Aflatoxin M1 detection method and kit

An aflatoxin and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high price, high detection cost, easy inactivation of biochemical reagents, etc., and improve detection sensitivity and accuracy. , the effect of eliminating interference

Active Publication Date: 2016-04-20
HUNAN UNIV OF SCI & TECH
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Problems solved by technology

Chromatography has high requirements for operation technology and high detection cost, while immunoassay needs to use more expensive enzymes, antibodies and other biochemical reagents, and these biochemical reagents are easily inactivated, etc.
The literature Materials Science and Engineering C33 (2013) 2229–2234 published an electrochemical assay method for aflatoxin M1 based on aflatoxin M1 nucleic acid aptamer. This method has high selectivity and detection sensitivity, but the electrode assembly is relatively complicated and requires professional talents. Finish

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  • Aflatoxin M1 detection method and kit
  • Aflatoxin M1 detection method and kit
  • Aflatoxin M1 detection method and kit

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Embodiment 1

[0022] The kit for detecting aflatoxin M1 in this embodiment includes: 40 μL of Tris buffer solution of 3.0 μmol aflatoxin M1 nucleic acid aptamer Apt, 40 μL of Tris buffer solution of 3.0 μmol single-stranded signal probe ssDNA, DNA amplification system, nucleic acid Exonuclease, silver ion reduction detection system. Aptamer Apt of aflatoxin M1 is 5'-ACTGCTAGAGATTTTCCACAT-3'. The single-stranded signal probe ssDNA is 5'-CCCCCCACACCCGATCCCCCCATGTGGAAAATCTCTA-3'. The DNA amplification system includes 10 μL amplification buffer solution, 18 μL deoxymononucleotide triphosphate mixed solution dNTP (10 mM), 2 μL Hi29 DNA polymerase (10 u / μl) and 10 μL NH 4 F (25mM)-NaCl (0.1M) solution; wherein, the composition of the amplification buffer solution is 50mM Tris-HCl, 10mM MgCl 2 , 10mM (NH 4 ) 2 SO 4 , pH7.5 (commercially available). The exonuclease was 2μL ExoIII exonuclease (20u / μL). The silver ion reduction detection system included 150 μL sodium citrate solution (10 mM, p...

Embodiment 2

[0024] The method for the detection of aflatoxin of the present embodiment, the specific operation process is as follows:

[0025]The Tris-HCl (50mM Tris-HCl, pH7.5) solution containing the aflatoxin M1 nucleic acid aptamer Apt and the Tris-HCl (50mM Tris-HCl, pH7.5) solution containing the single-stranded signal probe ssDNA were respectively incubated at 90°C After heating in a water bath for 5 minutes, it was quickly placed in an ice bath for 5 minutes. Take 40 μL of 3.0 μmol aflatoxin M1 nucleic acid aptamer Apt solution and 3.0 μmol signal probe ssDNA solution 40 μL respectively in a 2ml centrifuge tube after the above treatment, and hybridize at 37°C for 1 hour; ~50ng / mL of AFM1 was added to the above solution, and after 2 minutes, 10 μL of amplification buffer solution was added (the composition of amplification buffer solution was 50mM Tris-HCl, 10mM MgCl 2 , 10mM (NH 4 ) 2 SO 4 , pH7.5), 18 μL dNTP (10 mM) and 2 μLPhi29 DNA polymerase (10u / μl), reacted at 37 ° C fo...

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Abstract

The invention discloses an aflatoxin M1 detection method and a kit. The method includes: hybridizing aflatoxin M1 aptamer (Apt) and single-chain signal probe DNA (ssDNA) to form a hybrid chain Apt-ssCNA; putting the hybrid chain Apt-ssCNA into a to-be-tested sample, when AFM1 exists, allowing reaction between the AFM1 and an Apt segment of the hybrid chain Apt-ssCNA to generate an Apt-AFM1 compound, and releasing the single-chain signal probe DNA (ssDNA); subjecting the hybrid chain Apt-ssCNA to DNA amplification to generate double-stranded DNA, and quickly hydrolyzing the double-stranded DNA into mononucleotides under selective catalytic action of exonuclease, wherein the single-chain signal probe DNA are retained and not hydrolyzed; under ssDNA induction, allowing silver ion reduction to generate near infrared fluorescent silver nanoclusters; detecting system fluorescence intensity, and detecting AFM1 content of the to-be-tested sample according to a relation between the fluorescence intensity and the AFM1 content. The aflatoxin M1 detection method has the advantages of high sensitivity, simplicity in operation and low cost.

Description

technical field [0001] The invention belongs to the technical field of nanobiological sensing and biological detection, and in particular relates to a method and a kit for detecting aflatoxin M1. Background technique [0002] In 1993, aflatoxin was classified as a Class 1 carcinogen by the Cancer Research Institute of the World Health Organization (WHO). It is a highly toxic and highly toxic substance that is extremely harmful to humans and animals. Aflatoxins are metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are widely found in soil, soybeans, rice, corn, macaroni, condiments, milk and its products, edible oil, meat (fish) products, peanuts and walnuts and other animals and plants, and various nuts, especially peanuts and walnuts. Due to the wide range of aflatoxin pollution sources, it is impossible to completely eliminate aflatoxin pollution. After mammals ingest feed or food contaminated with aflatoxin, they can be hydroxylated in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2525/205C12Q2521/319C12Q2563/107
Inventor 曾云龙邓克勤刘婷易守军黄昊文夏晓东唐春然
Owner HUNAN UNIV OF SCI & TECH
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