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CN-121978326-A - Detection test strip, detection method and application of ochratoxin A

CN121978326ACN 121978326 ACN121978326 ACN 121978326ACN-121978326-A

Abstract

According to the invention, the quantum dot is loaded on the surface of the PS microsphere to construct the quantum dot microsphere composite signal marker, so that a novel fluorescent immunochromatography detection test strip based on a PS microsphere quantum dot loading technology is successfully developed and is used for rapid quantitative analysis of OTA in grain and oil crops and nuts. The ochratoxin A quantum dot fluorescence detection test strip constructed by the invention has the advantages of high sensitivity, good specificity, simple and convenient operation and quick analysis, is suitable for quick and accurate quantification of the ochratoxin A in grain and oil crops and nuts, and provides an effective technical means for on-site screening and monitoring of agricultural product mycotoxins.

Inventors

  • LI KE
  • QIAO XIANGJUN
  • ZHANG SHUXIA
  • Bahatiguri Manatiba
  • MIAO XIAOXING
  • ZHENG YUE

Assignees

  • 郑州海关技术中心

Dates

Publication Date
20260505
Application Date
20260127

Claims (10)

  1. 1. A detection test strip for ochratoxin A comprises a sample pad, a combination pad, an NC film, a T line, a C line and a water absorption pad which are sequentially stacked on a bottom plate, wherein QBs probes coupled with an OTA monoclonal antibody are sprayed on the combination pad, an OTA-OVA coating antigen diluent is sprayed on the T line, and a goat anti-mouse secondary antibody is sprayed on the C line.
  2. 2. The test strip for detecting ochratoxin a of claim 1, wherein the QBs probe conjugated with an OTA monoclonal antibody is prepared by the following method: S1, taking carboxylated PS microsphere suspension, adding MES, uniformly mixing, centrifuging, discarding supernatant, washing, then re-suspending in the MES, immediately adding EDC and NHS, activating at room temperature to obtain activated PS microsphere suspension, taking amino water-soluble CdSe/ZnS quantum dot solution, replacing with the MES, finally re-suspending in the MES to obtain amino quantum dot solution, quickly adding all the amino quantum dot solution into the activated PS microsphere suspension, stirring at room temperature for 2h, adding Tris, quenching at room temperature, centrifuging, discarding supernatant, washing to obtain QBs probes; S2, taking QBs probes, resuspending the probes in MES, adding EDC and NHS for room temperature activation reaction, centrifuging, discarding supernatant, resuspending the probes in PBS, immediately adding OTA monoclonal antibodies, oscillating and coupling for 2 hours at room temperature, centrifuging, discarding supernatant, washing for 2 times by PBS, resuspending the products in the MES, adding mPEG-NH2, EDC and NHS, reacting for 1 hour at room temperature, adding BSA to a final concentration of 1%, continuing to react for 1 hour for sealing, fully centrifuging and washing for 3 times by PBS, resuspending the probes in PBS containing 1% of BSA, and obtaining the QBs probes coupled with the OTA monoclonal antibodies, and storing the probes in a dark place at 4 ℃.
  3. 3. The test strip for detecting ochratoxin a of claim 1 or 2, wherein the test strip for detecting ochratoxin a is prepared by the following method: soaking the bonding pad in the pretreatment liquid, drying, spraying QBs probe solution coupled with the OTA monoclonal antibody on the bonding pad, and vacuum drying; spraying OTA-OVA coating antigen and goat anti-mouse secondary antibody on the T line and the C line respectively, and drying; the sample pad, the pretreated combining pad, the NC film and the water absorbing pad are sequentially stuck on a bottom plate, compacted and fixed, cut into single strips with the width of 4 mm, and stored at the temperature of 4 ℃ for standby after sealing.
  4. 4. The test strip for detecting ochratoxin a of claim 3, wherein the concentration of the OTA-OVA coated antigen is 0.1 mg/mL, and the dilution factor of the QBs probe solution coupled with the OTA monoclonal antibody is 1:15.
  5. 5. The test strip of ochratoxin a of claim 3, wherein PBS is used as a diluent for the OTA-OVA coated antigen.
  6. 6. The test strip for detecting ochratoxin a of claim 3, wherein the pretreatment liquid for treating the conjugate pad is selected from the group consisting of 0.01M PBS, 0.1% tween-20+0.01 mpbs, 0.1% peg200+0.01mpbs, 0.1% tween-20+0.1% peg200+0.01mpbs.
  7. 7. A method for detecting ochratoxin a, comprising: Weighing a sample, placing the sample into a sample tube filled with an extract, carrying out vortex oscillation and uniform mixing, taking out part of the sample, centrifuging, taking supernatant into a centrifuge tube, adjusting the pH value to 6.2-7.0, vertically placing the detection test strip of ochratoxin A according to any one of claims 1-6 into the centrifuge tube, reacting 3 min, taking out, reading fluorescent signal values of T line and C line by a fluorescent reader, and comparing with a standard curve to obtain the concentration of the sample.
  8. 8. The method for detecting ochratoxin A according to claim 7, wherein the detection method has a good linear relationship in the range of 2-20 ng/mL and the lowest detection limit is 0.5 ng/mL.
  9. 9. Use of the method for detecting ochratoxin a according to claim 7 or 8 for detecting ochratoxin a in grain and oil crops and nuts.
  10. 10. The use according to claim 9, wherein the grain and oil crop and nuts are selected from rice, pistachio, maize, cashew, peanut, soybean, walnut.

Description

Detection test strip, detection method and application of ochratoxin A Technical Field The invention belongs to the technical field of food detection, and particularly relates to an ochratoxin A detection test strip based on a polystyrene microsphere quantum dot loading technology, a detection method and application of the detection test strip in grain and oil crops and nuts. Background Ochratoxin a (Ochratoxin A, OTA) is a key secondary metabolite produced by the filamentous fungus aspergillus ochraceus (Aspergillus ochraceus), which is highly toxic, widely contaminated, stable in nature and not easily degraded by metabolism. Because ochratoxin a is widely existing in nature, the ochratoxin a is widely distributed in various animal and plant source foods, wherein the pollution of grain and oil crops, nuts, animal livers, blood and the like is most serious and the risk is prominent. The toxin has neurotoxicity and immunotoxicity, damages the livers and kidneys of human beings and animals, has teratogenic, oncogenic and mutagenic hazards, and has been evaluated by the international cancer research institute (International Agency for Research on Cancer, IARC) as a possible human carcinogen of class II (B), which constitutes a serious threat to food safety and public health. In order to effectively control the pollution level of OTA, a strict limit standard system is established by the global main regulatory agency and the state/region, the world health organization (World Health Organization, WHO) establishes the daily tolerable intake (Daily tolerable intake, DTI) of human beings based on risk assessment, the maximum daily intake of OTA per kilogram of body weight is regulated to be not more than 16 ng, the International food Commission (Codex Alimentarius Commission, CAC) sets a unified maximum limit standard of OTA for unprocessed grains (such as barley, wheat and rye) to be 5.0 mug/kg, the European Union (EU) sets more refined classification control on the OTA residual limit of up to 20 types of foods, wherein the limit of unprocessed grains and instant coffee is 5.0 mug/kg, the limit of finished products and baked coffee for end consumers is 3.0 mug/kg, the limit of wine is 2.0 mug/kg, the limit of special medical foods for infants, the limit of foods and infant foods is set to be the strict limit of 5.0 mug/kg for unprocessed grains (such as barley, wheat and rye), the limit of the special medical foods for infants is not strict limit of 5.0 mug/kg, the limit of the corresponding to be regulated in the European Union, and the limit of the limit is regulated in the maximum limit of the maximum limit standard of OTA is also exceeds the standard of 5.0 mug/kg for the maximum limit for detecting for 20 types of foods, and the maximum limit is regulated in the standard of current is regulated in the state of about 20, and the standard is allowed for the maximum limit of the standard is required for safety of 3.0 mug. To ensure food safety and meet the increasingly stringent OTA regulatory limit requirements worldwide, researchers have developed a variety of OTA detection techniques, mainly including high performance liquid chromatography (High Performance Liquid Chromatography, HPLC), liquid chromatography-tandem mass Spectrometry (Liquid Chromatography-TANDEM MASS Spectrometry, LC-MS/MS), thin-layer chromatography (Thin-Layer Chromatography, TLC), enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA), aptamer technology, immunochromatography technology, and the like. HPLC, LC-MS/MS methods are most commonly used at present, are suitable for high-precision quantitative analysis of complex matrix samples, but have complicated operation flow, long detection period, higher instrument cost and maintenance cost, are difficult to meet the requirements of on-site rapid detection, TLC can only realize primary qualitative analysis, has low sensitivity and poor accuracy, uses a large amount of harmful organic solvents, ELISA (enzyme-Linked immuno sorbent assay) methods are simple to operate, antibodies cross reaction causes insufficient specificity and high risk of false positives, the screening process of the aptamer technology is complicated and time-consuming, the success rate is greatly influenced by target properties, library design and screening strategies, and immunochromatography technology realizes qualitative and semi-quantitative analysis of OTA based on antigen-antibody competition inhibition combination mechanisms, is simple to operate, is economical and efficient, is suitable for on-site rapid screening of a large number of samples, but has the problems of poor stability, uneven signal intensity and the like of traditional marking materials due to high detection sensitivity, and quantitative restriction on accuracy. Disclosure of Invention Aiming at the problems in the prior art, the inventor fixes quantum dots on the surface of the functionalized PS microsphere through covalent bo