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Method for quantitatively detecting ochratoxin A

A technology for quantitative detection of ochratoxin, applied in the field of quantitative detection of ochratoxin A, aptamer biosensor combined with blood glucose meter for quantitative detection of ochratoxin A, achieving good specificity, low detection limit, and good application prospects Effect

Active Publication Date: 2016-03-16
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the blood glucose meter can only detect glucose as a substance, and the detection range is 0.6-33mmol / l (10-600mg / dl)

Method used

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  • Method for quantitatively detecting ochratoxin A
  • Method for quantitatively detecting ochratoxin A
  • Method for quantitatively detecting ochratoxin A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Synthesis of aptamer biosensor and detection of ochratoxin A in combination with a blood glucose meter

[0046] 1. Experimental method

[0047] 1.1 Synthesis of DNA-sucrase polymer

[0048] 1.1.1 Activation of sucrase molecules

[0049] Take 400 μl of 20 mg / ml sucrase (buffer B) and mix with 1 mg sulfo-SMCC, vortex and shake for 5 min, place on a constant temperature mixer, and react at room temperature for 2 h.

[0050] 1.1.2 Activation of DNA molecules

[0051] Take 100 μl of 100 μM complementary DNA (thiol-DNA, 5'-CCCACACCCGATCAAAAAAAAAAAA-SH-3'), 2 μl of 0.1M bufferB, 2 μl of 30mM TCEP (ultrapure water) and add it to a 1.5ml centrifuge tube, vortex and mix well, and place it on a constant temperature mixer. React at room temperature for 1 hour (1) Treatment of complementary DNA: Centrifuge the synthesized solid DNA (4°C, 12000 rcf, 5 min), add 345 μl of ultrapure water as required, and vortex slightly to obtain 345 μl of 100 μM DNA solution; 2) Preparat...

Embodiment 2

[0079] Synthesis of Example 2 Aptamer Biosensor

[0080] 1.1 Synthesis of DNA-sucrase polymer

[0081] 1.1.1 Activation of sucrase molecules

[0082] Take 300 μl of 20 mg / ml sucrase (buffer B) and mix with 0.5 mg sulfo-SMCC, vortex and shake for 5 min, place on a constant temperature mixer, and react at room temperature for 1 h.

[0083] 1.1.2 Activation of DNA molecules

[0084] Take 80 μl of 100 μM complementary DNA (thiol-DNA, 5'-CCCACACCCGATCAAAAAAAAAAAA-SH-3'), 1 μl of 0.1M bufferB, 1 μl of 30mM TCEP (ultrapure water) and add it to a 1.5ml centrifuge tube, vortex and mix well, and place it on a thermostatic mixer. Reaction at room temperature for 0.5h.

[0085] 1.1.3 Synthesis of DNA-sucrase polymer

[0086] Centrifuge the reaction solution of sucrase-SMCC and thiol-DNA (25°C, 12000rcf, 5min), draw the supernatant, and add them to ultrafiltration tubes (Amicon-100K for sucrase-SMCC; Amicon-100K for thiol-DNA) 3K), centrifuge (25°C, 12000rcf, 10min), wash 8 times with...

Embodiment 3

[0094] Example 3 Synthesis of Aptamer Biosensors

[0095] 1.1 Synthesis of DNA-sucrase polymer

[0096] 1.1.1 Activation of sucrase molecules

[0097] Mix 500 μl of 20 mg / ml sucrase (buffer B) with 2 mg of sulfo-SMCC, vortex for 5 min, place on a constant temperature mixer, and react at room temperature for 3 h.

[0098] 1.1.2 Activation of DNA molecules

[0099] Take 120 μl of 100 μM complementary DNA (thiol-DNA, 5'-CCCACACCCGATCAAAAAAAAAAAA-SH-3'), 3 μl of 0.1M bufferB, 3 μl of 30mM TCEP (ultrapure water) and add it to a 1.5ml centrifuge tube, vortex and mix well, and place it on a constant temperature mixer. Reaction at room temperature for 2h.

[0100] 1.1.3 Synthesis of DNA-sucrase polymer

[0101] Centrifuge the reaction solution of sucrase-SMCC and thiol-DNA (25°C, 12000rcf, 5min), draw the supernatant, and add them to ultrafiltration tubes (Amicon-100K for sucrase-SMCC; Amicon-100K for thiol-DNA) 3K), centrifuge (25°C, 12000rcf, 10min), wash 8 times with bufferA; ...

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Abstract

The invention discloses an aptamer of ochratoxin A and a nucleotide sequence of complementary DNA of the aptamer, further discloses a method for quantitatively detecting ochratoxin A, and belongs to the field of quantitative detection of ochratoxin A. The method includes the following steps that 1, an aptamer biosensor is prepared; 2, ochratoxin A in a sample to be detected is extracted to obtain sample extraction liquor, the sample extraction liquor is added into the aptamer biosensor and evenly mixed, and incubation is performed; 3, supernatant liquor is separated, and an excessive sucrose solution is added for a reaction; 4, quantitative detection is performed through a glucometer. The method for quantitatively detecting ochratoxin A in food or feed by combining the aptamer biosensor with the glucometer is good in specificity and repeatability, high in sensitivity, fast to implement and low in cost and provides a new means for quantitatively detecting ochratoxin A.

Description

technical field [0001] The invention relates to a method for quantitatively detecting ochratoxin A, in particular to a method for quantitatively detecting ochratoxin A with an aptamer biosensor combined with a blood glucose meter, and belongs to the field of quantitative detection of ochratoxin A. Background technique [0002] Mycotoxins mainly refer to the toxic metabolites produced by molds or fungi in their contaminated food, which can enter humans and animals through feed or food, causing acute or chronic toxicity to humans and animals, and damage the body's liver , kidney, nervous tissue, hematopoietic tissue and skin tissue, etc. Common mycotoxins include aflatoxin, ochratoxin, zearalenone, deoxynivalenol, T-2, HT-2 toxin, fumonisin, etc. Among them, ochratoxin A (OTA) mainly invades the liver and kidney of animals, mainly causing kidney damage, and a large amount of toxin may also cause inflammation and necrosis of animal intestinal mucosa. The International Agency ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/11G01N33/543
CPCC12N15/115C12N2310/16G01N33/54326
Inventor 郑楠文芳李松励张养东赵圣国王加启
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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