جستجوی مقالات مرتبط با کلیدواژه « Cell-SELEX » در نشریات گروه « دامپزشکی »

Aptamers are oligonucleotides that can be easily synthesized and bind to their targets with high affinity and specificity. Several aptamers specific to soluble factors of coagulation cascade have been produced, however, aptamers specific to platelet cell membrane molecules have not been reported yet. We aimed to discover DNA aptamers that specifically bind to human platelets. The cell-SELEX method was used for aptamer discovery. Synthetic 79 nucleotides length single-strand oligonucleotides were used as a library. Ultra-pure platelets were prepared using differential centrifugation steps and magnetic-bead-assisted removal of contaminating cells. The FITC-labeled forward primer was used for amplification of the selected oligonucleotides by PCR, and Lambda exonuclease was used for digestion of the lagging strand. After 12 rounds of cell-SELEX, selected oligos were amplified and cloned to pTG19-T vector, transfected into E. coli  (TOP10) and sequenced. Sequences of aptamers from 200 individual positive colonies were aligned and seven clusters were identified. Representative aptamers were amplified and their affinity, specificity, and digestibility of their targets were evaluated. Interferences of the aptamers to two platelet function tests were also investigated. Affinity (KD) of the representative aptamers were between 109 and 340 nM. Trypsin exposure of the platelets completely abolished the binding of the 7 aptamers to the targets. The binding of the four aptamers fully protected their target molecules from digestion. No one of the aptamers changed the parameters of the platelet function tests. Seven aptamers specific to platelets were identified and characterized. These aptamers may have potentially diverse applications in the diagnosis or treatment of platelet disorders.

Brucellosis is a zoonosis, caused by Brucella spp. which are small aerobic intracellular coccobacilli, localized in the reproductive organs of host animals, causing abortion and sterility. The diagnosis of this zoonosis is based on microbiological, serological or real time-polymerase chain reaction (RT-PCR) laboratory tests. Although the common microbiological and serological based assays have advantages, they are not able to solve the diagnosis problems.

To overcome some of the limitations of present techniques, in this study, we developed an aptamer through whole-cell systematic evolution of Ligands by EXponential enrichment (SELEX) procedures to detect Brucella.

We used mixture of Brucella melitensis and Brucella abortus as the target. In order to prepare the single-stranded DNA (ssDNA) aptamer, the DNA library was amplified with 5´-phosphorylated reverse primer and treated with lambda exonuclease. The SELEX procedure was performed by incubating the ssDNA pool with a bacterial suspension in a binding buffer. The selected procedures were monitored by flow cytometry using FITC-labelled forward primer. Aptamers with the highest binding affinity towards the target and the lowest to other strains were selected.

Two aptamers namely B20 and B21 showed significant binding affinity toward B. melitensis and B. abortus. The dissociation constant (Kd) for aptamers B20 and B21 was 40.179 ± 3.06 pM and 184.396 ± 465 pM, respectively.

The isolated aptamers were able to identify B. melitensis and B. abortus with a remarkable binding efficiency and appropriated Kd in a picoMolar range and therefore can be good candidates in the development of any rapid assay test implanted on routine brucellosis diagnoses.