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Detection method and detection kit for patulin

A technology of patulin and detection method, which is applied in the field of detection method and detection kit of patulin, can solve problems such as the further application of patulin detection, and achieve the effect of eliminating interference

Inactive Publication Date: 2016-05-04
HUNAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Chinese patent CN104593374A discloses an oligonucleotide aptamer that specifically recognizes patulin, which can be used for the detection of patulin, but there is no further application for the detection of patulin

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  • Detection method and detection kit for patulin

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Experimental program
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Effect test

Embodiment 1

[0025] A kit for detecting patulin at least includes: patulin aptamer, single-stranded signal probe DNA capable of hybridizing with patulin aptamer, DNA amplification system, exonuclease, silver Ion reduction detection system. The patulin aptamer is 5'-CAGCTCAGAAGCTTGATCCCGGCCCGCCAACCCGCATCATTCACTGATATTTACCTTGACTATCAGTCGTGC-3'. The single-stranded signal probe DNA is 5'-CCCCCCACACCCGATCCCCCCGCACGACTGATAGTC-3'. DNA amplification system includes buffer solution (buffer solution is made of Tris-HCl, MgCl 2 and (NH 4 ) 2 SO 4 Composition), deoxymononucleotide triphosphate mixed solution dNTP and Phi29 DNA polymerase. . The exonuclease is ExoIII exonuclease. The reducing agent in the silver ion reduction detection system is ascorbic acid.

Embodiment 2

[0027] A method for detecting patulin, the specific operation process is as follows:

[0028] Each DNA stock solution was heat-treated at 95°C for 5 min before use, and left at room temperature for 30 min. Then, take 40 μL of hybridization buffer solution containing 3.0 μmol of patulin aptamer (Apt) and 40 μL of hybridization buffer solution of 3.0 μmol of signal probe ssDNA (Sp) in a 2 ml centrifuge tube, and hybridize at 37°C for 1 Hours, a patulin aptamer-signal probe hybrid (Apt-Sp) was generated.

[0029] At 37°C, add patulin with a concentration of 0~50ng / mL to the Apt-Sp solution in turn, and the patulin reacts with the Apt segment in Apt-Sp to generate the nucleic acid aptamer-patulin , releasing Sp. At this time, there are substances such as Sp, remaining (unreacted) Apt-Sp and nucleic acid aptamer-patulin in the system.

[0030] Add 10 μL buffer solution (buffer solution composition is 50mM Tris-HCl, 10mM MgCl 2 , 10mM (NH 4 ) 2 SO 4 ,pH7.5), then add dNTP (10...

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Abstract

The invention relates to a detection method and detection kit for patulin. The method comprises the following steps that (A) a patulin aptamer (Apt) singlestrand signal DNA (ssDNA) probe is subjected to hybridization, and a hybridization chain is formed; (B) the hybridization chain is in reaction with a to-be-tested sample, and when patulin exists in the to-be-tested sample, the ssDNA probe is released by the reaction of the hybridization chain and the patulin; (C) the hybridization chain is changed into double chain DNA by the aid of DNA amplification, the double chain DNA hydrolyzed into mononucleotide under the selective catalysis of excision enzyme, and the ssDNA probe in the system is not hydrolyzed but reserved; (D) under ssDNA induction, silver ions are reduced to generate near infrared fluorescent silver nano clusters; the system fluorescence intensity is detected, so that the content of the patulin in the to-be-tested sample is measured. The method has the advantages that the sensitivity is high, the operation is simple, the cost is low and the like.

Description

technical field [0001] The invention relates to the field of nano-biological sensing and biological detection, in particular, it provides a detection method and a detection kit for patulin. Background technique [0002] Patulin is a toxic fungal metabolite produced by molds. Molds capable of producing patulin mainly include Penicillium, Aspergillus, and Clothes mould. These molds can widely infect a variety of fruits, vegetables, grains, feeds, and Chinese medicinal materials, resulting in the loss of these crops and their products. Contaminated by patulin. For apples infected by these molds, patulin can still be detected in the pulp 3 cm away from the moldy spots. Patulin is stable to heat, its structure cannot be destroyed by heating at 100°C for 15 minutes, and it can be preserved for a long time in acidic solution and organic solvent. Patulin has been used as an antibiotic because it inhibits the growth of a variety of bacteria. It has acute toxicity such as convulsio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2525/205C12Q2521/319C12Q2563/107
Inventor 曾云龙符方芬邓克勤杨凡黄昊文唐春然
Owner HUNAN UNIV OF SCI & TECH