Analysis method for rapidly detecting aflatoxin B1 on site

Abstract

The invention discloses a method for detecting aflatoxin B1 by using an on-site rapid test strip. According to the invention, a CRISPR / Cas12a system is combined with commercial early pregnancy test paper, the aflatoxin B1 extracted from food is specifically recognized by adopting a nucleic acid lock structure, a nucleic acid sequence in the lock structure is released to be combined with crRNA, the activity of CRISPR / Cas12a is started, and the concentration information of the aflatoxin B1 is converted into color information on the early pregnancy test paper by virtue of an hCG probe. And pictures are collected by photographing through the smart phone, and picture information is processed by software, so that the quantitative detection of the aflatoxin B1 in the food can be completed. According to the detection method, the detection cost and operation steps are greatly reduced, the sensitivity is high, the specificity is good, the lowest detection limit can reach 2.6 pg / mL, and the national detection standard is reached.

Description

Technical Field

[0001] The present invention belongs to the detection field of analytical chemistry, specifically relates to nucleic acid detection technology, and in particular to an analysis method for on-site rapid detection of aflatoxin B1 in food by combining an existing commercial early pregnancy test strip with CRISPR / Cas12a. Background Art

[0002] Toxigenic fungi are a major threat to food safety. They quietly secrete harmful contaminants during food storage, which makes food safety monitoring very important for human health. Aflatoxin B1 (AFB1) is the most toxic derivative component of the aflatoxin family. It has strong hepatotoxicity, nephrotoxicity and immunotoxicity, and has mutagenic, teratogenic and carcinogenic effects on humans, accompanied by severe liver and kidney damage. This toxin is easily produced by toxigenic fungi and often contaminates major crops such as peanuts and corn. Due to the widespread existence and strong stability of AFB1, it poses a great challenge to daily food safety. Therefore, sensitive detection of AFB1 has always been the focus of food safety, quality control, environmental analysis and health risk research. However, due to the widespread and latent nature of AFB1, there is an urgent need for AFB1 on-site rapid detection methods that can be popularized to downstream markets. The current mainstream detection methods for AFB1 include high-performance liquid chromatography and liquid chromatography-mass spectrometry. Although these methods have excellent accuracy and sensitivity, the limitations of expensive instruments and cumbersome operating steps are inevitable, and it is difficult to quickly obtain the desired test results in food samples. Subsequently, detection technologies such as enzyme-linked immunosorbent assay based on the principle of classical immunoassay have also gradually developed. However, these methods still face many challenges when it comes to on-site food sample testing. Considering practical applications, the unique characteristics of point-of-care testing (POCT) without complex laboratory settings or cumbersome testing procedures are very suitable for downstream food safety fields. The unique advantages of POCT include reliability, simple and time-saving steps, low cost and environmental protection. In recent years, with the vigorous development of molecular diagnostics, in order to develop portable detection methods, scientists have pioneered a new sensing concept, that is, using commercial POCT products to transduce and enhance the signal of the analyte; this method can not only reduce R&D costs and R&D cycles, but also truly realize the on-site rapid detection of multiple molecules. This method has been widely recognized and has become one of the most promising portable detection methods. However, early pregnancy test strips, as the commercial POCT product with the highest market share in the world, have rarely been studied. The cost of each early pregnancy test strip is as low as US$0.1, and the extremely low cost means extremely high application potential. Coincidentally, scientists have chosen smartphones, which are commonly used today, as a means of signal collection. At the same time, with the help of appropriate signal transduction and amplification methods and color signal processing methods, the POCT advantages of early pregnancy test strips can be retained, and the ability of accurate detection can be introduced to achieve quantitative detection of targets. Appropriate means of transduction and amplification of signals are key factors in solving the lack of sensitivity of commercially available early pregnancy test strips. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a powerful bioanalytical tool that may be an inevitable presence in signal transduction and amplification patterns.In recent years, CRISPR-associated protein (CRISPR-Cas) diagnostic technology has aroused great interest in the biosensor community. With the efforts of many researchers, many promising detection methods have been explored. Although most of these technologies are used for nucleic acid detection, the application of CRISPR systems in non-nucleic acid detection is also gradually developing. Despite the great success, CRISPR-based detection technology has hardly been applied to food safety. At present, the commonly used test strip method and CRISPR / Cas12a combination method are mainly to construct test strips to produce fluorescent signals; although the test strips used in this method can be commercialized and can also achieve the final portable detection, the price of the test strips is too high, which invisibly increases the cost of the entire detection and is not conducive to popularization in the downstream market. In view of this, the present invention uses the convenience of early pregnancy test strips to bring the excellent shearing performance of CRISPR / Cas12a into the field of food safety detection, develops an analysis method based on early pregnancy test strips for rapid on-site detection of aflatoxin B1, and intelligently collects and analyzes data. This method is convenient, fast and low-cost, and is expected to be used for downstream on-site detection of aflatoxin B1. Summary of the invention

[0003] The present invention develops an on-site rapid analysis method for detecting aflatoxin B1 in food based on early pregnancy test strips and CRISPR / Cas12a. The detection method has good specificity and high sensitivity, and greatly reduces the detection cost. The principle of the present invention is as follows Figure 1 As shown: relying on the specific binding between the nucleic acid aptamer and AFB1, the locked nucleic acid structure releases the activating nucleic acid (Activator) while capturing the AFB1 molecule. The Activator binds to the crRNA and activates the trans-cleavage activity of Cas12a. The activated Cas12a quickly and massively cleaves the hCG probe, converts the concentration information of AFB1 into the concentration information of free human chorionic gonadotropin (hCG), and then separates the uncut hCG probe with magnetic beads. The signal is detected with a commercially available pregnancy test paper, and then the pictures are collected with intelligent collection, and the Image J software is used to process and analyze the data. To achieve the above purpose, the present invention provides the following technical scheme: The principle of the lock structure prepared by the present invention is as follows Figure 2 As shown: APT1, APT2 and Activator single-stranded nucleic acid sequences are added at the same time, and the final concentrations after addition are 25, 25 and 12.5 μM. After heating to open the single-stranded structure, the temperature is slowly lowered, so that the three DNA chains form a sandwich hybrid structure, and the excess APT sequence allows the Activator to completely hybridize. When AFB1 is present, the lock structure will capture the AFB1 molecule and release the Activator sequence, which binds to the crRNA to activate the activity of Cas12; The principle of the hCG probe prepared by the present invention is as follows: the hCG probe consists of two parts, one part is the hCG protein, and the other part is the probe DNA sequence. The probe DNA is modified with a thiol group at the 5' end and with biotin at the 3' end. In order to combine the hCG protein and the probe DNA, a cross-linking agent sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is used to combine with the free amino groups on hCG. The combined hCG protein can form a covalent bond with the probe DNA modified with a thiol group, thereby preparing a conjugate of the hCG protein and the probe DNA. The biotin modified at the 3' end of the DNA probe is used to remove the uncut hCG probe in subsequent detection; The method of collecting and analyzing the results with a smartphone is as follows: after magnetic separation, immerse the pregnancy test strip in the supernatant for 15 seconds, then take it out and let it stand for 2 minutes. After taking a photo of the test strip with a smartphone, import it into Image J software and convert it into a grayscale image. After background processing and regional value selection, it is compared with the image value of the test strip with a known hCG concentration to obtain a relative value. By establishing a linear standard curve for the relative values ​​of the color development of the pregnancy test strips of different intensities corresponding to different concentrations of AFB1, the concentration of AFB1 in food samples can be quantitatively analyzed; The present invention has the following beneficial effects: The AFB1 detection method developed by the present invention combines commercially available pregnancy test strips and CRISPR / Cas12a systems, and specifically identifies AFB1 molecules through a lock structure, presents the concentration information of AFB1 on the pregnancy test strip in the depth of color, and collects and analyzes data through a portable smartphone, which greatly reduces the detection cost, not only ensures the specificity of the detection, but also increases the portability of the entire detection. It makes it possible to quickly detect AFB1 foods on site downstream. In addition, this method is expected to be used for other important molecules for food safety monitoring by modifying different nucleic acid sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 Schematic diagram of the principle of the analysis method of the present invention; Figure 2 This is a schematic diagram of the principle of the lock structure specifically recognizing AFB1 to activate Cas12a activity in the analysis method of the present invention; Figure 3 It is a feasibility analysis diagram in the analysis method of the present invention; Figure 4 It is a linear diagram and a specific selectivity analysis diagram of AFB1 in the analysis method of the present invention; Figure 5 This is a graph showing the test results of the analytical method of the present invention on actual food samples. Implementation

[0005] The present invention develops an analytical method for on-site rapid detection of aflatoxin B1 in food based on early pregnancy test strips and CRISPR / Cas12a. Researchers in this field can refer to the text content and appropriately improve the process parameters to achieve it. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified. The water used in the following embodiments is DEPC-treated water. All samples in the following examples were not purified before use; The present invention is carried out according to the following specific steps: a. Preparation of hCG probe a1 The preparation of hCG probe depends on the binding of hCG protein to nucleic acid sequence. In the present invention, a cross-linking agent is used to change the groups on the surface of hCG protein. First, 200 μL of PBS containing 0.125 mg / mL hCG and 20 μL of 1 mg / mL cross-linking agent sulfo-SMCC solution are added to the EP tube, and reacted at 25°C and 800 rpm for 1 hour. The obtained solution is washed and purified 4 times with PBS buffer through an Amicon-10K ultrafiltration tube at 12000 rpm for 10 minutes to remove excess cross-linking agent; a2 The disulfide bonds of the thiol-modified probe DNA need to be opened before cross-linking with the hCG protein. The probe DNA sequence used in this process is (5'-3'): SH-C6-CCC CCC CCC CCC CCC CCC CCC CCC CCC-Biotin. Take 150μL of 10μM probe DNA and mix it with 15μL of 10mM TCEP solution. The mixture is reacted at 25℃ and 800rpm for 1h to open the disulfide bonds between the DNA sequences. The solution is washed and purified 4 times with PBS buffer through an Amicon-3K ultrafiltration tube at 12000rpm for 20min to remove excess TCEP; a3: Mix the solutions obtained from a1 and a2, and react at 4°C for 36 hours, vortexing several times during the reaction. After the reaction, the obtained conjugate is washed and purified five times with NEB 2.1 buffer through an Amicon-10K ultrafiltration tube at 12000 rpm for 10 minutes to remove the unconnected probe DNA; b. Preparation of locked nucleic acid structure The nucleic acid sequence used in this process is as follows (5'-3'): APT1 TGC ACG TGT TGT CTC TCT GTG TCT CGT GCT TGT G APT2 TGT TGT GCA CGT GTT GTC TCT CTG TGT CTC GTG C Activator CAC GTG CAC AAC ACA CAA GCA CGA GA All three DNA sequences were dissolved in hybridization buffer, and then APT1, APT2, and Activator were taken in an EP tube with a total volume of 50 μL, and the final concentrations were 25, 25, and 12.5 μM, respectively. The resulting mixture was mixed and programmed with a thermocycler to allow the three DNA sequences to hybridize. Specifically, it was first heated to 90°C for 5 minutes, then slowly cooled to 25°C, and reacted for 1 hour; c. Analytical Procedure for AFB1 in Food Samples c1 Extraction steps of AFB1 in food samples. After crushing 1g of food sample (such as corn), add 3ml of extraction solution (methanol: water = 3:7) and stir evenly. After vigorous shaking for 5min, centrifuge at 12000rpm for 15min, and filter the collected supernatant with a 0.22μm membrane. The extracted extract is diluted with NEB2.1 to eliminate the influence of methanol; c2 Cas12a response to aflatoxin B1 in food. The crRNA sequence used in this process is as follows (5'-3'): UAA UUU CUA CUA AGU GUA GAU UCG UGC UUG UGU GUU GUG CA 30 μL of food extract sample; 20 μL of 500nM lock structure DNA; 8 μL of Cas12a / crRNA complex (NEB2.1 dissolved, containing 0.25pmol / μL Cas12a, 0.5pmol / μL crRNA); 22 μL of NEB 2.1 buffer solution were added to the EP tube and mixed. The mixed solution was reacted at 37°C for 25 minutes, and then heated at 65°C for 5 minutes to inactivate the enzyme; c3 Early pregnancy test strips to detect aflatoxin B1 concentration. Add 80 μL of 3 μL 10 mg / mL -1 The magnetic beads were mixed with 2× B&W buffer and reacted at 25°C and 1500 rpm for 30 min. After magnetic separation, the test strip was immersed in 150 μL of supernatant for 15 seconds, taken out and left to stand for 2 minutes, and the test strip was photographed with a smartphone, and image processing and analysis were performed using Image J software. The obtained color intensity value was substituted into the established linear standard curve to calculate the concentration. DETAILED DESCRIPTION

[0006] The following is further described in conjunction with the accompanying drawings of the specification, but the analysis method of the present invention is not limited to the following embodiments: Example 1: Feasibility study of the analytical method of the present invention This example explores the feasibility of combining commercially available pregnancy test strips with the CRISPR / Cas12a system for detecting AFB1 in food; In order to verify the feasibility of the method, it is first necessary to determine whether the hCG probe is successfully synthesized. By scanning with a UV-visible spectrophotometer, the peak relationship between the probe DNA, hCG protein, and the hCG protein coupled thereto can be preliminarily determined whether the hCG protein is successfully synthesized. Figure 3 A can clearly see that the red shift of hCG-probe relative to probe DNA is from 269nm to 271nm, and the peak shape has changed significantly compared with hCG, indicating that the DNA chain and hCG are successfully coupled. However, in order to verify the feasibility of the reaction of this scheme as a whole, 4 groups of reactions with different components were used for control experiments. Figure 3 B shows that only the activated Cas12a component can cleave the hCG probe to produce sufficient color development signals, while the components of the unactivated Cas12a and the hCG probe that are not cleaved, the hCG probe is removed by magnetic beads through magnetic separation without color development. This shows that the successful preparation of the hCG probe and the analytical method of the present invention are indeed feasible.

[0007] Example 2, the analytical performance experiment of the present invention for analyzing and detecting AFB1 in food, different concentrations of AFB1 were added to the reaction system and mixed, and reacted at 37°C for 25 minutes. After the reaction, 80 μL of 2×B&W buffer containing 3 μL of 10 mg / mL-1 magnetic beads was added, and the reaction was carried out at 25°C and 1500 rpm for 30 minutes. After magnetic separation, the test strip was immersed in 150 μL of supernatant for 15S, taken out and allowed to stand for 2 minutes, and the test strip photo was collected with a smart phone, and the image was processed and analyzed using Image J software.

[0008] Depend on Figure 4 As can be seen from A, a signal intensity curve positively correlated with the AFB1 concentration can be obtained based on the color signal intensity corresponding to a series of gradient concentrations. Figure 4 As can be seen from the inset of A, after taking the logarithm of the signal intensity, a linear standard curve of AFB1 concentration and color signal intensity can be obtained. The specific linear equation is Y = 2908.65 + 3477.29lgX (pg / mL), and the linear range is 1-10 5 pg / mL, and the detection limit was 2.6pg / mL (3σ), which showed that this analytical method had good sensitivity and excellent analytical ability.

[0009] Example 3: Specificity and selectivity of the analytical method of the present invention Using the above steps, 50 ng / mL of AFB1 (A) and 500 ng / mL of interfering fungal toxins were detected under the same conditions. The interfering fungal toxins included ochratoxin A (O), zearalenone (Z), and fumonisin B1 (F). Figure 4 As can be seen in B, only the components with AFB1 will eventually have sufficient signal display, while the color signal intensity of the components without AFB1 is almost the same as that of the blank group. This shows that the method of using nucleic acid structure specific recognition and capture of AFB1 is effective, and the sensing strategy of the present invention using early pregnancy test paper to detect AFB1 in food has high specificity and selectivity.

[0010] Example 4: Research on the application of the analytical method of the present invention in detecting AFB1 in actual food samples. The food samples, corn flour and peanut oil used in this analytical method were purchased from local supermarkets. Take AFB1 with a final concentration of 0.1, 1, 10, and 100 ng / g and add it to 1g of corn flour. After stirring evenly, add 3ml of extract. After vigorous shaking for 5min, centrifuge at 12000rpm for 15min, and filter the collected supernatant with a 0.22μm nylon membrane. The same method was used to extract 1mL of peanut oil samples containing 0.1, 1, 10, and 100ng / mL AFB1. The extracted extract was diluted with NEB2.1 to eliminate the influence of methanol. 30μL of diluted extracts of different concentrations were added to the reaction system respectively, and the reaction, data collection and analysis were carried out according to the above reaction steps; Depend on Figure 5 As can be seen from the table, the recovery ranges of AFB1 in corn flour and peanut oil are 84.98%-98.11% and 88.58%-96.58%, respectively, and the relative standard deviations (RSD) are 8%-18% and 3%-22%, respectively. This shows that the method of the present invention has good performance in the analysis of actual solid and liquid food samples, and has ideal potential, and can realize low-cost on-site rapid detection of AFB1 in food.

Claims

1. A method for rapid on-site detection of aflatoxin B1 in food, characterized in that: Follow these steps: (1) Preparation of locked nucleic acid structures: Two nucleic acid aptamers (Apt1 and Apt2) of aflatoxin B1 and an activating nucleic acid (Activator) were dissolved in a hybridization buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, pH 7.4), and the final concentrations of Apt1, Apt2 and Activator were 25, 25 and 12.5 μM, respectively; the obtained mixture was heated to 90°C for 5 min, and then slowly cooled to 25°C in a thermal cycler to allow the three DNAs to completely hybridize and construct a lock structure. The obtained lock nucleic acid structure was stored at 4°C for later use; (2) Preparation of hCG probe: (3) 150 μL of 10 μM probe DNA was mixed with 15 μL of 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution; the mixture was kept at room temperature for 1 hour to activate the thiol-modified probe DNA; b. The resulting solution was purified by Amicon-3K ultrafiltration tube using PBS buffer (10 mM PB, 137 mM NaCl and 2.7 mM KCl, pH 7.4); c. Mix 200 μL of PBS containing 0.125 mg / mL human chorionic gonadotropin (hCG) with 20 μL of 1 mg / mL sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) solution and incubate at room temperature for 1 h; d. The resulting solution was purified by Amicon-10K ultrafiltration tube with PBS to remove unreacted coupling agent; e. Mix the above two purified solutions and incubate at 4°C for 36 h; purify the obtained conjugate with Amicon-10K and NEB 2.1 buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9) to remove excess probe DNA; then dilute the obtained hCG probe to a final volume of 200 μL and store at 4°C for future use.

2. The analysis process of early pregnancy test strips to detect aflatoxin B1 in food: a. Add 30 μL of food extract sample and 20 μL of 500 nM lock structure DNA; 8 μL of Cas12a / crRNA complex (NEB2.1 dissolved, containing 0.25 pmol / μL Cas12a, 0.5 pmol / μL crRNA); 22 μL of NEB 2.1 buffer solution to the EP tube and mix; the mixed solution was reacted at 37 ° C for 25 min, and then heated at 65 ° C for 5 min to inactivate the enzyme; b. Add 80 μL of 3 μL 10 mg / mL to the mixture -1 The magnetic beads were added with 2× B&W buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.5) and reacted at 25 °C and 1500 rpm for 30 min; c. After magnetic separation, the test strip was immersed in 150 μL of supernatant for 15 seconds, taken out and allowed to stand for 2 minutes, and photos of the test strip were taken with a smartphone, and image processing and analysis were performed using Image J software.

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