Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.
Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Mojtaba
M
Hedayati Ch
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Jafar
J
Amani
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Hamid
H
Sedighian
Department of Drug and Food Control, and Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Mohsen
M
Amin
Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Jafar
J
Salimian
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Raheleh
R
Halabian
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Abbas Ali
AA
Imani Fooladi
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004768:Enterotoxins / Q000737:chemistry*; D006801:Humans; D052156:SELEX Aptamer Technique / Q000379:methods*; D013202:Staphylococcal Food Poisoning / Q000175:diagnosis* / Q000382:microbiology; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000472:pathogenicity
D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D004768:Enterotoxins; C031818:enterotoxin B, staphylococcal
SELEX; affinity chromatography; ssDNA-aptamer; staphylococcal enterotoxin B