovery test is carried out to verify the accuracy and stability of detecting the OTA in an actual sample by a triple helix DNA molecular switch probe. The added sample is pretreated according to the national standard (GB 5009.96-2016) method, namely: extracting, separating, and filtering. And 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.
4. The triple helix DNA molecular switch probe is applied to the colorimetric rapid detection of OTA.
The invention designs a novel G-quadruplex structure forming sequence to enhance the peroxidase activity of DNase, so that the DNase catalyzes H 2 O 2 Oxidation substrate ABTS 2- 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 beneficial effects of the invention are as follows:
1) The present invention has designed a G-rich base sequence GP26 that can form a G-quadruplex structure, and has been developed by simply adding one A base at its 3' -end (i.e.: GP 27) greatly enhances the catalytic activity of dnase formed thereby enhancing the sensitivity of detection;
2) Through ingenious design, the three-helix DNA molecular switch probe is formed by assembling two DNA chains of GP27 and AP9 through Watson-Crick and Hoogsteen base pairing. When detecting the OTA, the probe and the OTA are uniformly mixed in a buffer solution and react for 30 minutes at room temperature, so that the subsequent color reaction can be carried out, and the detection is finished. The preparation of the detection probe and the transduction of the detection signal do not need DNA amplification or any nano material amplification of the detection signal, and the steps of separation, cleaning and the like, so that the method has low cost and very simple operation steps of 'mixing-detection', and can finish the rapid detection of the OTA;
3) The three-helix DNA molecular switch probe based on the invention can be used for rapidly detecting OTA within 60 minutes, the speed is far faster than that of a high performance liquid chromatography detection method, the linear range of detection is 0.01 to 1.5mg/kg, the detection limit is 0.004mg/kg, and the detection limit is lower than the maximum residue limit (primary grains: 0.005mg/kg, 0.01mg/kg of soluble coffee);
4) Based on the three-helix DNA molecular switch probe for rapidly detecting the OTA, the aptamer has good specificity, the detection probe only responds to the target OTA, and the detection of the OTA is not interfered when mycotoxins such as AFB1, AFB2, AFG1, OTB and the like exist in the sample;
5) The designed and assembled triple-helix DNA molecular switch probe has expansibility, the aptamer sequence of the AP9 is changed into the aptamer sequences of other objects to be detected, and after proper optimization, the triple-helix DNA molecular switch probe capable of specifically responding to different objects to be detected can be assembled.
In general, the triple helix DNA molecular switch probe can be used for sensitive and rapid colorimetric detection of OTA, improves the detection sensitivity, avoids the steps of using DNA amplification or nanomaterial amplification to detect signals, has no steps of marking, separating, cleaning and the like in the detection process, greatly simplifies the detection steps while maintaining the detection sensitivity, has simple and rapid detection, low cost and good application prospect.
Drawings
FIG. 1 is a schematic diagram of assembly of a triple helix DNA molecular switch probe and its use for OTA rapid detection;
FIG. 2 is a schematic diagram showing base pairing when AP9 and GP27 are assembled to form a triple helix DNA molecular switch probe;
FIG. 3 shows the enhancement of DNase catalytic activity and the response of a triple helix DNA molecular switch probe to an object to be detected OTA. 3 (A) is the characterization of A base to enhance the catalytic activity of DNase; 3 (B) is A base to enhance the catalytic activity of DNase and accelerate the generation rate of detection signals; 3 (C) is to verify 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 ultraviolet-visible absorption spectrum; 3 (D) is 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 circular dichroism characterization;
fig. 4 is a chart showing the colorimetric detection of a sample solution at the time of OTA. C is a positive control, only contains GP27, does not contain a triple helix DNA molecular switch probe and does not contain OTA; s1 to S7 are samples containing triple helix DNA molecular switch probes and OTA with different concentrations, wherein the concentration of the OTA is gradually increased from 0 to 2mg/kg;
FIG. 5 shows the result of absorption spectrum response and the result of specific detection of colorimetric rapid detection of OTA using a triple helix DNA molecular switch probe. 5 (A) is an ultraviolet-visible absorption spectrum response chart of three-helix DNA molecular switch probe colorimetric rapid detection OTA, 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 increase of the concentration of the OTA; 5 (B) is that the absorbance value increase percentage of the sample detection reaction solution at 418nm increases with the increase of the OTA concentration; 5 (C) is a linear regression equation fit graph of the percentage increase of absorbance value of the sample detection reaction solution at 418nm and the logarithmic value of the OTA concentration when the concentration of the OTA is 0.01 to 1.5 mg/kg; 5 (D) is the specificity of the three-helix DNA molecular switch probe colorimetric rapid detection OTA.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples. It should be understood that the preferred embodiments described herein are presented for purposes of illustration and explanation only and are not intended to limit the present invention.
1. Three-helix DNA molecular switch probe
The triple helix DNA molecular switch probe is mainly formed by hybridization and assembly of a DNA strand GP27 capable of improving the peroxidase activity of G-quad-hemin and a DNA strand AP9 containing an ochratoxin A (OTA) aptamer sequence.
A DNA strand GP27 capable of improving the peroxidase activity of G-quad-hemin is rich in G bases to form a parallel G-quadruplex (G-quad) structure, and the base sequence is as follows: 5'-GGTGGTGGTGGTTGTGGAGGAGGAGGA-3', SEQ ID No.1.
As shown in FIG. 3, the 3' -end of the DNA strand GP27 is provided with an adenine base A, and the A base greatly enhances the peroxidase activity of G-quad-hemin, so that the detection signal is amplified without using DNA amplification or nano materials to amplify the detection signal, therefore, the used DNA strand does not need labeling, the detection process does not need 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 A base enhancing DNase catalytic activity; FIG. 3 (B) shows that A base enhances the catalytic activity of DNase, and accelerates the generation rate of detection signals; FIG. 3 (C) is a graph showing the assembly of a triple helix DNA molecular switch probe and its response to OTA using UV-visible absorption spectroscopy; FIG. 3 (D) is a graph depicting, using circular dichroism, the assembly of a triple helix DNA molecular switch probe and its response to OTA;
a DNA strand AP9 containing an OTA aptamer sequence, wherein 36 base sequences capable of selectively recognizing and combining the OTA are arranged in the middle, the sequences of the arm ends at the front end and the rear end are sequences composed of 6 cytosine C bases and 3 thymine T bases, and the base sequences are as follows: 5'-TCCTCCTCCGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACCT CCTCCT-3', 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 Watson-Crick and Hoogsteen base pairing with a TCCTCCTCC base sequence at the front arm end of a DNA strand AP9 and a CCTCCTCCT base sequence at the rear arm end of the DNA strand AP9 respectively and a AGGAGGAGG base sequence near the 3' end of a DNA strand GP27; the assembled triple-helix DNA molecular switch probe has a stem-loop structure, 36 base sequences GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA in the middle of a DNA strand AP9 form a loop part of the triple-helix DNA molecular switch probe and are used for identifying and combining an object to be detected OTA, TCCTCCTCC base sequences at the front arm end of the DNA strand AP9 and CCTCCTCCT base sequences at the rear arm end of the DNA strand AP9 are respectively matched with AGGAGGAGG base sequences near the 3' end of a DNA strand GP27 through Watson-Crick and Hoogsteen base pairing to form a stem part of the triple-helix DNA molecular switch probe, and the DNA strand GP27 is locked.
Too short or too long an arm end sequence will reduce the sensitivity of the detection. When the arm end sequence is too short, the triple helix DNA molecular switch probe is unstable and easy to unwind, and releases the DNA strand GP27, thereby generating a higher background signal and reducing the signal to noise ratio; when the arm end sequence is overlong, the triple helix DNA molecular switch probe is too stable, the object to be detected OTA 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 even impossible.
2. Preparation method of triple-helix DNA molecular switch probe
The method specifically comprises the following steps:
mixing the DNA strand GP27 and the DNA strand AP9 in PBS buffer solution with pH of 6.2, hybridizing for 30 min at normal temperature, assembling to obtain the triple helix DNA molecular switch probe for detecting the target object OTA, and preserving 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 strand AP9 to the DNA strand GP27 is 1:1, the three-helix DNA molecular switch probe is just assembled, and the DNA strand GP27 is completely locked. However, the assembly of the DNA strand AP9 and the DNA strand GP27 into the triple helix DNA molecular switch probe is a reversible chemical reaction, and analysis from the chemical reaction balance principle point of view, the increase of the quantity of the DNA strand AP9 is beneficial to promoting the combination and assembly of the DNA strand AP9 and the DNA strand GP27, so that the DNA strand GP27 is more completely combined, the free DNA strand GP27 is reduced, thereby reducing the background signal, further improving the signal to noise ratio when the probe detects the object to be detected, and generating higher sensitivity.
3. OTA rapid detection method based on triple-helix DNA molecular switch probe
The method specifically comprises the following steps:
uniformly mixing a triple helix DNA molecular switch probe solution with a sample solution containing an object to be detected OTA in PBS buffer solution to obtain a reaction solution, and reacting at 25 ℃ for 30 minutes; then adding HEPES buffer solution and Hemin into the reaction solution, uniformly mixing to obtain detection reaction solution, and reacting for 30 minutes at normal temperature; finally adding ABTS into the detection reaction solution 2- And H 2 O 2 The solution developed. The higher the OTA content in the sample, the darker the green color of the detection reaction solution, and the accurate quantification can be realized by measuring the absorbance value of the detection reaction solution at 418nm by using an enzyme-labeled instrument or an ultraviolet-visible spectrophotometer. Along with the increase of the concentration of the object to be detected OTA, 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 rapidly detect OTA. The detection of the target object OTA to be detected is completed within 60 minutes, and the detection speed is faster than that of the existing national standard method liquid chromatography and enzyme-linked immunosorbent assay.
As shown in FIG. 5 (A), the rate of increase in absorbance at 418nm of the detection reaction solution is linearly positive with the concentration of the object to be measured OTA from 0.01 to 1.5mg/kg.
1) The method for rapidly detecting the OTA content in the reaction buffer solution comprises the following steps:
incubating a triple-helix DNA molecular switch probe assembled by a DNA strand AP9 and a DNA strand GP27 with OTA in PBS buffer solution for 30 minutes at 25 ℃, and combining the OTA with an aptamer sequence in the DNA strand AP9 to open the triple-helix DNA molecular switch probe and release the DNA strand GP27; then adding HEPES buffer solution and Hemin, wherein the DNA strand GP27 forms a G-quadruplex structure and forms DNase with peroxidase-like activity with Hemin; finally add ABTS 2- And H 2 O 2 DNase-catalyzed H 2 O 2 Oxidizing ABTS 2- Making the color of the solution become deep, 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 the OTA in the peanut oil of the actual sample comprises the following steps:
peanut oil samples are purchased from a local supermarket, and no OTA is detected by high performance liquid chromatography, so 3 grades of OTA with different concentrations are added into the peanut oil samples, and an addition recovery test is carried out to verify the accuracy and stability of detecting the OTA in an actual sample by a triple helix DNA molecular switch probe. The added sample is pretreated according to the national standard (GB 5009.96-2016) method, namely: extracting, separating, and filtering. And 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.
4. The triple helix DNA molecular switch probe is applied to the colorimetric rapid detection of OTA.
The invention designs a novel G-quadruplex structure forming sequence to enhance the peroxidase activity of DNase, so that the DNase catalyzes H 2 O 2 Oxidation substrate ABTS 2- 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.
Preferred embodiments of the present invention are as follows:
example 1
As shown in fig. 1, a triple helix DNA molecular switch probe for colorimetric rapid detection of an object to be detected OTA is assembled by using a DNA strand GP27 and a DNA strand AP 9.
mu.L of the DNA strand GP27 having a concentration of 10. Mu.M and 7.2. Mu.L of the DNA strand AP9 having a concentration of 10. Mu.M were taken in 1 XPBS buffer (20 mM PBS, 20mM NaCl, 2.5mM MgCl) at pH 6.2 2 ) And (3) 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 preserved at room temperature for standby.
Example 2
And (3) rapidly detecting the OTA in the aqueous solution by using a triple helix DNA molecular switch probe.
S1: 100. Mu.L of the three-helix DNA molecular switch probe solution assembled in example 1 was taken, 4. Mu.L of an OTA standard solution (methanol as solvent) of a certain concentration was added thereto, and the mixture was stirred uniformly and reacted at 25℃for 30 minutes.
S2: to the samples were added 20. Mu.L of 10 XHEPES buffer (250 mM HEPES, 200mM KCl, 2000mM NaCl, 0.05% Triton X-100, pH 5.3), 4. Mu.L of 10. Mu.M Hemin and 32. Mu.L of ultrapure water (each sample volume was made to be 160. Mu.L), and the mixture was homogenized and reacted at room temperature for 30 minutes.
S3: to the sample was added 20. Mu.L of ABTS at 20mM concentration 2- And 20. Mu.L of H at a concentration of 100mM 2 O 2 And (5) uniformly mixing the solutions. Transferring the sample to a 96-well plate, and adding H to the sample 2 O 2 The timing was started after the solution, and the absorbance value of the sample solution at 418nm was measured by using an enzyme-labeled instrument at the time of the reaction until the 7 th minute.
S4: steps S1 to S3 were repeated using different concentrations of OTA standard solution (methanol as solvent) with final OTA concentrations of 0.002, 0.01, 0.05, 0.1, 0.3, 0.5, 1, 1.5, 2, 3, 4mg/kg in sequence, 3 samples were taken in parallel for each concentration.
S5: with the concentration of OTA as the abscissa, the Rate of increase of absorbance value Increasing Rate% = ((A-A) 0 ) /A 0 ) X 100% on the ordinate (A, A) 0 And respectively representing absorbance values of the reaction solution when the sample contains OTA and does not contain OTA), and establishing an OTA detection working curve.
Fig. 4 is a chart showing the colorimetric detection of a sample solution at the time of OTA. C is a positive control, only contains GP27, does not contain a triple helix DNA molecular switch probe and does not contain OTA; s1 to S7 are samples containing triple helix DNA molecular switch probes and OTA with different concentrations, wherein the concentration of OTA is gradually increased from 0 to 2mg/kg.
Detection result:
as shown in FIG. 5 (B), as the final concentration of OTA increases 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, 4mg/kg in this order), the absorbance value of the sample solution at 418nm gradually increases.
As shown in FIG. 5 (C), the absorbance increase rate of the sample solution at 418nm is fitted to the concentration of OTA (0.01 to 1.5 mg/kg), to obtain a linear regression equation IR=0.484C+0.131, C represents the concentration of OTA, and the linear correlation coefficient r 2 0.997, a linear range of 0.01 to 1.5mg/kg was detected. 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, 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 5g peanut oil, placing into a centrifuge tube, adding 1g sodium chloride and 25mL methanol extract (V) Methanol ：V Water and its preparation method =80: 20 Shaking for 30 minutes, centrifuging at 6000rpm for 10 minutes, removing 15mL of the upper layer extract, adding 30mL of phosphoric acid buffer solution, mixing uniformly, filtering for 2 times through a glass fiber filter paper membrane, and preparing OTA sample solutions with the concentration of 0, 0.01, 0.05 and 0.1mg/kg respectively from the filtrate.
S2: 100. Mu.L of the three-helix DNA molecule switch probe solution assembled in example 1 was taken, 4. Mu.L of the OTA sample solution of the above-mentioned certain concentration was added thereto, and the mixture was mixed and reacted at 25℃for 30 minutes.
S3: to the samples 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 (each sample volume was made to be 160. Mu.L), and the mixture was homogenized and reacted at room temperature for 30 minutes.
S4: to the sample was added 20. Mu.L of ABTS at 20mM concentration 2- And 20. Mu.L of H at a concentration of 100mM 2 O 2 And (5) uniformly mixing the solutions. Transferring the sample into a 96-well plate from the sampleAdding H into 2 O 2 The solution was started to time and absorbance values of the sample solution at 418nm were measured with an enzyme-labeled instrument at 7 minutes of the reaction.
S5: and 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 OTA concentration, the addition recovery rate and the relative standard deviation in each sample solution.
S6: OTA solutions with different concentrations are used for adding and recycling, so that the adding concentration of 3 grades is respectively 0.01mg/kg, 0.05 mg/kg and 0.1mg/kg, and 3 parallel samples are prepared for each grade of concentration. Repeating the steps S2 to S4, and calculating a 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 implementation of the present invention is not limited to this example. All alternatives which have similar effects as proposed in accordance with the principles and concepts of the invention are considered to be within the scope of the invention.
The DNA sequence of the present invention is as follows:
SEQ ID No.1：
name: DNA strand GP27 base sequence
The source is as follows: artificial sequence (Artificial Sequence)
GGTGGTGGTGGTTGTGGAGGAGGAGGA
SEQ ID No.2：
Name: DNA strand AP9 base sequence
The source is as follows: artificial sequence (Artificial Sequence)
TCCTCCTCCGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACC TCCTCCT。
Sequence listing
<110> academy of agricultural sciences in Zhejiang province
<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 (6)
1. The triple-helix DNA molecular switch probe is characterized by being formed by hybridization and assembly of a DNA strand GP27 capable of improving the peroxidase activity of G-quad-hemin and a DNA strand AP9 containing an ochratoxin A aptamer sequence;
the DNA chain GP27 capable of improving the peroxidase activity of the G-quad-hemin is rich in G bases to form a parallel G-quadruplex structure, and the base sequence is as follows: 5'-GGTGGTGGTGGTTGTGGAGGAGGAGGA-3', SEQ ID No.1;
the DNA strand AP9 containing the OTA aptamer sequence has 36 base sequences in the middle, wherein the 36 base sequences can be selectively identified and combined with OTA, the sequences of the arm ends 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', SEQ ID No.2.
2. The triple-helical DNA molecular switch probe of claim 1, wherein: the triple-helix DNA molecular switch probe has a stem-loop structure, 36 base sequences GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA in the middle of a DNA strand AP9 form a loop part of the triple-helix DNA molecular switch probe, TCCTCCTCC base sequences at the front arm end of the DNA strand AP9 and CCTCCTCCT base sequences at the rear arm end of the DNA strand AP9 respectively form a stem part of the triple-helix DNA molecular switch probe with AGGAGGAGG base sequences near the 3' end of a DNA strand GP27 through Watson-Crick and Hoogsteen base pairing.
3. A method for preparing a triple helix DNA molecular switch probe according to any one of claims 1-2, comprising the steps of:
mixing the DNA strand GP27 and the DNA strand AP9 in PBS buffer solution with pH of 6.2, hybridizing for 30 min at normal temperature, assembling to obtain the triple helix DNA molecular switch probe, and preserving at normal temperature.
4. The method for preparing a triple helix DNA molecular switch probe according to claim 3, wherein: the ratio of the amounts of the substances of the DNA strand AP9 to the DNA strand GP27 is equal to 1.8:1.
5. A method for colorimetric rapid detection of an object to be detected OTA using the triple helix DNA molecular switch probe according to any one of claims 1 to 2, comprising the steps of:
uniformly mixing a triple helix DNA molecular switch probe solution with a sample solution containing an object to be detected OTA in PBS buffer solution to obtain a reaction solution, and reacting at 25 ℃ for 30 minutes; then adding HEPES buffer solution and hemin into the reaction solution, uniformly mixing to obtain detection reaction solution, and reacting for 30 minutes at normal temperature; finally, adding 2, 2-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and H into the detection reaction solution 2 O 2 The solution developed.
6. Use of the triple helix DNA molecular switch probe according to claim 1, wherein: the application in the colorimetric rapid detection of OTA.
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