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Method for quantitatively detecting aflatoxin B1

A technology for quantitative detection of aflatoxins, applied in biochemical equipment and methods, measuring devices, instruments, etc., can solve problems such as unfavorable on-site detection, cumbersome operation, complicated pretreatment, etc., and achieve good application prospects and good repeatability. , good specificity

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)
[0004] At present, many detection methods for aflatoxin B1 have the disadvantages of requiring many reagents, cumbersome operation, long detection cycle, poor reproducibility, expensive equipment, complicated pretreatment, etc., which are not conducive to on-site detection

Method used

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  • Method for quantitatively detecting aflatoxin B1
  • Method for quantitatively detecting aflatoxin B1
  • Method for quantitatively detecting aflatoxin B1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Synthesis of aptamer biosensor and detection of aflatoxin B1 in combination with a blood glucose meter

[0047] 1. Experimental method

[0048] 1.1 Synthesis of DNA-sucrase polymer

[0049] 1.1.1 Activation of sucrase molecules

[0050] 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.

[0051] 1.1.2 Activation of DNA molecules

[0052] Take 100 μl of 100 μM complementary DNA (thiol-DNA, 3'-SH-A12-CAACCCGTGCACA-5'), 2 μl of 0.1M bufferB, 2 μl of 30mMTCEP (ultrapure water) into a 1.5ml centrifuge tube, vortex to mix, and place in a constant temperature mixer reaction at room temperature for 1 h (among them, ① treatment of complementary DNA: centrifuge the synthesized solid DNA (4°C, 12000 rcf, 5 min), add 345 μl ultrapure water as required, vortex slightly to obtain 345 μl 100 μM DNA solution; ② TCEP Preparation: TCEP molecular we...

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 80μl of 100μM complementary DNA (thiol-DNA, 3'-SH-A12-CAACCCGTGCACA-5'), 1μl of 0.1MbufferB, 1μl of 30mMTCEP (ultrapure water) and add it to a 1.5ml centrifuge tube, vortex and mix well, and place in a thermostatic mixer On, react 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), absorb 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; suck ...

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, 3'-SH-A12-CAACCCGTGCACA-5'), 3 μl of 0.1M bufferB, 3 μl of 30mMTCEP (ultrapure water) and add it to a 1.5ml centrifuge tube, vortex and mix well, and place in a constant temperature mixer On, room temperature reaction 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), absorb 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; suck out the thi...

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Abstract

The invention discloses an aptamer of aflatoxin B1 and a complementary DNA sequence of the aptamer, further discloses a method for quantitatively detecting aflatoxin B1, and belongs to the field of quantitative detection of aflatoxin B1. The method includes the following steps that 1, an aptamer biosensor is prepared; 2, aflatoxin B1 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 aflatoxin B1 in food by combining the aptamer biosensor with the glucometer is easy and convenient to implement, good in specificity and repeatability and high in sensitivity and provides a new means for quantitatively detecting aflatoxin B1.

Description

technical field [0001] The invention relates to a method for quantitatively detecting aflatoxin B1, in particular to a method for quantitatively detecting aflatoxin B1 with an aptamer biosensor combined with a blood glucose meter, and belongs to the field of quantitative detection of aflatoxin B1. 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. Aflatoxin B1 (AFB1) is identified as a primary carcinogen by the World Health Organization (WHO) International Agency for Research on Cancer, and the development of highly sensitive aflatoxin detection methods is the focus of interna...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/543
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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