Fluorescent quenching test paper for detecting field okadaic acid as well as a preparation method and application of the fluorescent quenching test paper

A technology of daesangic acid and fluorescence quenching, which is applied to the field of fluorescence quenching test strips for detecting daesic acid and their preparation, and can solve the problems of affecting the sensitivity of test strips, non-intuitive detection results, misjudgment by non-professionals, etc. , to achieve the effect that it is not easy to misjudge, the result is intuitive, and the specificity is strong.

Active Publication Date: 2019-04-19
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the test result of this method is not intuitive, that is, the signal on the T line indicates that the result is negative, otherwise it is positive, which can easily lead to misjudgment by non-professionals and cannot be quantified. Secondly, colloidal gold immunological lateral flow chromatography test paper The standard for judging the result of the strip is positive is that no signal can be observed on the T line, that is, a relatively large concentration of the analyte is required to make the T line have no signal at all, which greatly affects the sensitivity of the test strip.

Method used

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  • Fluorescent quenching test paper for detecting field okadaic acid as well as a preparation method and application of the fluorescent quenching test paper
  • Fluorescent quenching test paper for detecting field okadaic acid as well as a preparation method and application of the fluorescent quenching test paper
  • Fluorescent quenching test paper for detecting field okadaic acid as well as a preparation method and application of the fluorescent quenching test paper

Examples

Experimental program
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Effect test

Embodiment 1

[0048] This embodiment provides a fluorescence quenching test paper for detecting okadaic acid and a preparation method thereof.

[0049] (1) Preparation of OA artificial antigen

[0050] Take 4 mg OA, 0.62 mg NHS (N-hydroxysuccinimide, purchased from MACKLIN (Macklin)) and 1.14 mg DCC (N,N-dicycloethanecarbodiimide, purchased from MACKLIN (Macklin)) Mix in 240 μL of DMF (N,N-dimethylformamide, purchased from MACKLIN (Macklin)), incubate at room temperature for 2 h, and then add 3.8 mg of BSA (purchased from SIGMA, dissolved in 200 μL 0.1moL / L NaHCO 3 ), add 3 mg IgG (purchased from YESEN (YESEN Biology), and dissolve in 200 μL 0.1moL / L NaHCO 3 ), and continue to incubate at room temperature for 2 hours. Unreacted small molecules and by-products are removed by ultrafiltration (4°C, 8000rpm, 15min) in an ultrafiltration centrifuge tube (10K). The ultrafiltration centrifuge tube needs to be pretreated before use. Completely pass the pure water through the membrane, then ice b...

Embodiment 2

[0066] This embodiment provides a fluorescence quenching test paper for detecting okadaic acid and a preparation method thereof.

[0067] (1) Preparation of OA artificial antigen

[0068] Mix 4mg OA, 0.62mg NHS and 1.14mg DCC in 240μL of DMF, incubate at room temperature for 2h, then add 3.8mg BSA (dissolved in 200μL 0.1moL / L NaHCO 3 ), and 3 mg IgG (dissolved in 200 μL 0.1moL / L NaHCO 3 ), and continue to incubate at room temperature for 2 hours. Unreacted small molecules and by-products are removed by ultrafiltration (4°C, 8000rpm, 15min) in an ultrafiltration centrifuge tube (10K). The ultrafiltration centrifuge tube needs to be pretreated before use. Completely pass the pure water through the membrane, then ice bath, then pour out the water and put it on ice to pre-cool, and finally add the protein solution. The speed of centrifugation should not be too fast, and the centrifuge should be pre-cooled to 4°C before starting the centrifugation. The finally obtained conjugate ...

Embodiment 3

[0082] This embodiment provides a fluorescence quenching test paper for detecting okadaic acid and a preparation method thereof.

[0083] (1) Preparation of OA artificial antigen

[0084] Mix 4mg OA, 0.62mg NHS and 1.14mg DCC in 240μL of DMF, incubate at room temperature for 2h, then add 3.8mg BSA (dissolved in 200μL 0.1moL / L NaHCO 3 ), and 3 mg IgG (dissolved in 200 μL 0.1moL / L NaHCO 3 ), and continue to incubate at room temperature for 2 hours. Unreacted small molecules and by-products are removed by ultrafiltration (4°C, 8000rpm, 15min) in an ultrafiltration centrifuge tube (10K). The ultrafiltration centrifuge tube needs to be pretreated before use. Completely pass the pure water through the membrane, then ice bath, then pour out the water and put it on ice to pre-cool, and finally add the protein solution. The speed of centrifugation should not be too fast, and the centrifuge should be pre-cooled to 4°C before starting the centrifugation. The finally obtained conjugate ...

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Abstract

The invention belongs to the field of immunoassay technical methods, and particularly relates to fluorescence quenching test paper for detecting field okadaic acid as well as a preparation method andapplication of the fluorescence quenching test paper. The fluorescence quenching test paper comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate. An OA monoclonal antibody marked by colloidal gold particles is sprayed on the combination pad, detection lines on the nitrocellulose membrane are fluorescent microspheres and OA-BSA mixed solution, and quality control lines are fluorescent microspheres. The fluorescence quenching test paper is high in specificity and free of cross reaction, the sensitivity of the fluorescence quenching test paper is 1.56 ppb and is improved by 9.6 times compared with that of colloidal gold test paper with the same parameters, the detection limit of the fluorescence quenching test paper is 3.12-50 ppb, and the detection time is as short as 9 min. The obtained fluorescence quenching test paper has very high stability on interference of shellfish tissue, can achieve quantification of OA toxin by matching with a simple fluorescence immunochromatographic camera and data analysis software, result judgment is visual, and cause misjudgment of non-professionals can be provided. The fluorescence quenching test paper has bright development and application prospects.

Description

technical field [0001] The invention belongs to the field of immunoassay techniques and methods, and in particular relates to a fluorescence quenching test paper for detecting okadaic acid, a preparation method and application thereof. Background technique [0002] Algae biotoxins are another important factor that threatens the food safety of seafood after bacteria, parasites, viruses and chemical pollutants. Diarrheal shellfish toxins (DSP) are a class of macrolide or polyether compounds , are distributed in the oceans of the world. Ingestion of shellfish contaminated by DSP can cause gastrointestinal disorders, such as nausea, abdominal pain, diarrhea, and vomiting. Okadaic acid (OA) is the main component of DSP. In 2001, my country stipulated that the safety standard of OA in aquatic products was 20 μg / 100g edible tissue. In 2006, it was stipulated that OA should not be detected in pollution-free aquatic products. [0003] At present, the detection methods of red tide t...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486G01N2021/6432
Inventor 江天久陈效陈雨刘威刘建军黄新凤
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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