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Selection of Streptomycin-specific Single-stranded DNA Aptamers and the Application of Aptamers in the Detection of Streptomycin

Author: WangJingYuan
Tutor: ZhouZuoDi
School: Jiangnan University
Course: Biochemistry and Molecular Biology
Keywords: aptamer SELEX streptomycin gold nanoparticles UV-vis absorptionspectroscopy
CLC: TS207.5
Type: Master's thesis
Year: 2013
Downloads: 56
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Abstract


Streptomycin is an aminoglycoside antibiotic produced by Streptomyces griseus and it iswidely used as an antimicrobial drug. However, when it is overcommitted, streptomycinresidue will exist in food products, which can be hazard for human health. To establishefficient, accurate and economical methods for the detection of streptomycin residue is ofgreat importance in food safety.Single-stranded DNA (ssDNA) aptamers that bind to streptomycin with high specificityand affinity were separated and identified from an oligonucleotides library by the process ofsystematic evolution of ligands by exponential enrichment (SELEX).79-nucleootide (nt)ssDNA library containing35-nt random sequences was used as the original oligonucleotideslibrary, from which streptomycin-specific aptamers were selected by using streptomycin-coated epoxy magnetic beads as affinitive selection matrix. Initial random ssDNA wasincubated at90oC for10min and streptomycin-coated magnetic beads were added to ssDNAsolution quickly and incubated for30min at room temperature. The beads were washed withbinding buffer to remove unbound oligonucleotides completely. In order to recover theadsorbed oligonucleotides, the beads-DNA complex was incubated in elution buffer at80oC.Enrichment of streptomycin-specific aptamers eluted from the magnetic beads was achievedthrough the PCR amplification, with a reaction system as follows: Denaturation at95℃for30s, annealing at55℃for30s, elongation at72℃for1min, and20cycles were carried out.The selection was repeated for8rounds, and counter selection was performed after every twoselection rounds by using ethanolamine-coated epoxy magnetic beads. Thus ssDNA which arespecific to naked beads and ethanolamine groups were eliminated. ELISA was employed toanalyze the relative affinity of the selected ssDNA for the target streptomycin after each round.After8rounds of selection, the affinity of ssDNA for streptomycin no longer increased infurther selection round. Sixteen aptamers were obtained by cloning and sequencing. Thedissociation constant (Kd) was then measured through equilibrium osmosis. The sequenceSTR1, with Kdvalue of199.1nM, exhibits excellent affinity to the target.With this aptamer, detection of streptomycin was achieved by using gold nanoparticles(AuNPs)-based colorimetric method. In the presence of streptomycin, the competitive bindingof the target and aptamer decreases the stability of AuNPs in NaCl solution, triggers theaggregation, and exhibits visible color change of AuNPs solution. Through UV-visiblespectroscopic quantitative analysis, streptomycin can be detected in the range of0.2~1.2μM.The presence of other aminoglycoside antibiotics shows neglectable disturbance. Furthermore,the established method was utilized to detect streptomycin in different types of honey samplesand the same low detection limit and linear detection range were achieved. In theconcentration range of0.2~1.2μM, a linear relationship was also found between theabsorbance at520nm and the concentration of streptomycin in honey. The result showed thismethod has a wide application in detection of streptomycin residue in food samples.

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CLC: > Industrial Technology > Light industry,handicrafts > Food Industry > General issues > Food standards and testing > Determination of food contamination
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