Nucleic acid aptamer specifically combined with pethidine and application thereof
A nucleic acid aptamer and pethidine technology, which is applied in the field of nucleic acid aptamers that specifically bind pethidine, can solve the problems of complex antibody preparation process, batch differences, and inability to achieve fast and convenient detection
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Embodiment 1
[0036] Screening of nucleic acid aptamers specifically binding to pethidine
[0037] 1. Synthesize a random single-stranded DNA library and primers shown in the following sequences:
[0038] Random ssDNA library:
[0039] 5'-ATTGGCACTCCACGCATAGG-40N-CCTATGCGTGCTACCGTGAA-3',
[0040] Among them, "40N" represents a sequence formed by connecting 40 arbitrary nucleotide bases, and the library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;
[0041] The primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the primer information is shown in Table 1.
[0042] Table 1 Primers and their sequences
[0043]
[0044] Remarks: ①In the primer names, S1 is the forward primer, A2 is the reverse primer, S1-FAM is the fluorescently labeled forward primer, A2-ployA is the reverse primer connected with polyA tail, and polyA is the polyA tail. PolyA tail composed of nucleotide (A), S1CS is the complementary paired sequence of the forward primer;
[0045] ...
Embodiment 2
[0072] Detection of the affinity of meperidine-17 and meperidine with a microplate reader
[0073] Solution preparation:
[0074] Test solution: Take the nucleic acid aptamer meperidine-17 synthesized by Shanghai Sangong and dilute it with DPBS buffer, then add EvaGreen dye and mix well. %.
[0075] Control solution: Dilute Sequence A8 with DPBS buffer, then add EvaGreen dye and mix well, wherein the concentration of Sequence A8 is 1 μM, and the volume fraction of EvaGreen dye is 1%.
[0076] Meperidine solution: Take a 50 mM meperidine methanol solution and dilute it with DPBS buffer until the meperidine concentration is 10 μM.
[0077] Blank solution: take methanol and dilute 5000 times with DPBS buffer solution, which contains EvaGreen dye, and the volume fraction of EvaGreen dye is 1%.
[0078] operate:
[0079] Dispense the test solution into the orifice plate, 100 μl per well, and divide into 6 wells in total, among which, add 5 μl pethidine solution to 3 wells as te...
Embodiment 3
[0085] Meperidine-17 and Meperidine Affinity Detection by Isothermal Titration Calorimetry (ITC)
[0086] Solution preparation:
[0087] Test solution: Take the nucleic acid aptamer meperidine-17 synthesized by Shanghai Sangong and dilute it with DPBS buffer to a concentration of 10 μM to obtain an intermediate solution. Take 200 μl of the intermediate solution, 192 μl of DPBS buffer, and 8 μl of methanol to obtain a test solution. Among them, the nucleic acid aptamer The concentration of the ligand meperidine-17 was 5 μM.
[0088] Control solution: Dilute sequence A8 with DPBS buffer to a concentration of 10 μM to obtain an intermediate solution. Take 200 μl of the intermediate solution, 192 μl of DPBS buffer, and 8 μl of methanol to obtain a control solution. The concentration of random sequence A8 is 5 μM.
[0089] Pethidine solution: take 2 μl of meperidine methanol solution with a concentration of 5 mM and add it into 98 μl of DBPS buffer solution and mix well, wherein t...
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