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
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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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