Development of a DNA aptamer to detect Brucella abortus and Brucella melitensis through cell SELEX

Document Type : Full paper (Original article)


1 MSc in Microbial Biotechnology, Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran

2 Max Planck Institute for Polymer Research, Ackermannweg 10, 55128, Mainz, Germany

3 Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran



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.


Main Subjects

Abebe, G; Ike, A; Siegmund-Schultze, M; Mané-Bielfeldt, A and Valle Zárate, A (2010). Prevalence of mastitis and brucellosis in cattle in Awassa and the Peri-Urban areas of two smaller towns. Zoonoses Public Health. 57: 367-374.
Al Dahouk, S; Sprague, L and Neubauer, H (2013). New developments in the diagnostic procedures for zoonotic brucellosis in humans. Rev. Sci. Tech., 32: 177-188.
Alfavian, H; Mousavi Gargari, SL; Rasoulinejad, S and Medhat, A (2017). Development of a DNA aptamer that binds specifically to group A Streptococcus serotype M3. Can. J. Microbiol., 63: 160-168.
Almasi, F; Gargari, SLM; Bitaraf, F and Rasoulinejad, S (2016). Development of a single stranded DNA aptamer as a molecular probe for lncap cells using cell-selex. Avicenna J. Med. Biotechnol., 8: 104.
Bitaraf, F; Rasooli, I and Gargari, SM (2016). DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX. Eur. J. Clin. Microbiol. Infect. Dis., 35: 503-510.
Blind, M and Blank, M (2015). Aptamer selection technology and recent advances. Mol. Ther. Nucleic Acids. 4: e223.
Brody, EN and Gold, L (2000). Aptamers as therapeutic and diagnostic agents. J. Biotechnol., 74: 5-13.
Chen, Y; Munteanu, AC; Huang, YF; Phillips, J; Zhu, Z; Mavros, M and Tan, W (2009). Mapping receptor density on live cells by using fluorescence correlation spectroscopy. Chemistry. 15: 5327-5336.
Chen, C; Zhou, S; Cai, Y and Tang, F (2017). Nucleic acid aptamer application in diagnosis and therapy of colorectal cancer based on cell-SELEX technology. NPJ Precis. Oncol., 1: 1-7.
Christoforidou, S; Boukouvala, E; Zdragas, A; Malissiova, E; Sandalakis, V; Psaroulaki, A; Petridou, E; Tsakos, P; Ekateriniadou, L and Hadjichristodoulou, C (2018). Novel diagnostic approach on the identification of Brucella melitensis Greek endemic strains-discrimination from the vaccine strain Rev. 1 by PCR-RFLP assay. Vet. Med. Sci., 4: 172-182.
Ducrotoy, MJ; Muñoz, PM; Conde-Álvarez, R; Blasco, JM and Moriyón, I (2018). A systematic review of current immunological tests for the diagnosis of cattle brucellosis. Prev. Vet. Med., 151: 57-72.
Dunn, MR; Jimenez, RM and Chaput, JC (2017). Analysis of aptamer discovery and technology. Nat. Rev. Chem., 1: 1-16.
Dwivedi, HP; Smiley, RD and Jaykus, LA (2013). Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting. Appl. Microbiol. Biotechnol., 97: 3677-3686.
Fakhri, N; Hosseini, M and Tavakoli, O (2018). Aptamer-based colorimetric determination of Pb 2+ using a paper-based microfluidic platform. Anal. Methods. 10: 4438-4444.
Gewirtz, A (1999). Oligonucleotide therapeutics: clothing the emperor. Curr. Opin. Mol. Ther., 1: 297-306.
Gopinath, SC; Lakshmipriya, T and Awazu, K (2014). Colorimetric detection of controlled assembly and disassembly of aptamers on unmodified gold nanoparticles. Biosens. Bioelectron., 51: 115-123.
Hamula, CL; Le, XC and Li, XF (2011). DNA aptamers binding to multiple prevalent M-types of Streptococcus pyogenes. Anal. Chem., 83: 3640-3647.
Islam, M; Khatun, M; Saha, S; Basir, M and Hasan, MM (2018). Molecular detection of Brucella spp. from milk of seronegative cows from some selected area in Bangladesh. J. Pathog., 2018: 9378976.
Kaden, R; Ferrari, S; Alm, E and Wahab, T (2017). A novel real-time PCR assay for specific detection of Brucella melitensis. BMC Infect. Dis., 17: 230.
Ladju, RB; Pascut, D; Massi, MN; Tiribelli, C and
Sukowati, CH
(2018). Aptamer: A potential oligonucleotide nanomedicine in the diagnosis and treatment of hepatocellular carcinoma. Oncotarget. 9: 2951.
Lucero, N; Ayala, S; Escobar, G and Jacob, N (2008). Brucella isolated in humans and animals in Latin America from 1968 to 2006. Epidemiol. Infect., 136: 496-503.
Minda, AG and Gezahegne, MK (2016). A review on diagnostic methods of brucellosis. J. Vet. Sci. Technol., 7: 323.
Mirzakhani, K; Gargari, SLM; Rasooli, I and Rasoulinejad, S (2018). Development of a DNA aptamer for screening Neisseria meningitidis serogroup B by cell SELEX. Iran Biomed. J., 22: 193.
Morita, Y; Leslie, M; Kameyama, H; Volk, DE and Tanaka, T (2018). Aptamer therapeutics in cancer. Cancers (Basel). 10: 80.
Mostafavi, E and Asmand, M (2012). Trend of brucellosis in Iran from 1991 to 2008. IJE., 8: 94-101.
Mousa, ARM; Elbag, KM; Kbogali, M and Marafie, AA (1988). The nature of human brucellosis in Kuwait: study of 379 cases. Rev. Infect. Dis., 10: 211-217.
Ohuchi, S (2012). Cell-SELEX technology. BioRes. Open Access. 1: 265-272.
Peterson, LE; Blackburn, B; Peabody, M and O’Neill, TR (2015). Family physicians’ scope of practice and American Board of Family Medicine recertification examination performance. J. Am. Board Fam. Med., 28: 265-270.
Rasoulinejad, S and Gargari, SLM (2016). Aptamer-nanobody based ELASA for specific detection of Acinetobacter baumannii isolates. J. Biotechnol., 231: 46-54.
Shakerian, A and Nodargah, M (2018). Review of Brucella contamination in Milk and its products of Iran. J. food Microbiol., 5: 83-94.
Shangguan, D; Bing, T and Zhang, N (2015). Cell-SELEX: Aptamer selection against whole cells. In: Aptamers selected by cell-SELEX for theranostics. Berlin, Heidelberg, Springer. PP: 13-33.
Shipley, SL; White, E and Kim, SK (2010). Selection of aptamers against live E. coli cells using Cell SELEX. FASEB J., 24: 907-914.
Song, D; Qu, X; Liu, Y; Li, L; Yin, D; Li, J; Xu, K; Xie, R; Zhai, Y; Zhang, H and Bao, H (2017). A rapid detection method of Brucella with quantum dots and magnetic beads conjugated with different polyclonal antibodies. Nanoscale Res. Lett., 12: 179.
Tsao, SM; Lai, JC; Horng, HE; Liu, TC and Hong, CY (2017). Generation of aptamers from a primer-free randomized ssDNA library using magnetic-assisted rapid aptamer selection. Sci. Rep.,7: 45478.
Volk, DE and Lokesh, GL (2017). Development of phosphorothioate DNA and DNA thioaptamers. Biomedicines. 5: 41.
Yu, X; Chen, F; Wang, R and Li, Y (2018). Whole-bacterium SELEX of DNA aptamers for rapid detection of E. coli O157: H7 using a QCM sensor. J. Biotechnol., 266: 39-49.
Zhou, J and Rossi, J (2017). Aptamers as targeted therapeutics: current potential and challenges. Nat. Rev. Drug Discov., 16: 181.