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Test paper card for synchronously detecting ochratoxin, vomitoxin and T-2 toxin, preparation method and detection method

A technology of ochratoxin and vomitoxin, which is applied in the field of immunological detection, can solve problems such as result influence, and achieve the effects of saving cost, high stability and improving detection efficiency.

Inactive Publication Date: 2018-04-20
洛阳现代生物技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, in actual operation, there are still practical difficulties in how to prepare higher standards of test strips for the simultaneous determination of at least two mycotoxins based on fluorescent microspheres, because in the preparation process, the size and modification of fluorescent microspheres, antibody The selection of fluorescent microspheres and the coupling efficiency of antibodies and other factors will affect the results. Therefore, the preparation of test strips with higher sensitivity and stability for simultaneous detection of at least two mycotoxins is still a major research topic.

Method used

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  • Test paper card for synchronously detecting ochratoxin, vomitoxin and T-2 toxin, preparation method and detection method
  • Test paper card for synchronously detecting ochratoxin, vomitoxin and T-2 toxin, preparation method and detection method
  • Test paper card for synchronously detecting ochratoxin, vomitoxin and T-2 toxin, preparation method and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1. Preparation of a test strip for simultaneous detection of ochratoxin, deoxynivalenol and T-2 toxin in feed

[0026] The preparation process includes: the preparation of the fluorescent microsphere-antibody complex, the preparation of the binding pad, the preparation of the sample pad, the preparation of the detection pad and the assembly of the test strip.

[0027] 1. Preparation of fluorescent microsphere-antibody complexes:

[0028] (1) Cleaning: Take 50 μL of fluorescent microspheres into a 1.5 mL centrifuge tube, add 1 mL of 0.01M MES buffer, shake and mix, centrifuge at 15,000 r / min for 15 min, discard the supernatant, add 1 mL of 0.01M MES buffer, and ultrasonically Loose microspheres; Repeat this step three times to achieve the purpose of cleaning the microspheres;

[0029] (2) Activation: Add 250 μL of EDC solution to 1 mL of microsphere solution after washing, activate in the dark for 2 h, centrifuge at 15,000 r / min for 15 min, discard the supernat...

Embodiment 2

[0051] Embodiment two, the drawing of standard curve

[0052] 1. Configuration of standard products:

[0053] Use 0.1M pH7.4 PBS to dilute the ochratoxin standard to 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb; use 0.1M pH7.4 PBS to dilute the deoxynivalenol standard to 0.1 ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb; use 0.1M PBS pH7.4 to dilute the T-2 toxin standard to 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb;

[0054] 2. Reading:

[0055] Take 50 μL of the above-mentioned standard products, and after reacting for 10 minutes, read in the corresponding card slot of the portable fluorescence immunoassay analyzer. The reading results are shown in Table 1 below:

[0056]

[0057] 3. Verification of the calculation method:

[0058] (1) The four-parameter method, after inputting the corresponding parameters of the ochratoxin curve into the portable fluorescence immunoassay analyzer, select 0.5ppb and 2.5ppb to configure the standard within the standard curve range, click the card, react f...

Embodiment 3

[0062] Embodiment 3, the detection method of the test strips using the test paper card of the present invention to detect vomitoxin, ochratoxin and T-2 toxin in feed

[0063] The detection method of the test paper strips that utilize the test paper card of the present invention to simultaneously detect vomitoxin, ochratoxin and T-2 toxin in the feed comprises the following steps: weighing 5.0±0.05g of ground samples into 50mL polystyrene Add 25mL of methanol solution to the centrifuge tube, shake vigorously with an oscillator for 5min; put the shaken sample into a centrifuge, and centrifuge at 6000r / min for 5min; take 1mL of centrifuged supernatant and blow dry with a nitrogen blower. Add 1.0mL sample diluent (0.02M PBS, 0.5% methanol) to fully dissolve the coagulum to obtain the sample extract; dilute with the sample diluent at a ratio of 1:9 (for example: 100 μL sample extract + 900 μL sample diluent); Take 50 μL of the diluted liquid in the tube and drop it on the sample pa...

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Abstract

The invention relates to a test paper card for synchronously detecting ochratoxin, vomitoxin and T-2 toxin, a preparation method and a detection method, and belongs to the field of immunological detection. The test paper card comprises a card shell and a test paper strip, wherein the test paper strip comprises a base plate, as well as an absorbent pad, a detection pad, a conjugate pad and a samplepad which are sequentially lapped and stuck to the base plate; the detection pad is an NC film provided with a quality control line, a detection line T1, a detection line T2 and a detection line T3;the quality control line coats a goat anti-mouse secondary antibody; the detection line T1 coats OTA-BSA; the detection line T2 coats DON-BSA; the detection line T3 coats T-2-BSA; the conjugate pad isa glass cellulose film, labeled by embedding time-resolved fluorescent microspheres, of an ochratoxin monoclonal antibody, a vomitoxin monoclonal antibody and a T-2 toxin monoclonal antibody; the sample pad is a dried glass cellulose film soaked by sample pad treating fluid. The invention provides a novel method for systematic, convenient and normalized quantitative synchronous detection of at least two fungaltoxins. The novel method has the advantages of good stability and high sensitivity.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to a test paper card for simultaneous detection of ochratoxin, deoxynivalenol and T-2 toxin, a preparation method and a detection method. Background technique [0002] Ochratoxin, deoxynivalenol and T-2 toxin are three common mycotoxins. Corn-based feed and agricultural products are easily contaminated by them. After animals ingest mildewed feed, it will seriously affect the growth and reproduction of animals. rate and offspring survival. Ochratoxin (OTA) is produced by ochratoxin and sulfur Aspergillus, which can pollute almost all grains such as corn and wheat. It has strong acute toxicity. Experiments have proved that ochratoxin is teratogenic to animals. The main component of vomitoxin is deoxynivalenol (DON), which is mainly derived from the genus Fusarium, which can cause vomiting in pigs. When humans ingest food contaminated with DON, it will cause anorexia...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 任雪建吴彬李茂龙程果郭育培石方方
Owner 洛阳现代生物技术研究院有限公司
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