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Aptamer capable of simultaneously recognizing aflatoxins B1, B2, G1 and M1 and application thereof

An aflatoxin and aptamer technology, which can be used in DNA/RNA fragments, biochemical equipment and methods, material testing products, etc. The effect of stable performance, consistent and efficient performance

Inactive Publication Date: 2020-03-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the aptamers obtained only by screening usually have a large number of bases, and the synthesis cost is high, and the redundant bases form a space barrier to techniques such as trace detection and material application. Therefore, it is necessary to tailor the aptamers obtained Optimization and precise retention of its action site are the inevitable requirements for improving the efficiency of aptamer action, reducing synthesis cost, and broadening the scope of application

Method used

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  • Aptamer capable of simultaneously recognizing aflatoxins B1, B2, G1 and M1 and application thereof
  • Aptamer capable of simultaneously recognizing aflatoxins B1, B2, G1 and M1 and application thereof
  • Aptamer capable of simultaneously recognizing aflatoxins B1, B2, G1 and M1 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1. Synthesis of aflatoxin aptamer original sequence and tailoring optimized sequence (completed by Shanghai Shenggong Company)

[0035] Aptamer original sequence:

[0036] 5’-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCACA-3’

[0037] According to the secondary structure of the aptamer fitted by the DNAMAN software and the Mfold software, the trimming and optimization were simultaneously performed in three ways from the 5' end, 3' end, and both ends of the aptamer. Simultaneously cut from both ends of the original aptamer, first remove the redundant bases other than the stem-loop structure to obtain the sequence Apt-1, and then cut it in units of 3 bases until the stem-loop structure is destroyed, and then obtain the sequence Apt-2, Apt-3, Apt-4. Starting from the 5' end of the original aptamer, after removing the redundant bases at the 5' end that constitute the stem-loop structure of the original sequence, it is trimmed in units of 3 bases ...

Embodiment 2

[0041] The processing of embodiment 2 aflatoxins

[0042] According to the molecular structure of 6 common types of aflatoxin, select and aflatoxin B 1 The other 3 toxins with only one difference in molecular structure, aflatoxin B 2 , G 1 , M 1 . aflatoxin B 1 , B 2 , G 1 , M 1 The solid powder was dissolved by adding DMSO to a final concentration of 2 mg / mL, shaken evenly, and stored at -4°C for later use.

Embodiment 3

[0043] Embodiment 3 is based on the aflatoxin B of isothermal titration calorimetry 1 , B 2 , G 1 , M 1 Aptamer optimization

[0044] (1) Preparation of reagents and samples: first, prepare 0.1M Tris, adjust the pH to 7.4 with HCl, filter, and degas by ultrasonic. Take 975 μL Tris-HCl, add 25 μL DMSO, the buffer finally contains 2.5% DMSO. Aflatoxin B 1 , B 2 , G 1 , M 1 Prepare 64μM Tris-HCl system containing 2.5% DMSO respectively. Each optimized tailoring aptamer was prepared in 5 μM Tris-HCl system containing 2.5% DMSO.

[0045] (2) Setting of titration conditions: When measuring the dissociation equilibrium constant, the volume of each needle is generally small and the method of titrating multiple times is generally adopted. The initial selection is 0.5 μL, and each subsequent drop is 2 μL. The total number of titrations is 18 drops. The duration of each drop in the experiment was 4 s except the titration duration of the first drop which was 1 s. The interval...

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Abstract

The invention discloses an aptamer capable of simultaneously recognizing aflatoxins B1, B2, G1 and M1. The aptamer has a nucleotide sequence shown in the formula of SEQ ID NO.1. The end 5' or end 3' of the aptamer is marked with FAM, a mercapto group, FITC or biotin for recognizing aflatoxins B1, B2, G1 and M1. The aptamer can also form an aptamer affinity column. According to the invention, an existing aptamer is used as a main chain, according to the secondary structure, affinity and specificity inspection on the 12 clipped aptamers is carried out by an isothermal titration calorimetric method so that the oligonucleotide aptamer capable of recognizing aflatoxins B1, B2, G1 and M1 with high affinity and high specificity is selected, has the characteristics of high stability, convenience in synthesis, easiness in marking functional groups and the like, and can be widely applied to rapid detection of aflatoxins B1, B2, G1 and M1 in foods.

Description

technical field [0001] The invention relates to the field of food safety biotechnology, in particular to a method of tailoring and optimizing an isothermal titration calorimetry method, which can specifically identify aflatoxin B at the same time. 1 , B 2 , G 1 , M 1 Optimal oligonucleotide aptamers and their applications. Background technique [0002] Aflatoxin (Aflatoxin, AFT) is a class of strong carcinogenic and mutagenic compounds produced by Aspergillus parasiticus and Aspergillus flavus. Now it has been found that there are 18 types of AFT, the most important type of which is B 1 , B 2 , G 1 , G 2 and metabolite M 1 ,M 2 . AFT is the most common toxin in food and food crops, and it can contaminate food after it is grown or harvested. Due to the stable nature of AFT, it is difficult to destroy its structure during food processing to reduce its toxicity or inactivate it. In order to prevent humans from being poisoned by AFT, many countries have stipulated AF...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115C12N2310/16G01N33/5308
Inventor 王周平梁瑶段诺
Owner JIANGNAN UNIV
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