Detection method of fungaltoxin DON (deoxynivalenol) and detection kit

The technology of deoxynivalenol and detection kit is applied in biological testing, biochemical equipment and methods, measuring devices, etc., and can solve the problems of high price, easy occurrence of false positives, easy inactivation of biochemical reagents, and the like, Achieve the effect of improving detection sensitivity and precision and eliminating the interference of background fluorescence

Active Publication Date: 2016-06-22
HUNAN UNIV OF SCI & TECH
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Problems solved by technology

[0003] At present, the methods for detecting DON mainly include thin-layer chromatography, enzyme-linked immunosorbent assay, gas chromatography, liquid chromatography, and immunoassay, etc. Chromatography requires high operating technology and high detection cost, while immunoassay requires the use of more expensive enzymes and antibodies and other biochemical reagents, and these biochemical reagents have shortcomings such as easy inactivation
Among them, the enzyme-linked immunosorbent assay (ELISA) has the advantages of rapidity, convenience, and simple pretreatment, but the DON required by ELISA is highly efficient, and the preparation process of specific monoclonal or polyclonal antibodies is complicated, and the antibodies used are generally different from the acetylated analogs of DON. Cross reaction, prone to false positives; patent CN102559686A discloses a deoxynivalenol nucleic acid aptamer and is applied to the detection of DON. This method has the characteristics of high selectivity and rapidity for DON detection, but it needs to use Modified DNA

Method used

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  • Detection method of fungaltoxin DON (deoxynivalenol) and detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0029]A kit for detecting mycotoxin deoxynivalenol, comprising: deoxynivalenol nucleic acid aptamer DONApt, single-stranded signal probe ssDNA, DNA amplification system, exonuclease III, silver ion Restoration of the detection system. DONApt is 5'-GCATCACTACAGTCATTACGCATCGTAGGGGGGATCGTTAAGGAAGTGCCCGGAGGCGGTATCGTGTGAAGTGC-3'. The single-stranded signal probe ssDNA is 5'-CCCCCCACACCCGATCCCCCCGCACTTCACACGATA-3'. DNA amplification system includes deoxygenated mononucleotide triphosphate mixture dNTP solution, Phi29 DNA polymerase, NH 4 F-NaCl solution and amplification buffer solution, the amplification buffer solution consists of Tris-HCl, MgCl 2 , (NH 4 ) 2 SO 4 composition. The exonuclease is ExoIII. The silver ion reduction detection system comprises sodium citrate solution and reducing agent vitamin C solution.

Embodiment 2

[0031] A method for detecting mycotoxin deoxynivalenol, the specific operation process is as follows:

[0032] Heat the Tris-HCl (50mM, pH 7.5) solution of the deoxynivalenol nucleic acid aptamer DONApt and the Tris-HCl solution of the single-stranded signal probe ssDNA at 90°C for 5 minutes, and quickly place it in an ice bath 5 minutes, remove and store at room temperature for later use.

[0033] Take 40 μL of the DONApt solution containing 3.0 μmol of deoxynivalenol nucleic acid aptamer and 40 μL of 3.0 μmol of signal probe ssDNA solution that have been treated above, respectively, and place them in a 2ml centrifuge tube, and perform hybridization reaction at 37°C for 1 hour; follow the above method Prepare several hybridization solutions, and then add 5 μL of deoxynivalenol with a concentration of 0 to 1000 ng / mL to the above hybridization solution, so that the concentration (DON concentration calculated when the volume is 470 μL) is 0 ng / mL, 0.001ng / mL, 0.005ng / mL, 0.01n...

Embodiment 3

[0037] (1) Sample processing

[0038] Flour sample 1 was purchased from a supermarket, and the background DON value was 0.927mg / Kg. The flour used in Example 3 was all flour sample 1; the DON recovery was measured in the flour sample.

[0039] Take nine 10mL clean small beakers, numbered A1, A2, A3, B1, B2, B3, C1, C2, and C3; accurately weigh 3 copies of 0.1g flour samples, and place them in the beakers numbered A1, A2, and A3 respectively. Then add 15 μL of DON solution with a concentration of 30 μg / mL to the beakers numbered A1, A2, and A3 respectively; then accurately weigh 3 parts of 0.25 g flour samples, and place them in the beakers numbered B1, B2, and B3 respectively Then add 15 μL of DON solution with a concentration of 15 μg / mL to B1, B2 and B3 respectively; also accurately weigh 3 parts of 0.5 g flour samples and place them in beakers numbered C1, C2 and C3 respectively. Then add 15 μL of DON solution with a concentration of 5 μg / mL to C1, C2, and C3 respectively;...

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Abstract

The invention discloses a detection method of fungaltoxin DON (deoxynivalenol) and a detection kit. The method comprises steps as follows: DONApt and a single-stranded signal probe ssDNA are hybridized, and a DONApt-ssDNA (single-stranded deoxyribonucleic acid) hybrid chain is formed; a to-be-detected sample is added to the DONApt-ssDNA hybrid chain, and when the to-be-detected sample has DON, DONApt-ssDNA reacts with DON to generate DONApt-DON and release ssDNA; remaining DONApt-ssDNA hybrid chain in the system forms double-stranded DNA through DNA amplification; double-stranded DNA is hydrolyzed into mononucleotide under catalysis of exonuclease and is removed, and ssDNA in the system is reserved; silver ions and a reducing agent are added to the system, and silver ions are reduced to generate near infrared fluorescence silver nano-clusters under induction of ssDNA; finally, the content of DON in the to-be-detected sample is calculated according to the relation between the near infrared fluorescence intensity and the quantity of DON.

Description

technical field [0001] The invention relates to the field of nano-biological sensing and biological detection, in particular to a detection method and a detection kit for mycomycin deoxynivalenol. Background technique [0002] Deoxynivalenol (DON), also known as vomitoxin, is a B-type trichothecene compound, mainly produced by Fusarium graminearum and Fusarium pink, mostly distributed in wheat, barley, Corn and other grain seeds also contaminate food products. DON can produce a wide range of toxic effects on humans and animals. It is highly toxic or moderately toxic and can cause acute poisoning symptoms such as vomiting, diarrhea, and fever. It is also closely related to anemia, immunosuppression, esophageal cancer, and Keshan disease. In addition, it also has It often contaminates crops together with other mycohalotoxins such as aflatoxin, and can interact with each other after entering the human body. DON is worldwide, especially in China, Japan, USA, Argentina, South A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53G01N21/64
CPCC12N15/115C12N2310/16G01N21/6486G01N33/5308
Inventor 曾云龙陈佳鑫林雨欢邓克勤易守军黄昊文夏晓东唐春然
Owner HUNAN UNIV OF SCI & TECH
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