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Fungaltoxin detection method and detection kit based on double signal amplification

A mycotoxin and dual-signal technology, which is applied in biological testing, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of expensive, difficult to popularize and apply, and high detection cost, and achieve simple operation and high sensitivity , the effect of low detection cost

Active Publication Date: 2021-07-02
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high price of commonly used imported kits and the high cost of detection, it is difficult to popularize and apply

Method used

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  • Fungaltoxin detection method and detection kit based on double signal amplification
  • Fungaltoxin detection method and detection kit based on double signal amplification
  • Fungaltoxin detection method and detection kit based on double signal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A kit for detecting zearalenone toxin, comprising mycotoxin capture probes S1-Z1-MB, S2-MB, Nt.AlwI enzyme, Cas12a-crRNA complex, single-stranded DNA R1, and fluorescent signal substrate;

[0058] Wherein, the mycotoxin capture probe S1-Z1-MB is prepared by the following method:

[0059] (1) Preparation of Z1-MB:

[0060] ①Take 20 μL of monodisperse streptavidin-modified magnetic microspheres MB (the concentration of the magnetic microspheres is 5 mg / mL, purchased from the Pharmaceutical Analysis and Quality Evaluation Laboratory of Tianjin University School of Pharmacy, Tianjin Basele Chromatography Technology Development Center) Mix and disperse 50 μL of zearalenone aptamer Z1 with 10 μM zearalenone aptamer Z1 whose 5’ end is linked with biotin molecules, and disperse in 500 μL Tris-HCl buffer (10 mM Tris-HCl, 1 mM EDTA, 1M NaCl, 0.01%~0.1% In Tween-20), vortex at room temperature for 30 minutes, so that the nucleic acid aptamer Z1 of zearalenone is modified on the s...

Embodiment 2

[0087] A method for detecting zearalenone toxin with double signal amplification, the steps are as follows:

[0088] (1) Add 100 μL of the zearalenone toxin capture probe S1-Z1-MB prepared in Example 1 to the sample solution to be tested, and the total volume is 500 μL; place the centrifuge tube on a rotary mixer at room temperature, Rotate and mix at room temperature for 30 minutes to allow the mycotoxin to fully react with the probe, and the mycotoxin in the sample to be tested will combine with the corresponding nucleic acid aptamer on S1-Z1-MB to form Z1-MB-mycotoxin and release the S1 sequence; After magnetic separation of the reacted solution, the supernatant containing S1 was obtained; transferred to a new EP tube and kept at 4°C for use.

[0089] (2) Add 100 μL (excessive amount) of S2-MB prepared in Example 1 to the supernatant containing S1 in step (1), vortex and mix well, react in an air-bath shaker at room temperature for 30 min, and S1 passes through The base co...

Embodiment 3

[0100] The specificity of embodiment 3 zearalenone toxin detection method

[0101] Select aflatoxin, ochratoxin, fumonisin, T-2 toxin and vomitoxin five kinds of mycotoxins and zearalenone toxin as interfering to test at the same time, so as to verify Example 2 zearalen of the present invention The specificity of the ketone toxin detection method, the concentration of each of the above toxins is 10ng / mL. Detection method is with embodiment 2; Specificity result is as Figure 4 Shown: It can be seen from the fluorescence value that the zearalenone toxin value is significantly higher than the results of other toxins. Therefore, the zearalenone toxin detection method in Example 2 of the present invention has a high detection rate for zearalenone toxin specificity.

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Abstract

The invention discloses a Fungaltoxin detection method and detection kit based on double signal amplification, which belong to the technical field of fungaltoxin detection. According to the fungaltoxin detection method based on double signal amplification, the nicking enzyme signal amplification and the CRISPR / Cas12a technology are combined, and twice signal amplification in fungaltoxin detection is realized; the fungaltoxin detection method based on double signal amplification is simple and convenient to operate, does not need to carry out complex pretreatment on a sample to be detected, and is low in detection cost; the method can be used for detecting low-concentration fungaltoxin and is high in sensitivity; the method can be used for specific detection of fungaltoxin in food, and qualitative and quantitative analysis of target fungaltoxin is realized.

Description

technical field [0001] The invention belongs to the technical field of mycotoxin detection, and in particular relates to a double signal amplification mycotoxin detection method and a detection kit. Background technique [0002] At present, there are more than 400 kinds of mycotoxins known, which can contaminate almost all crops. Food contaminated by mycotoxins will not only cause food corruption and deterioration, but also cause food poisoning in severe cases, threatening human health and life safety. [0003] Mycotoxins have carcinogenicity, toxicity, teratogenicity and other hazards, and can cause nephrotoxicity, hepatotoxicity and reproductive abnormalities in humans and animals. Some foodborne mycotoxins can cause acute episodes, in which severe disease symptoms develop rapidly after ingestion of food contaminated with the mycotoxin. Other mycotoxins can cause long-term health effects, including cancer and immune deficiency. [0004] Among the existing detection metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12Q1/682
CPCG01N33/5308C12Q1/682C12Q2521/313C12Q2521/327C12Q2525/161C12Q2563/107Y02A50/30
Inventor 吴薇姚锡京俞鑫杨庆利
Owner QINGDAO AGRI UNIV
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