Z Nosaz1,2, S Rasoulinejad3,2, S L Mousavi Gargari4. 1. MSc in Microbial Biotechnology, Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran. 2. These authors contributed equally to this work. 3. Max Planck Institute for Polymer Research, Ackermannweg 10, 55128, Mainz, Germany. 4. Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran.
Abstract
BACKGROUND: 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. AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: 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. AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Entities:
Keywords:
Brucella; Cell SELEX; DNA aptamer; Flow cytometry
Authors: Marie J Ducrotoy; Pilar M Muñoz; Raquel Conde-Álvarez; José M Blasco; Ignacio Moriyón Journal: Prev Vet Med Date: 2018-01-31 Impact factor: 2.670