Rapid detection method for ochratoxin A

A technology for ochratoxin and detection method, applied in the field of rapid detection method and detection kit for ochratoxin A, can solve the problems of expensive equipment, high price, complicated operation, etc. Effect

Inactive Publication Date: 2016-05-04
HUNAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] Usually, chromatography is not sensitive enough for the detection of trace fungi in food. Although the liquid-mass spectrometry method has high sensitivity and fast speed, the equipment is expensive, and only professional technicians should be required to perform the detection. The technical requirements are high, and it is not suitable for popularization and However, the detection method of ochratoxin based on nucleic acid aptamer described in the literature may need to use expensive labeled (biological materials, etc.) DNA, or the operation is complicated and other deficiencies

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  • Rapid detection method for ochratoxin A

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Embodiment 1

[0030] The kit for detecting ochratoxin A of the present invention at least includes: ochratoxin A nucleic acid aptamer, single-stranded signal probe ssDNA, DNA amplification system, exonuclease, and copper ion reduction detection system. The ochratoxin A nucleic acid aptamer is 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAC-3'. The single-stranded signal probe ssDNA is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGTCCCATGCTCC-3'. Described DNA amplification system comprises buffer solution, and buffer solution is made of Tris-HCl, MgCl 2 , (NH 4 ) 2 SO 4 Composition, deoxymononucleotide triphosphate mixed solution (dNTP) and Phi29 DNA polymerase. The exonuclease is ExoIII exonuclease.

Embodiment 2

[0032] The rapid detection method of ochratoxin A, the specific operation process is as follows:

[0033] Each DNA stock solution was heat-treated at 95°C for 5 min before use, and left at room temperature for 30 min. Then, 40 μL of hybridization buffer solution containing 3.0 μmol ochratoxin A nucleic acid aptamer (Apt) and 3.0 μmol signal probe ssDNA hybridization buffer solution 40 μL were placed in a 2 ml centrifuge tube, and hybridized at 37 ° C for 1 hour to generate Ochratoxin A nucleic acid aptamer-signaling probe hybrid (Apt-ssDNA).

[0034] At 37°C, ochratoxin A with a concentration of 0-50 ng / mL was added to the Apt-ssDNA solution in turn, and ochratoxin A reacted with the ochratoxin A nucleic acid aptamer to generate the nucleic acid aptamer-ochratoxin A, Release of ssDNA. At this time, there are substances such as ssDNA, remaining (unreacted) Apt-ssDNA and nucleic acid aptamer-ochratoxin A in the system.

[0035] Add 10 μL buffer solution (buffer solution compo...

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Abstract

The invention relates to a rapid detection method for ochratoxin A. The method comprises the following steps that A, an aptamer (Apt) of the ochratoxin A is hybridized with single-stranded signal probe DNA (ssDNA) to form a hybridized chain; B, the hybridized chain is made to make contact with a sample to be tested, and when the ochratoxin A exists in the sample to be tested, the hybridized chain and the ochratoxin A are subjected to a reaction to release the single-stranded signal probe DNA (ssDNA); C, DNA amplification is utilized for making the hybridized chain into a double-stranded DNA, then, an excision enzyme is used for selectively catalyzing the hydrolysis of the double-stranded DNA to form mononucleotide, and the single-stranded signal probe DNA (ssDNA) is not hydrolyzed and reserved; D, under the induction of the ssDNA, copper ions are reduced to generate near infrared fluorescence copper nanometer particles; the system fluorescence strength is detected, and therefore the content of the ochratoxin A in the sample to be tested is tested. The method has the advantages of being high in sensitivity, easy to operate, low in cost 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 rapid detection method of ochratoxin A and a detection kit. Background technique [0002] Ochratoxins include 7 compounds with similar structures, general structural formula: R1=Cl or H; R2=H, CH3 or C2H5. Among them, ochratoxin A (R1=C1, R2=H) is the most toxic and is most common in moldy grains and feed. [0003] Ochratoxin A is produced by fungi such as Aspergillus and Penicillium, and it widely contaminates grains such as wheat, corn, barley, oats, rye, rice and millet, as well as peanuts and vegetables (beans). The toxin can show up in the meat of animals that ingest moldy feed. Humans will directly or indirectly absorb ochratoxin. Ochratoxin mainly damages the liver and kidneys, and can also cause intestinal mucosal inflammation and necrosis, and has teratogenic effects. Therefore, the detection of ochratoxin A is very necessar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/115
CPCC12Q1/6816C12N15/115C12N2310/16C12Q2525/205C12Q2521/319C12Q2563/137
Inventor 曾云龙刘婷黄昊文徐合意邓克勤易守军唐春然
Owner HUNAN UNIV OF SCI & TECH
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