Aptamer for detecting aflatoxin M1, sensor, kit and application thereof

A kind of technology of aflatoxin and nucleic acid aptamer, applied in the direction of biological testing, instruments, measuring devices, etc., can solve the problems of limitation and difficulty in guaranteeing antibody stability, and achieve the effect of lower detection limit, high sensitivity and good selectivity

Active Publication Date: 2017-09-15
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, it is difficult to ensure the stability of antibodies during transportation and storage, which limits the application of this method.

Method used

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  • Aptamer for detecting aflatoxin M1, sensor, kit and application thereof
  • Aptamer for detecting aflatoxin M1, sensor, kit and application thereof
  • Aptamer for detecting aflatoxin M1, sensor, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of embodiment 1 sensor solution

[0055] Mix DNA Sequence A and DNA Sequence B reactions. Wherein, the concentrations of DNA sequence A and DNA sequence B are both 10 nM.

[0056] Wherein, the sequence of DNA sequence A is:

[0057] 5'-GGTGTGACGGATAATCTGGTTTAGCTACGCCTTCCCCGTGGCGATGTTTCTTAGCGCCTTAC-3'.

[0058] The sequence of DNA sequence B is:

[0059] 5'-ATCCGTCACACCTGCTCTGACGCTGGGGTCGACCCG-Biotin-3'.

[0060] Prepare PCR system solution:

[0061] PCR primer 1, PCR primer 2, Premix Ex Mix with ROX Reference Dye II (50×) reaction solution; wherein, the concentrations of PCR primer 1 and PCR primer 2 are both 10 μM.

[0062] Wherein, the sequence of PCR primer 1 is:

[0063] 5'-GTAAGGCGCTAAGAAACATCG-3'.

[0064] The sequence of PCR primer 2 is:

[0065] 5'-AATCTGGTTTAGCTACGCCTTC-3'.

Embodiment 2

[0066] Embodiment 2 makes standard curve

[0067] Treat the PCR tube with 50 μL of 0.8% glutaraldehyde solution at 37°C for 5 hours; after washing with ultrapure water three times, add 50 μL of streptavidin dissolved in 0.01M carbonate buffer and incubate for 2 hours (37°C);

[0068] After washing twice with phosphate buffer, the aptamer and complementary DNA 1:1 (v / v) were thoroughly mixed, and 50 μL of the mixture was added to each tube and incubated for 1 h (37°C);

[0069] Hybridization buffer was washed three times, and 50 μl of aflatoxin M with different concentration gradients (concentrations were 0.1ng / L, 1ng / L, 0.01μg / L, 0.1μg / L, 1μg / L) 1 Standard;

[0070] After washing with Tris buffer three times, add the PCR system solution, detect the fluorescent signal by quantitative PCR, and make a standard curve, see figure 2 .

Embodiment 3

[0071] Aflatoxin M in the detection sample of embodiment 3 1 Content

[0072] Weigh 0.5g of the tested sample, add 2.5mL 0.05ng mL -1 Aflatoxin M 1 The standard solution was mixed well with it, and then 2.5mL of 70% methanol was added. The mixture was vortexed for 5 min and then centrifuged at 10000 g for 10 min. The supernatant was collected and concentrated to 0.5 mL by nitrogen blowing. Add 2mL of 5% methanol to redissolve; quantitative PCR detects the fluorescent signal.

[0073] Quantitative PCR detection of aflatoxin M 1 For the amplification curve see image 3 , according to the standard curve measured in Example 2, determine the aflatoxin M in the detection sample 1 The content is 0.045ng mL -1 .

[0074] SEQUENCE LISTING

[0075] Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences

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Abstract

The invention discloses an aptamer for detecting aflatoxin M1, a sensor, a kit and application thereof. The aptamer for detecting aflatoxin M1 has the nucleotide as shown in SEQ ID No:1. The invention further discloses a sensor for preparing the nucleotide aptamer and a detection kit containing the sensor solution. Signal amplification is carried out by using PCR technology as signal transduction, the detection limit is greatly reduced and reaches 0.03ng/L. The nucleotide aptamer is used as an identification unit, the sensor prepared from the nucleotide aptamer is used for detecting the content of aflatoxin M1 in a sample and has high sensitivity and wide linear range when detecting the aflatoxin M1, and accurate quantitative detection can be realized.

Description

technical field [0001] The invention relates to the detection of aflatoxin M 1 The nucleic acid aptamer of the present invention further relates to the sensor containing this nucleic acid aptamer and the detection aflatoxin M prepared by it 1 The kit, the present invention also relates to their detection of aflatoxin M 1 Application in the category of aflatoxin M 1 detection field. Background technique [0002] Aflatoxin M 1 (AFM 1 ) is one of the most toxic mycotoxins in milk products, which poses a huge hazard to human health. AFM 1 It is designated as a class 1 carcinogen by the World Health Organization (WHO) International Agency for Research on Cancer. When cows eat food containing AFB 1 After the moldy feed, the metabolite AFM will be produced in the milk 1 . In order to prevent food safety scares and economic losses caused by the recall of contaminated feed and food, many countries have imposed AFM 1 Limited. Different food AFM 1 The limit range is 0.05-0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53C12Q1/68
CPCC12N15/115C12N2310/16C12Q1/6851G01N33/5308C12Q2531/113C12Q2563/107
Inventor 郑楠文芳郭晓东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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