Iranian Journal of Veterinary Research, Shiraz University 294

Original Article

Development of a DNA aptamer to detect Brucella abortus and

Brucella melitensis through cell SELEX
Nosaz, Z.1**; Rasoulinejad, S.2** and Mousavi Gargari, S. L.3*
1MSc in Microbial Biotechnology, Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran; 2Max Planck
Institute for Polymer Research, Ackermannweg 10, 55128, Mainz, Germany; 3Department of Biology, Faculty of Basic Sciences,
Shahed University, Tehran, Iran

*Correspondence: S. L. Mousavi Gargari, Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran. E-
mail: [email protected]
**These authors contributed equally to this work

(Received 2 Apr 2020; revised version 10 Sept 2020; accepted 21 Oct 2020)

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.

Key words: Brucella, Cell SELEX, DNA aptamer, Flow cytometry

Introduction diagnosis based on bacteriology counts as a standard test,

prolongs cultivation periods which at least needs 4-5
Brucellosis is a common human and animal disease days for detection; also, difficulties of bacterial isolation
affecting a wide range of livestock and all age groups. and unsuccessful culturing are the worst drawbacks of
Brucellosis is still counted as a major general hygiene common methods (Mousa et al., 1988). In serological
concern in the world and is common as a zoonosis. The assays, body fluid and antibodies are used as a common
major clinical manifestations of brucellosis in animals approach. The possibility of cross reactivity with other
can be found as arthritis, mastitis and lameness, abortion pathogens makes the results of serological assays less
and inflammation of the epididymis (Abebe et al., 2010). trustworthy. Although antibodies play an important role
In humans, Brucella melitenis is the most known acute in detection and diagnosis, low stability and high
disease and Brucella abortus can create subacute forms production costs are some of the limitations of antibody-
(Peterson et al., 2015). In Iran, brucellosis is an endemic, based detections. The tests are based on
severe health issue that has increased recently (Shakerian lipopolysaccharide (LPS) detection and because of the
and Nodargah, 2018). Annual cases of brucellosis are low specificity of the immunodominant epitope of
reported to be about 27500, mostly caused by B. Brucella and its similarity to other equivalent epitopes in
melitenis (Mostafavi and Asmand, 2012). In humans, it Escherichia coli, Salmonella (sp), Yersinia (sp), Vibrio
causes infectious or noninfectious manifestations, which (sp), and some other spices, they cannot count as
make the early diagnosis difficult (Lucero et al., 2008). accurate assays. Molecular methods like polymerase
Bacteriological, serological, and molecular assays are the chain reaction (PCR) and real time-PCR (RT-PCR) could
three common methods in the diagnosis of brucellosis somehow overcome the aforementioned drawbacks of
infections. In most of these common assays, isolating the serological and culture based diagnostic methods but still
causative agent by culture is necessary. Although in some cases sensitivity and specificity are not

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295 Iranian Journal of Veterinary Research, Shiraz University

satisfying (Ducrotoy et al., 2018). Due to the small detection of B. melitensis and B. abortus.
amount of Brucella present in clinical samples, the direct
detection of DNA in brucellosis is another challenging Materials and Methods
issue. Ultimately, the efficiency of the molecular
detection can be affected by the quality of the extracted
DNA and the type of clinical samples (Minda and Bacterial strains preparation
Gezahegne, 2016; Ducrotoy et al., 2018; Shakerian and All bacterial species used in this study are listed in
Nodargah, 2018). Since brucellosis is a zoonosis, Table 1. All strains were cultured in Luria-Bertani broth
developing an accurate, fast, efficient and cost effective (LB) and maintained in LB agar, except Haemophilus
assay will be useful, especially in rural life, due to the influenza, Streptococcus pneumonia, and Neisseria
difficulty in accessing advanced equipment. Aptamers meningitidis type B, cultured in 5% defibrinated sheep’s
are short, single-stranded oligonucleotides with a length blood agar in aerobic conditions at 37°C. Cells were
between 60-100 bp (Tsao et al., 2017). The single cultured and counted using a standard plate counting
stranded molecule can fold and twist into spatial method. Brucella melitensis and B. abortus (OD600=0.4)
structures and specifically bind to the target. The were mixed in a suspension with a 1:1 ratio. A total cell
dissociation constant (Kd) of aptamers toward their density of 108 cell/ml of this mixture was centrifuged at
targets is ranged from micro to picoMolar (Gewirtz, 1000 × g for 10 min. Cells were washed 3 times with
1999; Brody and Gold, 2000). Their target can differ phosphate buffer saline 1X, 0.02% Twee-20 (PBST).
from small molecules to large macromolecules such as The supernatant was discarded and the pellet was
large proteins and even whole cells. In 2017, the Food suspended in 300 µL of binding buffer (PBST plus 1%
and Drug Administration approved the therapeutic bovine serum albumin) to be used in the SELEX
application of six aptamers (Volk and Lokesh, 2017; procedure. Likewise, other strains, for counter SELEX,
Ladju et al., 2018). To date, numerous aptamer were prepared in a suspension with a 1:1 ratio (108
sequences have been characterized with different cell/ml) l ml in a total volume of 10 ml. One ml of
purposes in diagnostic and therapeutic fields (Blind and homogeneous cell suspension was centrifuged, washed 3
Blank, 2015; Chen et al., 2017; Dunn et al., 2017; Zhou times with PBST and resuspended in 300 µL of PBS.
and Rossi, 2017). Furthermore, the combination of Escherichia coli TOP10 competent cells were used for
aptamers with some chemicals can help enhance their the transformation of the enriched library.
activity or stability (Ladju et al., 2018). The binding
affinity of an aptamer can be enhanced by engineering DNA library amplification
their sequences (Volk and Lokesh, 2017). The backbones The DNA library and primers were purchased from
of aptamers are flexible enough to conjugate with drugs, Metabion (Steinkirchen, Germany). The initial library
nanoparticles with functional groups, providing an (10 µM) was amplified by PCR using the following
adjustable approach for detection (Gopinath et al., 2014; primers:
Fakhri et al., 2018; Morita et al., 2018). Forward: 5´-GCCTGTTGTGAGCCTCCTAAC-3´
In order to overcome some of the aforementioned Reverse: 5´-GGGAGACAAGAATAAGCA-3´
limitations, we developed an aptamer through a whole The amplification was performed at 95°C for 5 min
cell systematic evolution of Ligands by EXponential as an initial denaturation, then 30 cycles of 94°C
enrichment (SELEX) procedure to detect Brucella. We denaturation, 62°C annealing, and 72°C extension all for
used B. melitensis and B. abortus as the mixture target. 30 s, and 72°C for 5 min as the final extension. All PCR
Using a mixture of bacteria as target provides a wide products were checked on 1.5% agarose in Tris-borate-
detection range toward B. melitensis and B. abortus. The EDTA buffer (TBE 0.5X) and were purified with ethanol
selected aptamer can be used in any tool designed for the precipitation assay. The purified DNA concentration was

Table 1: Bacterial species used in this study

SELEX target Collection center
Brucella melitensis 16M ATCC 11649 Razi Vaccine and Serum Research Institute
Brucella abortus 544 ATCC 21749
Counter SELEX and Cross test
Vibrio cholorae clinical strain Shahed University
Pseudomonas aeruginosa ATCC 27853
Shigella sonnei clinical isolated
Listeria monocytogenes ATCC 7644
Staphylococcus aureus ATCC 25923
Acinetobacter baumannii ATCC 19606
Escherichia coli ATCC 25922
Haemophilus influenza type b ATCC 10211
Streptococcus pneumonia ATCC 33400
Neisseria meningitidis type B ATCC 13090
Salmonella typhi ATCC 700931
Yersinia enterocolitica ATCC BAA-1511D-5
Competent cell
Escherichia coli TOP10

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Iranian Journal of Veterinary Research, Shiraz University 296

measured by Nano drop (Pico200 Spectrophotometer, aptamers was measured by flow cytometry.
UK). Subsequent purification of the PCR products used
in SELEX procedures was carried by gel purification kits Maintenance of obtained ssDNA pool
according to the manufacturer’s protocol. The recovered outcome of the 14th round of SELEX
was amplified with the PCR. In order to introduce the 3´
Single stranded DNA (ssDNA) library “A” overhang added by Taq DNA polymerase, the final
preparation extension at 72°C was carried out for 10 min. The PCR
The DNA library was amplified with 5´- product was cloned on a pTZ57R/T vector and
phosphorylated reverse primer in order to prepare the transformed to E. coli TOP10 competent cells. Colonies
ssDNA library. The purified 5´-phosphorylated dsDNA were tested using colony PCR with free-labelled primers.
was incubated at 37°C with 5 units of lambda Positive clones were kept in glycerol and saved at -80°C
exonuclease in a total volume of 20 µL reaction for 25 for further studies.
min. The reaction was terminated at 80°C for 15 min
followed by 5 min incubation on ice. Affinity selection
Each positive clone was used as a template to amplify
SELEX and counter SELEX processes aptamers with FITC-labelled primer. The labeled
The SELEX procedure was performed as described aptamers, made as ssDNA then were incubated with B.
previously (Rasoulinejad and Gargari, 2016). The melitensis and B. abortus. The percentage of binding
ssDNA library (2 nM) was incubated at 95°C for 10 min, affinity of each aptamer was measured. The DNA
then transferred into an ice bath. The ssDNA pool was aptamers with highest binding affinity were selected for
added to 300 µL of a suspension of B. melitensis and B. counter- and cross-tests. The counter-test was performed
abortus bacteria, in a binding buffer and incubated for 1 by incubating counter bacterial strains with high binding
h at room temperature with gentle shaking. The mixture aptamers toward the target. Four aptamers with the
was centrifuged at 1000 × g for 10 min. The supernatant highest binding affinities toward the target were selected
was removed and the cells were washed with 1 ml of the for a cross-reactivity test. All four aptamers were
wash buffer (PBST). Incubation time was gradually incubated individually with E. coli, Salmonella typhi,
reduced from 60 min to 10 min and washing times were Yersinia enterocolitica, and Vibrio cholerae. Finally, the
increased from 1 to 10 times from 1st to 14th SELEX aptamers with the highest binding affinity toward the
rounds. In order to recover the binding aptamers, the target and the lowest towards other strains were selected
cells were resuspended in 100 µL of miliQ water, heated for further investigation.
at 95°C for 10 min and centrifuged at 5000 × g for 30 s.
The recovered ssDNA was amplified with PCR and used Measurement of dissociation constant
for the next round of SELEX. In total, fourteen rounds of Determination of Kd was carried out by the procedure
SELEXs were carried out and the selection processes described previous by (Alfavian et al., 2017). In brief,
were monitored by flow cytometry. different concentrations of FITC labeled aptamer (0-200
pM) were incubated with fixed concentrations of
Counter SELEX bacterial cocktail (B. melitensis and B. abortus) at room
The 3rd, 6th, 7th and 11th rounds were proceeded as temperature for 45 min. The bacteria pellets were
counter SELEX. Each counter SELEX included two collected, washed with the washing buffer and suspended
steps; a negative selection followed by positive selection. in 1 ml binding buffer. The fluorescent intensity of each
The ssDNA pool obtained from previous rounds of aptamer concentration was measured by flow cytometry.
SELEX was added to the 300 µL suspension of all The Kd was calculated and the curve was plotted as
counter strains in the binding buffer. This mixture was follows:
incubated for 60 min at room temperature with gentle
shaking. Cells were precipitated and the supernatant was Y = BmaxX / (Kd + X) equation
transferred to another tube and used as a source of
(Jandel, San Rafael, CA, USA) by Sigmaplot 12.0.
aptamer for further SELEX rounds.

Sequencing and secondary structure prediction

Enrichment of ssDNA aptamers pool
The selected aptamers, with the highest affinity
In order to monitor the selection procedures, the
toward B. melitensis and B. abortus and the lowest
outcome of 2nd, 5th, 11th, 13th and 14th SELEXs were
toward other strains was sent for sequencing (Bioneer,
used as templates for PCR using forward FITC labelled
Daejeon, South Korea). The alignments were performed
primer. The assay was carried out by incubating 50 pM
by CLC bio workbench (CLC) sequence alignment 7.8.1
of ssDNA from each round with a mixture of B.
software. The secondary structures of aptamers was
melitensis and B. abortus at room temperature for 60
analyzed by the Zuker algorithm, using Oligo Analyzer
min. Cells were collected and washed with the wash
3.1 web server. The prediction analysis was carried out
buffer. The pellet of cells and bound ssDNA aptamers
under 144 mM Na+ and 1 mM mg+2 ion conditions at
was resuspended with 1 ml of binding buffer. The
room temperature.
percentage of fluorescent intensity of bound ssDNA

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297 Iranian Journal of Veterinary Research, Shiraz University

Results PCR showed 24 positive clones analyzed using agarose

electrophoresis.
DNA aptamer pool and SELEX Procedures
The PCR amplification reactions after each selection Highest binding affinity selection
round were optimized. Factors influencing the results of Nine out of 24 colonies showed the highest binding
the PCR reactions were concentration templates, MgCl2, affinity toward B. melitensis and B. abortus. Two
and annealing temperature. The binding affinity of aptamers, namely B20 and B21, showed significant
aptamer pools toward B. melitensis and B. abortus, binding affinity toward B. melitensis and B. abortus
measured by flow cytometry, showed significant increase compared to the initial library.
at round 5, and the increscent continued to the 11th Out of 24 selected aptamers, the average binding
round of SELEX. The percentage binding affinity of affinity of 9 aptamers toward counter bacteria were less
rounds 2nd, 5th, 11th, 13th and 14th is demonstrated in than 2%, where the binding affinity of B21 toward B.
Fig. 1A. The comparison of binding affinity between melitensis and B. abortus was about 51% (Figs. 2A and
SELEX 13th and 14th indicated that the SELEX process B). Based on the binding affinity to B. melitensis and B.
can be ended in the 14th selection round (Figs. 1A and abortus and counter cocktail (Fig. 2A), 4 aptamer were
B). selected for cross reactivity test. Aptamer B20 and B21
demonstrated the lowest binding affinity to bacterial
Transformation species selected for the cross-reactivity test (Figs. 3A-B)
The aptamers obtained from the 14th selection round and therefore these 2 aptamers were selected as high
were cloned in the pTZ57R/T vector and transformed to binders for further studies. The result of the cross test is
E. coli TOP10 competent cells. The results of colony shown in Fig. 4.

Fig. 1: Binding affinity toward target cells through SELEX procedures. (A) Development of Aptamer pool from beginning to end,
and (B) Improvement of flow cytometry data through SELEX. The numbers in the legend referring to each SELEX cycle

Fig. 2: Representation of aptamers’ binding affinity. (A) Binding affinity of isolated aptamers toward B. melitensis and B. abortus,
and (B) Selected flow cytometry result of isolated aptamer, B21 and B20

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Fig. 3: Binding affinity and selectivity of aptamer candidates. Each chart illustrates, (A) Counter, and (B) Cross reactivity,
representing the binding affinity of aptamer candidates toward other competitor targets

Fig. 4: Flow cytometry of B20 and B21 against selected bacteria for cross-test. The measurement of flow cytometry indicated that
B20 and B21 had moderate but non-significant affinity toward the bacteria that always cause false positives in common serological
tests. The consequence of counter- and cross-reactivity tests shows the high specificity of the isolated aptamers. The results refer to
B20 and B21 from left to right

Fig. 5: Dissociation constant curve. Using the binding affinity of each point concentration and ligand binding one site saturation
equation with the calculated Kd

Dissociation constant calculation (Fig. 5).

The binding affinity of B20 and B21 aptamers toward
B. melitensis and B. abortus was measured using Discussion
different concentrations of aptamers (0, 50, 100, 150, and
200 pM). The calculated Kd for B20 and B21 were Brucella species as a zoonotic pathogen can be
40.179 ± 3.06 pM and 184.396 ± 465 pM, respectively transmitted from animals such as sheep, goats, and cattle,

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299 Iranian Journal of Veterinary Research, Shiraz University

resulting in brucellosis. This disease, as a common attributed to the accuracy of cell SELEX as well as a
zoonotic infection, can cause significant endemic dual selection performed in counter SELEX steps. To
damage to the livestock industry, hence it is considered eliminate non-specific and weak binders, ascending
as an obstacle in the trade of livestock and their products. stringency through selection rounds could also play a key
Unfortunately, Brucella does not show specific clinical role to access highly efficient aptamers. In cell SELEX,
symptoms making the diagnosis difficult. The the aptamers are selected against whole live cells as a
development of an accurate, quick, easy and, low cost target. In whole cell SELEX, aptamers bind to cell
method is necessary for the detection and diagnosis of surface proteins in their physiological condition, and we
this disease. Recent fluorescent polarization assays do not need to have information about the target toward
(FPA) are promising methods that have paved the way to which the aptamer is developed. However, in the cell
overcoming some of the previous problems encountered SELEX method, aptamers do not bind to their target with
with B. melitensis and B. abortus detection. More studies full efficiency. This is because the expression levels of
are needed to prove the reproducibility of the assay. The cell surface proteins in the bacterial pool are different
real time-polymerase chain reaction has been used for (Dwivedi et al., 2013). In protein SELEX, aptamers bind
the detection of B. melitensis and B. abortus as a rapid to their target with a high specificity of nearly 100%, but
method. However, the genomic analysis of different there are several issues concerning protein SELEX. The
Brucella species revealed high conservation between aptamers may bind to domains of the target(s) which are
them, which makes designing species-specific RT-PCR not exposed to physiological conditions, especially in the
assays difficult. Even probes sometimes result in a signal case of membrane proteins. Moreover, the cost of protein
for all Brucella as well as non-melitensis species (Kaden purification is prohibitive and has its own problems.
et al., 2017). In addition, PCR-based methods require Aptamers B20 and B21 showed the highest specificity
accurate and expensive instruments and professional toward B. melitensis and B. abortus without having a
operators (Kaden et al., 2017). significant affinity for counter-cells and other bacteria
The immunological method is also widely used for which affect the false positive results in serological tests.
the detection of Brucella. Although this method is quick, Counter-SELEX is a strategy to eliminate nonspecific
shows cross-reactivity (Song et al., 2017). The 19KD bindings when an aptamer encounters a large amount of
antibody against outer membrane protein, used for the non-target proteins on a cell surface. The combination of
detection of brucellosis, did not show high sensitivity a positive selection and counter-SELEX promote the
and specificity compared to PCR (Islam et al., 2018). In developmental procedure and reduce the cross-reactivity
addition, S-LPS antigen is not present in all Brucella spp. of selected aptamers to structures that are closely related
and in the majority, false negative results are reported to that of the main target. Previous reports indicate that
due to the presence of a single subgroup of cell-SELEX can increase the selection rounds without
immunoglobulin (Al Dahouk et al., 2013; Christoforidou applying counter selection (Ohuchi, 2012; Shangguan et
et al., 2018). al., 2015). Data obtained by flow cytometry after counter
Due to their special folding and 3D structure, SELEX was performed at the 3rd, 6th, 7th, and 11th
aptamers can specifically bind toward their targets and rounds of selection showed a significant increase in the
are compatible with antibody-target interactions, hence binding affinity with a steep slope. Therefore, we can
being good candidates to replace with antibodies. assume that the selected aptamers are able to detect the
Brucella melitensis and B. abortus are two of the Brucella family, specifically melitensis and abortus.
important species that cause brucellosis, and therefore In conclusion, the aptamers we developed were able
detecting these two in single assays can reduce expenses to identify B. melitensis and B. abortus with a
as well as the time for detection. The characterization of remarkable binding efficiency and appropriated K d in a
aptamers B20 and B21 to prevent B. melitensis and B. picoMolar range. Therefore, the isolated aptamers herein
abortus demonstrated a high binding affinity according can be good candidates for further studies on the
to flow cytometry data and low Kd of 40.179 ± 3.06 pM development of any rapid assay test implanted on routine
and 184.396 ± 465 pM, respectively. The finding is brucellosis diagnosis.
consistent with our previous reports and comparable to
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IJVR, 2020, Vol. 21, No. 4, Ser. No. 73, Pages 294-300

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