A portable detection device and method based on affinity sensor method for detecting mycotoxins

Abstract

The application discloses a ricin toxin detection sensor based on competitive binding of ricin to mitochondrial adenine nucleotide translocator (ANT) and a detection method, which comprises ANT protein, hollow dialysis fiber, chemical cross-linking agent and fluorescently labeled ATP. When in use, the ANT is first fixed on the inner wall of the hollow fiber dialysis membrane and the fluorescently labeled ATP (cy5-12-ATP or cy3-12-ATP or DEPC-12-ATP) is added; then, it is placed in the to-be-detected solution for 30-60 seconds, and then is placed under a fluorescence detection instrument (such as a fluorescence microscope) for observation, so that the presence or absence of the ricin toxin and the approximate concentration of the toxin are determined; then, the relative intensity of the fluorescence of the to-be-detected solution is determined; a standard curve of the corresponding relationship between the concentration of the fluorescent group and the concentration of the toxin is established; and the concentration of the ricin toxin in the to-be-detected solution is obtained by substituting the concentration of the fluorescent group of the to-be-detected solution into the above standard curve and performing calculation. The application can effectively improve the detection efficiency, simplify the detection method and be widely applied in the society.

Description

TECHNICAL FIELD

[0001] The present application relates to the detection of bongkrekic acid, in particular, to provide a portable detection device based on affinity sensor method of bongkrekic acid toxin. BACKGROUND

[0002] Food safety is highly valued by the public. With the improvement of people's living standards, bongkrekic acid (BA) poisoning caused by various foods occurs frequently. Bongkrekic acid is a mitochondrial toxin produced by pseudomonas cocovenenans, which is an extremely strong toxin, and a very small dose can cause human poisoning and death. Bongkrekic acid has strong heat resistance, so cooked food can also be poisoned. Rapid and accurate detection of the presence and concentration of bongkrekic acid is a very worthwhile topic to explore.

[0003] At present, there are many detection techniques for bongkrekic acid, such as Chinese patent application CN201510907266.9: A kind of liquid chromatography-tandem mass spectrometry detection method of bongkrekic acid: The method needs complicated sample pretreatment before detection, the detection steps are more complicated, and the detection time is long. The detection instrument is heavy and expensive, and the technical requirements for the detector are high, which limits the popularization and use of the detection technology. For example, Chinese patent application CN2021102231903: Test paper strip and method for detecting bongkrekic acid: Bongkrekic acid monoclonal antibody, bongkrekic acid hapten and other materials are needed, which involves the use of bongkrekic acid with strong toxin. The preparation process is difficult, the safety requirement coefficient is high, which increases the cost and safety cost of preparation to some extent. At present, the technology for detecting bongkrekic acid by small instrument in China is not perfect. Therefore, the applicant team is committed to realizing the simple detection of bongkrekic acid toxin, improving the operability, and achieving more efficient detection effect. The technology does not need to use large experimental instruments, and does not need to perform complex sample pretreatment before detection. The preparation process is relatively safe and simple, and the technical requirements for the detector are not high. The detection result is intuitive and visible, so it is conducive to the popularization and use of the detection technology. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide a portable detection device based on affinity sensor method of bongkrekic acid toxin.

[0005] The technical solution adopted by the present application: A portable detection device based on affinity sensor method of bongkrekic acid toxin, including expression of ANT protein, chemical cross-linking on the inner surface of hollow dialysis fiber, and fluorescence-labeled ATP based on affinity sensor method of bongkrekic acid competitive binding of ATP binding site of ANT protein.

[0006] The ANT-related sequence includes an ANT gene sequence (SEQ ID NO: 1) and an ANT protein sequence (SEQ ID NO: 2).

[0007] The portable detection device for aflatoxin B1 based on the affinity sensor method is applied to the detection of the concentration of aflatoxin B1.

[0008] The method for detecting the concentration of aflatoxin B1 based on the portable detection device for aflatoxin B1 based on the affinity sensor method comprises the following steps:

[0009] S1, fixing ANT on the inner wall of a hollow fiber dialysis membrane through a chemical crosslinking agent (glutaraldehyde);

[0010] S2, adding ATP with a fluorescent marker (cy5-12-ATP or cy3-12-ATP or DEPC-12-ATP) to the solution surrounded by the dialysis membrane.

[0011] S3, placing the aflatoxin B1 detection sensor in the to-be-detected solution for 30-60 seconds;

[0012] S4, placing the sensor under a fluorescence detection instrument (such as a fluorescence microscope) for observation;

[0013] S5, detecting the color or the content of the fluorescent group in the solution to determine whether aflatoxin B1 exists and the approximate concentration of the toxin.

[0014] The method for detecting the concentration of aflatoxin B1 based on the portable detection device for aflatoxin B1 based on the affinity sensor method comprises the following steps:

[0015] S6, determining the relative intensity of fluorescence of the to-be-detected solution by using software;

[0016] S7, detecting fluorescence under a fluorescence microscope, and establishing a standard curve according to the corresponding relationship between the concentration of the fluorescent group and the concentration of the toxin;

[0017] S8, substituting the concentration of the fluorescent group of the to-be-detected solution into the above standard curve to obtain the concentration of aflatoxin B1 contained in the to-be-detected solution.

[0018] This invention is based on the competitive binding principle of a substance (ATP) competing with a specific metabolite (bongkrekic acid) and a fluorescent group (fluorescein-labeled mimic) to the specific metabolite and labeled ligand receptor sites. It utilizes the adenosine monophosphate transporter (ANT), a specific binding protein of bongkrekic acid in cells, which is chemically cross-linked and immobilized on the inner surface of a container made of dialysis fibers. This allows the ANT to bind to ATP modified with a fluorescent label, assembling a special sensor. If bongkrekic acid is present in the test solution, it can pass through the dialysis fibers into the container, competing with the ATP with the fluorescent group already bound to the ANT, displacing the ATP. The detection of bongkrekic acid can then be achieved by observing the fluorescence or color of the solution within the container. This invention, by constructing a sensor for detecting bongkrekic acid, effectively improves detection efficiency, simplifies the detection method, and has high practicality and feasibility, making it easy to promote in society and facilitate the practical application of bongkrekic acid detection technology. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the method principle;

[0020] Figure 2 This is a diagram showing the sensor's external dimensions.

[0021] Figure 3 This is a schematic diagram of sensor detection. Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0023] A portable detection device for bongkrekic acid toxin based on an affinity sensor method includes the expression and chemical cross-linking of ANT protein on the inner surface of hollow dialysis fibers. Based on the affinity sensor method, bongkrekic acid competitively binds to fluorescently labeled ATP at the ATP-binding site of the ANT protein. The ANT-related sequences include the ANT gene sequence (Sequence Listing Identifier 1) and the ANT protein sequence (Sequence Listing Identifier 2).

[0024] Referring to the SLC25A6 gene sequence published by NCBI: the upstream primer plus the start codon, FLAG tag, and pET-23a(+) plasmid sequence EcoR The 16 bp base sequence at the 5' end of site I, and the downstream primer with the reverse complementary sequence Xba of the pET-23a(+) plasmid. The 16 bp base sequence at the 3' end of site I.

[0025] PCR amplification was performed with the purchased SLC25A6 gene as a template, and the reaction system was as follows: gold medal Mix, 10 μM upstream primer 1 μl, 10 μM downstream primer 1 μl, 100 ng / μl SLC25A6 gene 1 μl. The PCR reaction conditions were as follows: 98 ℃ for 2 min, 98 ℃ for 10 s, 55 ℃ for 10 s, 72 ℃ for 10 s, 72 ℃ for 1 min, for a total of 30 cycles.

[0026] The primer pair comprises an upstream primer (SEQ ID NO: 3) and a downstream primer (SEQ ID NO: 4).

[0027] The DNA amplification product was recovered and purified by 1% agarose gel electrophoresis.

[0028] The extracted pET-23a (+) and pUAST were digested with Xba Ⅰ, EcoR Ⅰ, and incubated at 50 ℃ for 1 h, and the gel was recovered and purified.

[0029] The recovered target gene fragment and the double enzyme digestion product were mixed at a ratio of 2.5:1, 10 μl of seamless cloning Mix was added, then sterile water was added to 20 μl, and the reaction was performed for 20 min.

[0030] The ligation product was transformed into E. coli Top 10 competent cells, and the cells were cultured in LB liquid medium containing ampicillin at 37 ℃ and 280 r / min for 12-16 h. The plasmid was extracted, and colony PCR verification and sequencing identification were performed.

[0031] The plasmid was transfected into fruit fly cells by liposome, and after 3-4 days of culture, the cells were washed with PBS and centrifuged to collect the cells.

[0032] The fruit fly cells were lysed with a cell lysis solution to extract proteins, and then purified by anti-short peptide labeled antibody magnetic beads to obtain the desired ANT protein fragment.

[0033] ANT was fixed on the inner wall of hollow fiber dialysis membrane by chemical crosslinking (glutaraldehyde), and ATP with a fluorescent marker (cy5-12-ATP or cy3-12-ATP or DEPC-12-ATP) was added to the solution surrounded by the dialysis membrane.

[0034] The sensor was completed.

[0035] The purchased rice mycotic acid was configured into standard solutions of different concentrations, and the sensor was used to detect a fixed volume of standard sample. The sensor was placed under a fluorescence detection instrument (handheld fluorescent protein observer) for observation. Rice mycotic acid would compete with the fluorescent ATP on the wall of the tube to the lumen, so that the lumen contained free fluorescence.

[0036] Select a cross section of the sensor and take a picture of the observed results. Upload the photo to the software and analyze the fluorescence intensity of the middle position of the cross section. Use the concentration of mycotoxin as the horizontal coordinate and the fluorescence intensity as the vertical coordinate to create a standard curve.

[0037] Take a certain amount of fermented agaric and cut it into small pieces. Place it in 100 ml of sterile water and stir thoroughly to suspend the mycotoxin on the surface of the agaric in the water as the test solution.

[0038] Place the sensor in the test solution for 30-60 seconds. Place the sensor under the fluorescence detection instrument for preliminary observation. If the sensor lumen does not contain free fluorescence, it is preliminarily determined that the solution does not contain mycotoxin. If the lumen contains free fluorescence, it is determined that the solution contains mycotoxin. Take a picture of the results and upload it for fluorescence intensity analysis. According to the position of the fluorescence intensity on the standard curve, the concentration of mycotoxin is determined.

Claims

1. A method for detecting the concentration of mycophenolic acid toxin based on an affinity sensor method, characterized by, It comprises the following steps: S1, fixing the ANT protein SLC25A6 on the inner wall of the hollow fiber dialysis membrane by a chemical cross-linking agent glutaraldehyde, the gene sequence of the ANT is shown in SEQ ID NO: 1, and the protein sequence is shown in SEQ ID NO: 2; S2, adding Cy5-12-ATP or Cy3-12-ATP with a fluorescent marker in the solution surrounded by the dialysis membrane; S3, placing the ricin toxin detection sensor in the solution to be detected for 30-60 seconds; S4, placing the sensor under a fluorescence detection instrument for observation; S5, detecting the color or the content of the fluorescent group in the solution to determine whether the ricin toxin exists and the concentration of the toxin.

2. The detection method according to claim 1, characterized in that, It comprises the following steps: S6, determining the relative intensity of the fluorescence of the solution to be detected by software; S7, detecting the fluorescence under a fluorescence microscope, and establishing a standard curve according to the corresponding relationship between the concentration of the fluorescent group and the concentration of the toxin; S8, substituting the concentration of the fluorescent group of the solution to be detected into the above standard curve to calculate the concentration of the ricin toxin contained in the solution to be detected.

3. Application of the detection method according to claim 1 in the detection of the concentration of ricin toxin.

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