CN117265079A - P-ERCA-based ochratoxin A visual detection method and kit - Google Patents
P-ERCA-based ochratoxin A visual detection method and kit Download PDFInfo
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Abstract
The invention discloses a P-ERCA-based ochratoxin A visual detection method and a kit, wherein the method comprises the following steps: ochratoxin a is specifically bound with a specific optimized aptamer thereof, so that a specific primer sequence partially complementary to the aptamer is released, and further, high-sensitivity padlock-type exponential rolling circle amplification is initiated. The single-chain amplification product has a specific enzyme cutting site to form amplification and cutting effects, and the cut short chain can further excite the hanging lock type level rolling ring amplification to form exponential amplification. And then, visually detecting the cut single-stranded DNA product subjected to exponential amplification by using a signal probe marked by colloidal gold and a test strip fixed with a detection probe. According to the invention, the ochratoxin A is used for identifying and inducing the exponential roller amplification to amplify the signal, and is combined with the test strip for the first time, so that the high-sensitivity and visual detection of the ochratoxin is realized, and the method has the advantages of low cost, convenience in operation, high sensitivity, good specificity, short detection time and the like.
Description
Technical Field
The invention relates to a detection method of ochratoxin A, in particular to a padlock-based visual detection method and a kit of ochratoxin A by exponential rolling circle amplification (P-ERCA), and belongs to the technical field of biochemical detection.
Background
Ochratoxin a (OTA) is widely found in crops such as barley, corn, oat and soybean contaminated with mold, and when the animal ingests the moldy feed, ochratoxin a accumulates in the animal and enters the human body along with the processed bacon product to attack the liver and kidney, and also damages the immunity of the human body. Because ochratoxin A has the strongest toxicity, the widest distribution and the closest relation with human health in mycotoxin, the OTA trace detection method with high sensitivity has great significance for monitoring the quality safety of animal feed and protecting human health.
At present, the instrument method for detecting ochratoxin A in grains and feeds mainly comprises an instrument method and a test strip method. The instrument methods comprise high performance liquid chromatography, gas chromatography, mass spectrometry, fluorescence photometry and the like, the methods have higher sensitivity and stable and repeatable results, but the required instruments are more expensive, the pretreatment steps of the samples are complicated, the time is long, the method is not suitable for the field detection of a large number of samples to be detected, and the application of the method is limited. The test strip method is mainly a flow direction test strip method, and in practice, the principle of an immunoassay is mainly applied, namely, the high specificity of antigen and antibody is adopted to detect ochratoxin A, but the time required for preparing the antibody is long and the price is high, so that the requirements of low cost, high flux and the like required by on-site detection are difficult to meet.
Disclosure of Invention
The invention mainly aims to provide a P-ERCA-based ochratoxin A visual detection method, which has the advantages of low cost, convenience in operation, high sensitivity, good specificity, short detection time and the like, thereby overcoming the defects of the prior art.
The invention also provides a P-ERCA-based ochratoxin A visual detection kit.
One aspect of the invention provides a P-ERCA-based ochratoxin A visual detection method, which comprises the following steps:
s1, enabling a first modifier modified on an ochratoxin A aptamer to be specifically combined with a second modifier fixed on a solid carrier, and then enabling the ochratoxin A aptamer to be hybridized and combined with a single-stranded DNA primer subjected to affinity and competitive release capacity screening to obtain a first liquid-phase reaction system;
s2, adding ochratoxin A into the first liquid-phase reaction system, and enabling the ochratoxin A to be specifically combined with the ochratoxin A aptamer, so that at least part of the single-stranded DNA primer is separated from the ochratoxin A aptamer and is free in the liquid-phase reaction system, and a second liquid-phase reaction system is obtained;
s3, separating supernatant from the second liquid phase reaction system, constructing a padlock type exponential rolling circle amplification reaction system based on the supernatant, and performing padlock type exponential rolling circle amplification to obtain a third liquid phase reaction system containing target DNA;
s4, detecting the third liquid phase reaction system by using a signal probe modified with a chromogenic label and a test strip fixed with a detection probe, thereby realizing the visual detection of ochratoxin A, wherein the nucleotide sequences of the signal probe and the detection probe are respectively complementary with part of the nucleotide sequence of the target DNA.
The invention provides a P-ERCA-based ochratoxin A visual detection kit, which comprises an ochratoxin A aptamer, a single-stranded DNA primer, a padlock-type exponential rolling circle amplification reaction component, a signal probe modified with a chromogenic mark, a test strip and the like; the padlock type exponential rolling circle amplification reaction component comprises DNA ligase, deoxynucleotide triphosphate, DNA polymerase and padlock type template probes; and a detection probe and a quality control probe are respectively fixed on a detection line and a quality control line of the test strip.
Compared with the prior art, the method and the device for detecting ochratoxin by utilizing the exponential roller amplification amplify the signal, combine the signal with the test strip for the first time, realize high-sensitivity and visual detection of ochratoxin, and have the advantages of low cost, convenience in operation, high sensitivity, good specificity, short detection time and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram of an ochratoxin A visualization detection method in some embodiments of the invention;
FIG. 2 is a feasibility test result of a visual inspection method of ochratoxin A according to an embodiment of the invention;
FIG. 3 is a sensitivity test result of an ochratoxin A visual inspection method according to an embodiment of the invention;
FIG. 4 shows the results of a specific assay for ochratoxin A visual inspection in accordance with one embodiment of the invention;
FIG. 5 is a graph showing OTA competition value assays for different single stranded DNA primers complementary to ochratoxin aptamers according to one embodiment of the invention.
Detailed Description
Aiming at the defects of the existing ochratoxin A detection technology, the inventor of the present invention has provided a technical scheme through long-term research and a large amount of practice, and mainly discloses a padlock-based method for detecting ochratoxin A by using exponential rolling circle amplification and gold-labeled test paper strip visualization, which has the advantages of good specificity, high sensitivity, simple and convenient operation and low detection cost, and meets the requirements of on-site rapid detection. The technical scheme, the implementation process, the principle and the like are further explained as follows.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as described herein, and therefore the scope of the present invention is not limited to the specific embodiments disclosed below.
Some embodiments of the present invention provide a method for detecting ochratoxin A based on P-ERCA, comprising:
s1, enabling a first modifier modified on an ochratoxin A aptamer to be specifically combined with a second modifier fixed on a solid carrier, and enabling the ochratoxin A aptamer to be hybridized and combined with a single-stranded DNA primer to obtain a first liquid-phase reaction system;
s2, adding ochratoxin A into the first liquid-phase reaction system, and enabling the ochratoxin A to be specifically combined with the ochratoxin A aptamer, so that at least part of the single-stranded DNA primer is separated from the ochratoxin A aptamer and is free in the liquid-phase reaction system, and a second liquid-phase reaction system is obtained;
s3, separating supernatant from the second liquid phase reaction system, constructing a padlock type exponential rolling ring amplification reaction system based on the supernatant, and performing padlock type exponential rolling ring amplification, wherein an amplification product is rich in enzyme cutting sites, and can form an amplification and cutting effect at the same time, so that a third liquid phase reaction system containing target DNA is obtained, wherein the target DNA can continuously participate in excitation padlock type exponential rolling ring amplification.
S4, detecting the third liquid phase reaction system by using a signal probe modified with a chromogenic label and a test strip fixed with a detection probe, thereby realizing the visual detection of ochratoxin A, wherein the nucleotide sequences of the signal probe and the detection probe are respectively complementary with part of the nucleotide sequence of the target DNA.
In one embodiment, the nucleotide sequence of the ochratoxin A aptamer is shown in SEQ ID NO. 1. The nucleotide sequence of the single-stranded DNA primer is shown as SEQ ID NO.2, and is specifically designed and optimized, so that the sensitivity of the method and the specificity of downstream padlock type exponential rolling circle amplification can be ensured.
In one embodiment, the first modification comprises biotin and the second modification comprises streptavidin.
In one embodiment, the solid support is an inner wall of a vessel for containing the first liquid phase reaction system. The vessel may be a PCR tube or the like. In some cases, the immobilization carrier may also be, for example, a magnetic microsphere or the like.
In one embodiment, the padlock-type exponential rolling circle amplification reaction system further comprises a DNA ligase, a deoxynucleotide triphosphate, a DNA polymerase, a padlock-type template probe, and the like. The padlock-type exponential rolling circle amplification reaction system may also comprise other conventional components such as buffer salts, as will be appreciated by those skilled in the art.
In one embodiment, the 5' end of the padlock type template probe is modified with a phosphate group, so that the 5' end and the 3' end of the padlock type template probe are connected into a ring through DNA ligase under the condition of primer complementation pairing combination. Furthermore, three nicking endonuclease cleavage sites are designed in the nucleotide sequence of the padlock type template probe, and can be specifically identified under the condition of generating double chains by rolling circle amplification, so that enzyme cleavage reaction occurs. Compared with a padlock type probe containing less than two enzyme cutting sites, the padlock type template probe can remarkably improve the amplification efficiency, thereby effectively improving the detection sensitivity and specificity.
Preferably, the nucleotide sequence of the padlock type template probe is shown as SEQ ID NO. 3.
In one embodiment, the nucleotide sequence of the signaling probe is shown as SEQ ID NO. 4.
In one embodiment, the chromogenic tag comprises a nano gold particle, or colloidal gold, which may be provided in a particle size of 20 to 35nm. For example, a thiol group may be modified at the 5' end of the detection probe, and the thiol group may be used to covalently link the gold nanoparticle to the detection probe, thereby making the detection probe also referred to as a gold-labeled probe.
In one embodiment, the test strip has a detection line (T line) and a quality control line (C line), the detection line is fixed with the detection probe (also referred to as T line probe), the quality control line is fixed with the quality control probe (also referred to as C line probe), the nucleotide sequence of the detection probe is shown as SEQ ID No.5, and the nucleotide sequence of the quality control probe is shown as SEQ ID No. 6.
Further, the test strip may be a test strip commonly used in the art, and may include conventional components such as a sample absorbing pad, a gold-labeled pad, a nitrocellulose membrane, a water absorbing pad, and a bottom plate. The nitrocellulose membrane is coupled with a T line probe and a C line probe, and the gold label pad is coupled with a gold label probe. In some cases, the sample absorption pad, the gold-labeled pad, the nitrocellulose membrane and the water absorption pad are sequentially stuck on the bottom plate, and the gold-labeled pad is covered under the sample absorption pad in the length direction by about 1/3-1/2, and sequentially assembled into the test strip.
In one embodiment, the 3' -end of the detection probe is modified with biotin, which can be bound to streptavidin and thus immobilized on the T-line, and the detection probe can specifically bind to the target DNA. More specifically, the target DNA is a padlock-type exponential rolling circle amplified single-stranded DNA product, a part of which is complementary to the detection probe and the other part of which is complementary to the signaling probe.
In one embodiment, the 5' end of the quality control probe is modified with biotin, which can be combined with streptavidin to be fixed on a C line, and the quality control probe is specifically combined with the signal probe.
In one embodiment, the ochratoxin a is isolated from contaminated cereal grains.
Referring to fig. 1, the visual detection method of the present invention mainly includes an amplification step and a detection step. Wherein the amplification step comprises two parts, namely aptamer competition and primer priming padlock-type exponential rolling circle amplification (P-ERCA).
The aptamer competition step refers to that ochratoxin A competes for part of complementary strands (primers) of the aptamer, dropped single-stranded DNA primers are suspended in a competition system, and the primers are sucked to trigger subsequent amplification reaction. Illustratively, after adding a biotin-modified ochratoxin a aptamer to a PCR tube having streptavidin immobilized thereon, the aptamer is immobilized in the PCR tube due to the specific binding of biotin to streptavidin, and then when a single-stranded DNA primer having a nucleotide sequence complementary to a part of the nucleotide sequence of the aptamer is added thereto, the aptamer hybridizes to the single-stranded DNA primer, and then when ochratoxin a is added to the PCR tube, the aptamer specifically binds to ochratoxin a, and at least a part of the single-stranded DNA primer is competed for release in the reaction system.
The padlock-type exponential rolling circle amplification step initiated by the primer comprises the processes of connecting into a circle, rolling circle amplification and repeatedly alternating nicking by nicking enzyme. Specifically, the process of ligation into a loop is to ligate padlock type template probes into a circular DNA by DNA ligase under the combination of single-stranded DNA primers, so as to trigger isothermal amplification. Then, under the existence of padlock type template probes, a large amount of amplified single-stranded products are nicked by nicking endonuclease, and under the action of DNA polymerase, single-stranded products (namely target DNA) are continuously amplified, so that exponential rolling circle amplification is realized.
Illustratively, the ochratoxin aptamer is modified at the 5 'end with biotin, which binds to a streptavidin-immobilized PCR tube, and the primer is partially complementary to the aptamer 3' end sequence. In the ochratoxin recognition stage, the ochratoxin is specifically combined with an aptamer, the primer is competed down and is specifically combined with the 5 'end and the 3' end of a padlock type template probe, in the ligation reaction stage of isothermal amplification (RCA), DNA ligase is used for ligating the 5 'end and the 3' end of the padlock type template probe together, and the padlock type template probe is ligated into a ring; in the RCA extension stage, DNA polymerase synthesizes a new chain along the direction from 5 'to 3' of the padlock type template probe chain, when the nicking endonuclease reaches the recognition site of the nicking endonuclease, nicking reaction occurs, amplification is continued under the action of the DNA polymerase, and the dropped single-chain fragment is continuously combined with the ring type padlock type template probe to perform further amplification-nicking-amplification circulation, so that exponential amplification is realized.
Wherein, the working concentration of the ochratoxin aptamer can be 1-100 nM; the working concentration of the single-stranded DNA primer can be 1-100 nM, and the volume concentration ratio of the single-stranded DNA primer to the ochratoxin aptamer can be 1:1, a step of; the working concentration of the padlock type template probe can be 0.1-1 mu M.
Wherein, the reaction procedure of isothermal amplification may comprise: the ligation reaction was carried out at about 22℃for about 30min, and the cleavage reaction was carried out at about 30℃for about 4 h.
And after the amplification step is finished, analyzing and detecting the amplified product by using a test strip, and obtaining a detection result of the sample to be detected according to the depth degree of the T line of the test strip, thereby realizing the visual detection of the sample to be detected.
Some embodiments of the present invention provide a P-ERCA-based ochratoxin A visual detection kit comprising:
the nucleotide sequence of the ochratoxin A aptamer is shown as SEQ ID NO. 1;
a single-stranded DNA primer, the nucleotide sequence of which is shown as SEQ ID NO. 2;
the padlock type exponential rolling circle amplification reaction assembly comprises DNA ligase, deoxynucleotide triphosphate, DNA polymerase and padlock type template probes, wherein the nucleotide sequence of the padlock type template probes is shown as SEQ ID NO. 3;
the nucleotide sequence of the signal probe modified with the chromogenic label is shown as SEQ ID NO. 4;
the test strip is characterized in that a detection probe and a quality control probe are respectively fixed on a detection line and a quality control line of the test strip, the nucleotide sequence of the detection probe is shown as SEQ ID NO.5, and the nucleotide sequence of the quality control probe is shown as SEQ ID NO. 6.
In one embodiment, the chromogenic label comprises a nanoparticle of gold covalently bonded to the 5' end of the signaling probe.
In one embodiment, the test strip comprises a bottom plate, and a sample absorption pad, a gold-labeled pad, a nitrocellulose membrane, a water absorption pad and the like which are sequentially stuck on the bottom plate. The upper end surface of the bottom plate can also be considered to comprise a loading area, a marking area, a scribing area and a water absorbing area which are sequentially arranged along the set direction. The labeling zone is combined with a signal probe modified with a chromogenic label, such as a signal probe coupled with nano gold particles, namely the gold-labeled probe. And a detection line and a quality control line are arranged in the scribing area, and the detection line and the quality control line are respectively combined with a biotin modified detection probe and a biotin modified quality control probe through streptavidin.
Wherein, the material of the bottom plate can comprise plastics and the like, the material of the sample loading area can comprise glass fiber or polyester film and the like, and the material of the scribing area can comprise nitrocellulose film or pure cellulose film and the like; the material of the water absorption area can comprise filter paper and the like.
Illustratively, a method of preparing the test strip may include:
coupling the signal probe modified by sulfhydryl group with colloidal gold to prepare the colloidal gold coupled with the probe, namely the gold-labeled probe;
applying a gold-labeled probe to a gold-labeled region, and respectively coupling a biotin-modified detection probe and a quality control probe with streptavidin to prepare a detection line and a quality control line of a scribing region;
and sequentially arranging the loading area, the gold marking area, the scribing area and the water absorbing area on the bottom plate along the set direction.
Wherein, the working concentration of the gold-labeled probe can be 1.0-2.0 mu M; the particle size of the colloidal gold can be 20-35 nm; the concentration of the streptavidin can be 2.5-5.0 mg/mL; the working concentration of the T line probe is 0.2-0.8 mu M; the working concentration of the C line probe is 0.2-0.5 mu M.
The qualitative detection limit of the visual detection method is 0.0001-10000pg/mL. Compared with the existing instrument method or test strip method for detecting OTA, the method of the invention gets rid of the dependence on expensive instruments (such as fluorescent quantitative PCR instrument and the like) and precise operation, simplifies the detection means of OTA, improves the sensitivity, has lower cost, is more convenient and fast, has higher sensitivity and stronger specificity, effectively improves the detection accuracy, and can fully meet the detection requirements of on-site and high flux.
The technical scheme of the present invention is further described in detail below with reference to several preferred embodiments and the accompanying drawings, the embodiments are implemented on the premise of the technical scheme of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.
Unless otherwise indicated, all of the experimental materials used in the examples below were purchased from conventional biochemistry reagent companies. The nucleotide sequences of the ochratoxin A aptamer, the single-stranded DNA primer 1-primer 6, the padlock type template probe, the colloidal gold modified signal probe, the detection probe and the quality control probe are shown in the following table 1.
TABLE 1 nucleotide sequences of partial reagents used in the examples of the present invention
Example 1 an ochratoxin a visual detection method comprises the following steps:
(1) Immobilization of aptamer-primer complexes on PCR tubes:
20. Mu.L of 0.8wt% glutaraldehyde was added to a PCR tube and incubated for 5 hours at 37℃followed by aspiration of glutaraldehyde, washing with ultrapure water for 3 times, followed by addition of 10. Mu.g/mL of streptavidin and incubation for 2 hours at 37℃followed by aspiration of streptavidin, washing with PBST solution for 2 times, followed by addition of 2. Mu.L of 1. Mu.M 5' -end modified biotin aptamer and 18. Mu.L of 50mM PBS, shaking incubation for 2 hours at room temperature, followed by aspiration of aptamer mixture, washing with PBST solution for 3 times, followed by addition of 2. Mu.L of 1. Mu.M single-stranded DNA primer 1 (hereinafter referred to as primer 1) and 18vL binding solution (containing 10mM Tris-HCl,120mM NaCl,10mM KCl,20mM MgCl) 2 0.05% PEG 1000, pH 7.4), incubating at 37deg.C for 1 hr, sucking out unbound primer mixture, adding PBST solution, washing for 4 times to obtain aptamer-primer complex immobilized on PCR tube wall, adding 20 μl of 20wt% sucrose solution for activity protection, and placing in a refrigerator at 4deg.C for use.
(2) Competition reaction of primers in PCR tube: and (3) sucking redundant sucrose solution into the PCR tube coupled with the aptamer-primer complex obtained in the step (1), adding 10 mu L of OTA aqueous solution with different concentrations (0, 0.001pg/mL, 0.01pg/mL, 0.1pg/mL, 10pg/mL, 1ng/mL and 10 ng/mL) and 10 mu L of binding solution respectively, uniformly mixing at room temperature for 1h, performing primer competition reaction, sucking 2 mu L, and performing subsequent padlock-type exponential rolling circle amplification. Wherein, the OTA aqueous solution is formed by dissolving OTA in DEPC water.
(3) Hanging lock type exponential rolling circle amplification (P-ERCA)
Firstly, adding 2 mu L of the supernatant containing the primer 1 in the step (2) respectively, adding 1 mu L of padlock type template probes, 2 mu L of 10 xT 4 DNA ligase buffer solution and 1 mu L T4 DNA ligase (5U/. Mu.L), and incubating at 22 ℃ for 30min to form a rolling circle system; secondly, adding 2 mu L of 25mM dNTPs, 1 mu L of Phi 29DNA polymerase (1000U/mL), 2 mu L of Phi 29DNA polymerase buffer solution, 1 mu L of Nb.BbvCI nicking endonuclease (1000U/mL) and 2 mu L of rCut Smart, supplementing the system to 20 mu L by DEPC water, and incubating for 4 hours at 37 ℃, wherein the rolling circle amplification is triggered to generate a large amount of single-stranded DNA with nicking endonuclease recognition sites, and short target DNA is formed under the action of the nicking endonuclease, and the rolling circle amplification is continuously participated, so that the exponential increase of the number of the target DNA is realized; finally, the mixture was heated at 65 ℃ for 10min to inactivate the enzymes.
Wherein the composition of the P-ERCA reaction system is shown in Table 2 (20. Mu.L).
TABLE 2 composition of the P-ERCA reaction System in this example
| Component (A) | Dosage of |
| T4 DNA ligase | 5U |
| 10×T4 DNA ligase buffer | 2μL |
| dNTPs | 2.5mM |
| Phi 29DNA polymerase | 0.5U |
| Phi 29DNA polymerase buffer | 2μL |
| Nb.BbvCI nicking endonucleases | 0.5U |
| rCutSmart | 2μL |
| DEPC pure water | 5μL |
| Hanging lock type template probe | 1μL |
| Primer(s) | 2uL |
(4) Preparing colloidal gold dispersion liquid:
the conical flask is soaked in aqua regia, and the double distilled water is repeatedly boiled and cleaned, and then is dried in an oven for standby; during preparation, a conical flask is filled with about 200mL of deionized water, 2.55mL of chloroauric acid solution with the mass fraction of 0.5% and a stirrer, the mixture is stirred at a medium speed and then is kept stand, 3mL of trisodium citrate solution with the mass fraction of 1% is added when the mixture is heated to micro-boiling, the mixture is stirred while being heated until the color of the solution is changed from dark purple to reddish wine, and when the color is no longer changed, the heating is turned off, and the stirring is continued for 5min to complete the reaction; finally, preparing colloidal gold dispersion liquid, and preserving at 4 ℃ for standby.
(5) Preparing a colloidal gold-labeled signaling probe:
adding 1mL of prepared colloid Jin Fensan solution and 4 mu L of 10% Tween 80 into each 1.5mL of centrifuge tube, shaking and incubating for 30min, centrifuging at 13000rpm for 15min, discarding supernatant, and adding 100 mu L of deionized water for redissolution; 10 mu L of a colloidal gold-labeled signaling probe and 5 mu L of 1mM TCEP are respectively added into each 5mL glass bottle for activation, and the mixture is kept stand for 1h at room temperature; adding the re-dissolved colloid Jin Fensan solution and 100 mu L of PBS solution into each glass bottle, uniformly mixing, and placing the mixture in a water bath kettle at 50 ℃ for water bath for 2 hours; 100. Mu.L of 100. Mu.M dATP was added to each flask for 1h at room temperature; after the coupling is completed, the mixture is centrifuged at 10000rpm for 10min, the supernatant is discarded, the volume is restored to 1/10 of the original volume by using the heavy suspension, the colloidal gold-labeled signaling probe compound is formed, and 6 mu L of the colloidal gold-labeled signaling probe compound is dripped into a gold-labeled pad for drying for standby.
(6) Preparation of detection line and quality control line probe:
6 mu L T line probe, 2.5 mu L of 5mg/mL streptavidin and 2 mu L C line probe, 2.5 mu L of 5mg/mL streptavidin are respectively added with 50mM PBS to 20 mu L, and the mixture is rotated and incubated for 2h in a dark place; and respectively and uniformly spraying the coupled T line probe and C line probe onto the nitrocellulose membrane at a spraying speed of 2cm/s, and drying in a baking oven at 30 ℃ for later use.
(7) And (3) after the step (3) is finished, dripping each P-ERCA reaction amplification product into the sample loading area of the test strip prepared in the step (6), and observing the color development conditions of the T line and the C line on the test strip to realize detection of OTA.
Referring to fig. 2, when the sample does not contain OTA (the OTA concentration is 0, and is actually DEPC water, namely, a negative control group), the primer competition reaction cannot be performed, the primer cannot appear in the solution, the subsequent exponential rolling circle amplification cannot be initiated, and the detection line T line has no signal response; when OTA, especially OTA with a concentration above 0.01pg/mL, is present in the sample, the primer competition reaction proceeds normally, the primer appears in the solution, the subsequent exponential rolling circle amplification is initiated, and the T line has a signal response.
Meanwhile, the detection result of the sample can be judged by observing the color depth of the detection line on the test strip by naked eyes. Specifically, referring to FIG. 3, when the OTA concentration in the sample is 0.01pg/mL, the color development intensity of the T line is significantly enhanced compared with that of the negative result. The detection method of the embodiment has good sensitivity. Moreover, as the concentration of OTA in the sample increases gradually, the color development intensity of the T line increases gradually, which means that the detection method of the embodiment is also suitable for quantitative detection of samples containing concentrations of 0.01 pg/mL-10 ng/mLOTA.
In addition, to test the selectivity of the detection method of this example, 10 μl of the OTA aqueous solution in step (2) was replaced with 10 μl of 5ng/mL AFM1, ZEN, DON, OTB, OTC, mixture and OTA aqueous solution, respectively, as positive control, and 10 μl of DEPC water was used as negative control, and experiments were performed using the test strips described above, and the experimental results are shown in fig. 4. It can be seen that when the sample does not contain OTA, the T line of the test strip has no signal response, which indicates that the detection method of the embodiment has good specificity for OTA, and the final result can be directly observed by naked eyes, thereby being beneficial to improving the accuracy and convenience of OTA detection.
Examples 2 to 6
The method for detecting ochratoxin A in the embodiments 2 to 6 is basically the same as that in the embodiment 1, and the difference is that: the primer 1 was replaced with the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6, respectively. Wherein under the reaction conditions provided in example 1, the Tm values of the DNA double strand formed by binding the primer 1 to the primer 6 and the aptamer are 62.3 ℃, 60.2 ℃, 62.3 ℃, 66.9 ℃, 60.2 ℃, 52.4 ℃, respectively, and the OTA competition value F/F 0 370.6, 275, 274.4, 151, 109.1, 26, F/F respectively 0 Is measured when OTA was added to the reaction system at a concentration of 0.01 pg/mL. The test results of examples 2 to 6 show that the specificity and sensitivity for detection of OTA are far inferior to those of example 1 when primers 2 to 6 are used.
In addition, the inventors designed a series of other primers by using Primer Premier software, and also conducted experiments in the manner of example 1, and as a result, it was revealed that when these primers were used, the visual detection effect of high sensitivity and high specificity for OTA could not be obtained at the same time.
While the present application has been described with reference to an illustrative embodiment, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the present application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the application without departing from the scope thereof. Therefore, it is intended that the present application not be limited to the particular embodiments disclosed for carrying out this application, but that the application will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A P-ERCA-based ochratoxin A visual detection method is characterized by comprising the following steps of:
s1, enabling a first modifier modified on an ochratoxin A aptamer to be specifically combined with a second modifier fixed on a solid carrier, and enabling the ochratoxin A aptamer to be hybridized and combined with a single-stranded DNA primer to obtain a first liquid-phase reaction system;
s2, adding ochratoxin A into the first liquid-phase reaction system, and enabling the ochratoxin A to be specifically combined with the ochratoxin A aptamer, so that at least part of the single-stranded DNA primer is separated from the ochratoxin A aptamer and is free in the liquid-phase reaction system, and a second liquid-phase reaction system is obtained;
s3, separating supernatant from the second liquid phase reaction system, constructing a padlock type exponential rolling circle amplification reaction system based on the supernatant, and performing padlock type exponential rolling circle amplification to obtain a third liquid phase reaction system containing target DNA;
s4, detecting the third liquid phase reaction system by using a signal probe modified with a chromogenic label and a test strip fixed with a detection probe, thereby realizing the visual detection of ochratoxin A, wherein the nucleotide sequences of the signal probe and the detection probe are respectively complementary with part of the nucleotide sequence of the target DNA.
2. The visual inspection method according to claim 1, wherein the nucleotide sequence of the ochratoxin a aptamer is shown in SEQ ID No. 1; the nucleotide sequence of the single-stranded DNA primer is shown as SEQ ID NO. 2.
3. The visual inspection method of claim 1, wherein the padlock-type exponential rolling circle amplification reaction system further comprises a DNA ligase, a deoxynucleotide triphosphate, a DNA polymerase, and a padlock-type template probe.
4. The visual inspection method according to claim 3, wherein the padlock type template probe has a phosphate group modified at the 5' end, and three nicking endonuclease cleavage sites are present in the nucleotide sequence of the padlock type template probe.
5. The visual inspection method according to claim 3 or 4, wherein the padlock type template probe has a nucleotide sequence shown in SEQ ID NO. 3.
6. The visual inspection method according to claim 1, wherein the nucleotide sequence of the signaling probe is shown in SEQ ID NO. 4.
7. The visual detection method according to claim 1, wherein the test strip is provided with a detection line and a quality control line, the detection probe is fixed on the detection line, the quality control probe is fixed on the quality control line, the nucleotide sequence of the detection probe is shown as SEQ ID NO.5, and the nucleotide sequence of the quality control probe is shown as SEQ ID NO. 6.
8. The visual inspection method of claim 1, wherein the chromogenic label comprises a nano-gold particle; and/or, the first modification comprises biotin and the second modification comprises streptavidin; and/or the solid phase carrier is the inner wall of a container for accommodating the first liquid phase reaction system; and/or, the ochratoxin a is isolated from contaminated cereal grains; and/or the qualitative detection limit of the visual detection method is 0.0001-10pg/mL.
9. An ochratoxin a visual detection kit based on P-ERCA, comprising:
the nucleotide sequence of the ochratoxin A aptamer is shown as SEQ ID NO. 1;
a single-stranded DNA primer, the nucleotide sequence of which is shown as SEQ ID NO. 2;
the padlock type exponential rolling circle amplification reaction assembly comprises DNA ligase, deoxynucleotide triphosphate, DNA polymerase and padlock type template probes, wherein the nucleotide sequence of the padlock type template probes is shown as SEQ ID NO. 3;
the nucleotide sequence of the signal probe modified with the chromogenic label is shown as SEQ ID NO. 4;
the test strip is characterized in that a detection probe and a quality control probe are respectively fixed on a detection line and a quality control line of the test strip, the nucleotide sequence of the detection probe is shown as SEQ ID NO.5, and the nucleotide sequence of the quality control probe is shown as SEQ ID NO. 6.
10. The kit of claim 9, wherein the chromogenic label comprises a nanoparticle of gold covalently bonded to the 5' end of the signaling probe.
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