Aflatoxin B1 aptamer, DNA sensor, kit and application

A technology of aflatoxin and nucleic acid aptamer, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of cumbersome operation and high cost, and achieve simple operation, low cost, and portability Effect

Active Publication Date: 2016-04-20
SHENZHEN KIVITA INNOVATIVE DRUG INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, these methods are costly and require the use of large and expensive instruments, which are cumbersome to operate

Method used

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  • Aflatoxin B1 aptamer, DNA sensor, kit and application
  • Aflatoxin B1 aptamer, DNA sensor, kit and application
  • Aflatoxin B1 aptamer, DNA sensor, kit and application

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0056] Preparation Example 1: Preparation of DNA sensor solution.

[0057] Mix DNA(A), DNA(B), DNA(C), dNTP and RCA reactions. Wherein, the concentrations of DNA (A), DNA (B), DNA (C), and dNTP are 40 nM, 60 nM, 40 nM, and 0.5 nM, respectively.

[0058] Wherein, the sequence of DNA (A) is:

[0059] 5'-ACTGATATTAACCCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3'.

[0060] The sequence of DNA(B) is:

[0061] 5'-GTTCCCGTGCTGTTGTCTCTCTGTGTCTGCACGGGTTCGCTAGGCCCACA-3' (SEQ ID NO: 7).

[0062] The sequence of DNA(C) is:

[0063] 5'-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3'.

Embodiment 1

[0065] (1) Use 70% methanol to configure 50 μl of aflatoxin B1 standard substances with different concentration gradients (500 μg / L, 50 μg / L, 5 μg / L, 500 ng / L, 50 ng / L respectively);

[0066] (2) Add 50 μl of DNA sensor solution and 10UPhi29 polymerase to it, mix well, incubate at 30°C for 60 minutes, and heat at 90°C for 5 minutes to terminate the reaction;

[0067] Add hemin solution and 2×HEPES buffer solution, incubate at room temperature for 30 min, add ABTS reduced colorless substrate and 30% hydrogen peroxide, the final concentration of the hemin is 0.5 μM; the final concentration of the ABTS reduced colorless substrate is 2mM; the final concentration of the hydrogen peroxide is 0.1mM; read the color signal after 5min.

[0068] Make a standard curve.

Embodiment 2

[0070] Take 10 g of the sample to be tested (the content of aflatoxin B1 is known to be 1 μg / L), add 500 μl of aflatoxin extract (70% methanol) and mix it thoroughly, centrifuge, and extract 50 μl of the supernatant;

[0071] Add 50 μl of DNA sensor solution and 10UPhi29 polymerase to the supernatant, mix well, incubate at 30°C for 60 minutes, and heat at 90°C for 5 minutes to terminate the reaction;

[0072] Adding hemin solution and HEPES buffer solution, incubating at room temperature for 30 minutes, adding ABTS reduced colorless substrate and 30% hydrogen peroxide; the final concentration of the hemin is 0.5 μM; the final concentration of the ABTS reduced colorless substrate is 2 mM; The final concentration of the hydrogen peroxide is 0.1 mM. After 5 minutes, the color change can be seen with the naked eye. The color signal was read, and the content of aflatoxin B1 in the tested sample was determined to be 1 μg / L according to the standard curve measured in Example 1.

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Abstract

The invention provides an aflatoxin B1 aptamer, a DNA sensor, a kit and application. The aflatoxin B1 aptamer has the nucleotide sequence as follows: 5'-GTTmmmmmmmTGTTGTCTCTCT GTGTCTnnnnnnnTTCGCTAGGCCCACA-3', each m and n independently are A or T or C or G, and the m chain segment and the n chain segment achieve complementary pairing. According to the aflatoxin B1 aptamer, the DNA sensor, the kit and the application, no large instrument equipment is needed when the aflatoxin B1 is measured, and therefore the cost is low; a constant-temperature reaction is achieved, qualitative detection of naked eyes can be achieved, and quantitative detection can also be achieved; in addition, no fluorochrome is needed, and therefore the cost is greatly reduced.

Description

technical field [0001] The present invention relates to the field of biological detection, and more specifically, relates to an aflatoxin B1 nucleic acid aptamer, a DNA sensor comprising the nucleic acid aptamer, a test kit comprising the DNA sensor, and the DNA sensor and the test kit. Background technique [0002] Aflatoxins are secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus, and the probability of aflatoxins appearing in food and feed in hot and humid areas is the highest. They exist in soil, animals, plants, and various nuts, especially grain and oil products such as peanuts, corn, rice, soybeans, and wheat. They are the most toxic mycotoxins and are extremely harmful to human health. In natural food, aflatoxin B1 is the most common and most harmful. [0003] Currently available aflatoxin detection methods mainly include: [0004] (1) TLC [0005] Thin layer chromatography is the most widely used separation technique in the stu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68
CPCC12N15/115C12Q1/6844C12Q2531/125C12Q2525/205C12Q2565/607
Inventor 张晶珠毛瑜
Owner SHENZHEN KIVITA INNOVATIVE DRUG INST
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