Analytica Chimica Acta 1112 (2020) 54e61

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Analytica Chimica Acta

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Yersinia pestis detection using biotinylated dNTPs for signal

enhancement in lateral flow assays
Salah Kortli a, 1, Miriam Jauset-Rubio b, 1, Herbert Tomaso c,
Mohammed Nooredeen Abbas d, Abdulaziz S. Bashammakh e,
Mohammad S. El-Shahawi e, Abdulrahman O. Alyoubi e, Mounir Ben-Ali a, **,
Ciara K. O’Sullivan b, f, *
a
University of Sousse, Higher Institute of Applied Science and Technology of Sousse- ISSAT, NANOMISENE Lab, LR16CRMN01-CRMN, 4003, Sousse, Tunisia
b
INTERFIBIO Consolidated Research Group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007, Tarragona, Spain
c
Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Strasse 96a, 07743, Jena, Germany
d
Electroanalytical Laboratory, Applied Organic Chemistry Department, National Research Centre, El Bohouth St., Dokki, 12622, Giza, Egypt
e
Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, 21589, Jeddah, Saudi Arabia
f  Catalana de Recerca I Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010, Barcelona, Spain
Institucio

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Ultrasensitive molecular detection of

Yersinia pestis using biotinylated
dNTPs and tailed primer is
demonstrated.

Optimisation of ratio of biotinylated
dNTPs: natural dNTPs is carried out
using PCR and ELONA.

Optimised parameters were exploi-
ted using RPA.

Lateral flow assays for detection of
single tailed amplicon and bio-
tinylated dNTPs with SA-AuNPs was
developed.

Real samples were tested.

a r t i c l e i n f o a b s t r a c t

Article history: Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent,
Received 19 August 2019 which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid
Received in revised form detection at extremely low levels is required, in order to facilitate a timely intervention for containment.
1 March 2020 Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplifi-
Accepted 30 March 2020
cation with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The
Available online 1 April 2020
polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to opti-
mise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and
Keywords: 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely
Yersinia pestis
recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of
Lateral flow assay
Biotinylated dNTPs
amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then

* Corresponding author. INTERFIBIO Consolidated Research Group, Department

of Chemical Engineering, Universitat Rovira I Virgili, 43007, Tarragona, Spain.
** Corresponding author.
E-mail addresses: mounirbenaliissat@gmail.com (M. Ben-Ali), ciara.osullivan@
urv.cat (C.K. O’Sullivan).
1
These authors contributed equally to this work and are both first authors.

https://doi.org/10.1016/j.aca.2020.03.059
0003-2670/© 2020 Elsevier B.V. All rights reserved.
 S. Kortli et al. / Analytica Chimica Acta 1112 (2020) 54e61 55

Enzyme linked oligonucleotide assay
(ELONA)
Nucleic acid amplification tests (NAAT)
Recombinase polymerase amplification
(RPA)
explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively.
The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection
of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude
of diverse real life applications.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
Yersinia pestis is a high-risk pathogen categorised as a highly
hazardous organism that can be used as a biological weapon since it
is relatively easy and inexpensive to obtain, and is difficult to
distinguish from naturally occurring infectious disease outbreaks
[1]. Y. pestis is responsible for plague, a zoonotic disease, with
dissemination of the infection occurring through direct contact
with infected animals, animal tissue, bodily fluids, ingestion of the
meat from infected animals, drinking contaminated water, bites
from arthropods or inhalation of infectious droplets [2].
Different methodologies have been reported for the detection of
Y. pestis, offering a more rapid alternative to the gold standard
technique of culture-based methods, whilst having the same or
even higher sensitivity, including the use of molecular techniques
[3e7], immunochemistry [8e10] or the use of nanoconstructs [11].
However, these techniques cannot be deployed for use in situ at the
point of need and in order to reduce the impact of this potential
biowarfare agent, an accurate, rapid, inexpensive and easy-to-use
analytical tool is essential.
Lateral flow assays (LFAs) have been developed for rapid
detection at the point of need. These tests are single-use and the
vast majority of those reported to date are based on immuno-
chromatographic assays generating detectable colored bands with
a qualitative yes or no answer, with the best known assay being the
pregnancy test [12], and LFAs have evolved to be semi-quantitative
or quantitative [13].
Various commercial kits are available exploiting antibodies,
including the SMART II Yersinia pestis Anti-F1 detection kit (New
Horizons Diagnostics), Plague Biothreat Alert (Tetracore), BADD
Plague (Advnt Biotechnologies) and the ABICAP classic test kit e Y.
pestis (Senova). However, none of these immunotests have been
demonstrated to have both high sensitivity and high specificity, and
thus there is still a mature need for the development of reliable
diagnostic kits for use at the point of need (Table S1) [14].
As an alternative to immunodetection, molecular detection can
combine both high sensitivity and specificity. Nucleic acids can be
easily amplified producing multiple copies of DNA and conse-
quently, improving the limit of detection. Furthermore, a set of
primers can be designed to amplify a unique region specific for the
microorganism of interest and avoid cross-reactivity with other
related species. Moreover, molecular detection could also be
deployed as lateral flow assays [6].
There are a wide range of paper analytical devices that have
been developed for the detection of amplified products, commonly
referred to as nucleic acid amplification tests (NAAT). There are two
main types of NAAT: nucleic acid lateral flow immunoassays
(NALFIA) and nucleic acid lateral flow assays (NALF). NALFIA is the
most commonly reported and uses primers modified with small
molecules, including digoxigenin (Dig), carboxyfluorescein (FAM),
fluorescein isothiocyanate (FITC) or biotin, which are detected us-
ing antibodies or streptavidin, either immobilised at the test and
control lines, or linked to reporter molecules [15e19]. NALF, on the
other hand, does not use hapten labelled amplicons and instead
detects DNA using capture and reporter oligonucleotide probes.
There are far less reports of NALF, which can be attributed to more
complicated assays requiring dT or dA probes [20,21], asymmetric
PCR [22,23], or alternative methods for the post-amplification
generation of single stranded DNA to hybridise with the capture
and reporter probes.
Specifically referring to the detection of Yersinia pestis, the
combination of different isothermal DNA amplification techniques
including loop-mediated isothermal amplification (LAMP), recom-
binase polymerase amplification (RPA) and thermophilic helicase-
dependent isothermal DNA amplification (tHDA) combined with
lateral flow dipsticks for the detection of biothreat agents based on
NALFIA assays was recently reported. The authors demonstrated
that either LAMP or RPA gave 100% specificity against a wide range
of related microorganisms tested without the need for a thermo-
cycler, further approaching the realisation of diagnostics for
deployment to the point-of-need [24]. However, the NALFIA assay
they used requires hapten labelled primers for immunobased
detection, which is not only more expensive than the primers,
capture and reporter probes needed for NALF assays, but also in-
troduces issues of protein stability [25]. Due to this reason, NALF
assays were developed by our group based on isothermal RPA
amplification combined with the use of tailed primers, which result
in an amplicon comprising the target duplex flanked by two single
stranded DNA tails designed to be complementary to capture and
reporter probes, facilitating direct detection via hybridization
[25,26]. This unique type of amplicon is obtained thanks to an in-
ternal modification on the oligonucleotides consisting in a propyl
chain (C3), positioned between the primer binding site and the
single stranded tail, acting as “stopper”. Most recently, this
approach was applied for the simultaneous duplex detection of
Yersinia pestis and Francisella tularensis in a multiplex lateral flow
assay, achieving LODs of 4 fg and 243 fg, respectively [27].
In the work described here, we have extended our previous
work to develop a more sensitive NALF assay, using the detection of
Yersinia pestis as a model system. We combined the use of a forward
tailed primer which hybridises to the immobilised capture probe
with the incorporation of biotinylated dNTPs to enhance the signal
of the assay. Assay parameters were optimised using synthetic DNA
and then applied to the detection of genomic DNA. The developed
platform is generic in nature and can be applied to the detection of
all types of nucleic acids using optical or electrochemical trans-
duction by just applying a specific set of primers for each target of
interest.
2. Materials and methods
2.1. Materials
Phosphate-buffered saline (PBS; 10 mM phosphate, 137 mM
NaCl, 2.7 mM KCl, pH 7.4), PBS-Tween (10 mM phosphate, 137 mM
NaCl, 2.7 mM KCl, 0.05% v/v Tween 20, pH 7.4), Streptavidin
-Horseradish Peroxidase (SA-HRP), 6-mercapto-1-hexanol (MCH),
Skimmed milk powder, sodium citrate, sulphuric acid, gold (III)
chloride trihydrate (HAuCl4), 3,30 ,5,50 -tetramethylbenzidine (TMB)
and all other reagents were purchased from Sigma (Barcelona,
56 S. Kortli et al. / Analytica Chimica Acta 1112 (2020) 54e61
Spain). Maleimide activated plates, 8-well strips, were from Fischer
Scientific (Madrid, Spain) and Tfi DNA polymerase and 10 bp DNA
ladder from Life Technologies (Barcelona, Spain). Certified™ Low
Range Ultra Agarose was purchased from Bio-Rad (Barcelona,
Spain). Streptavidin Poly-HRP80 was supplied from SDT-reagents
(Baesweiler, Germany). Biotin-16-dCTP, Biotin-16-dUTP and Biotin
PCR Labeling Core Kit were purchased from Jena Bioscience (Jena,
Germany). All DNA oligonucleotides were purchased from Biomers
(Germany). All solutions were prepared in high-purity water ob-
tained from the Milli-Q RG system (Barcelona, Spain).
2.2. Genomic DNA extraction
The genomic DNA from Yersinia pestis was prepared at the
Friedrich-Loeffler-Institut (FLI), Institute of Bacterial Infections and
Zoonoses (Germany). The strain used was Yersinia pestis EV76. The
DNA was extracted using the High Pure PCR Template Preparation
kit from Roche following the manufacturer’s instructions.
2.3. Primers design
Primers and synthetic sequences for the detection of Y. pestis
were designed to be specific for targeting gene pla using multiple
primer analyzer from ThermoFischer Scientific and BLAST tools.
The size of the amplified region is 120 bp, but as a DNA tail and
biotinylated dNTPs are incorporated during amplification, a band
above 120 bp is expected (around 150 bp). Primers and probe se-
quences can be found in Table S2.
2.4. Optimisation of biotinylated dNTPs’ratio
2.4.1. Sample preparation: incorporation of biotinylated dNTPs
In order to obtain the highest signal, different ratios of bio-
tinylated dNTPs were studied (0, 20, 40, 60, 80 and 100%) using
different polymerases (Tfi, Therminator and Kapa2G Robust poly-
merases). Commercial Biotin-16-dCTP and Biotin-16-dUTP bio-
tinylated dNTPs were used, with the biotin linked via biotin-16-
Propargylamino-dCTP attached to the C5 position of cytidine and
biotin-16-5-aminoallyl-dUTP attached to the C5 position of uridine
respectively.
The introduction of biotinylated dNTPs in the amplicon was
carried out using the Polymerase Chain Reaction (PCR) with 50 pM
of template DNA. All the PCR reactions were performed in a total
volume of 100 mL with 200 nM of primers, 5 mM of MgCl2, 200 mM
of dNTPs, 1x PCR buffer and 1 U polymerase. The program used was
based on a previous heated step at 95  C for 2 min, followed by 25
rounds of PCR, with 30 s denaturation at 95  C, 30 s annealing at
58  C, and 30 s elongation at 72  C, followed by a final extension
step at 72  C for 5 min. Double-stranded PCR products were
visualised using gel electrophoresis, via addition of 5 mL of ampli-
fied sample with 4 mL of 6x loading buffer to a 3% (w/v) agarose gel
stained with GelRed nucleic acid stain (VWR, Spain) and imaging
with a UV lamp (l ¼ 254 nm). Finally, the amplicons were purified
with the DNA Clean and Concentrator kit (Ecogen, Barcelona, Spain)
according to the manufacturer’s instructions and were evaluated
using an enzyme linked oligonucleotide assay (ELONA).
2.4.2. Evaluation of biotinylated dNTPs using an enzyme linked
OligoNucleotide assay (ELONA)
Maleimide plates were functionalised by pipetting 100 mL of
200 nM thiolated capture probe in PBS into each well, and the
plates were then incubated overnight at 4  C, and subsequently
washed with PBS-Tween. Any remaining maleimide groups were
blocked by addition of 200 mL (per well) of 100 mM 6-mercapto-1-
hexanol in deionised water and incubation for 30 min, before
washing the plate thoroughly with PBS-Tween. A second blocking
step was carried out by adding 200 mL (per well) of 5% w/v skimmed
milk powder in PBS-Tween buffer, which was left to incubate for
15 min, followed by thorough washing.
Subsequently, PCR amplicons were added to the functionalised
maleimide plates (50 mL per well) for 30 min at room temperature
under shaking conditions. This was followed by a washing step
with PBS-Tween, and subsequent addition of 50 mL of a 1 in 20,000
dilution of 1 mg mL1 streptavidin bioconjugated to horseradish
peroxidase (SA-HRP) or streptavidin linked to a polymer containing
five identical colvalent HRP homopolymer blocks that are, also
covalently, coupled to multiple streptavidin molecules (SA-pol-
yHRP80), to each well and then incubated for a further 30 min.
After a final washing step, the presence of HRP was measured
following addition of 50 mL of TMB substrate, with the enzymatic
reaction being stopped via addition of 50 mL 1 M H2SO4 5 min later.
The absorbance was read at 450 nm (SpectraMax 340PC384, bio-
Nova Scientifics S.L.).
A range of concentrations of amplified Yersinia pestis synthetic
DNA were tested (50 nM, 5 nM, 0.5 nM, 0.05 nM, 0.005 nM,
0.0005 nM, 0.00005 nM and 0 nM) using the optimised ratio of
modified: natural dNTPs. The signals obtained were plotted with
GraphPad Prism software and fitted to a sigmoidal 4 PL model. The
limit of detection (LOD) was defined as the lowest signal obtained
(blank; no target) plus three times its standard deviation
(Blank þ 3x SD Blank) and the value was interpolated from the
fitted curve. Triplicate measurements were performed for each
concentration.
2.5. Isothermal recombinase polymerase amplification (RPA)
RPA was performed according to the manufacturer’s in-
structions (TwistAmp Liquid Basic kit, TwistDX, Cambridge, UK).
Briefly, master mix was prepared in a tube with 1 x reaction buffer,
480 nM of each primer, 1x Basic E-mix, 200 mM dNTPs (using 60% of
biotinylated dCTP), 1x Core reaction mix, 14 mM magnesium ace-
tate, the desired concentration of the synthetic or genomic DNA,
and adjusted to a final volume of 50 mL with Milli Q water.
Amplification was carried out at 37  C for 15 min. Double-stranded
RPA products were purified with DNA Clean and Concentrator kit
(Ecogen, Barcelona, Spain) according to the manufacturer’s in-
structions and checked using electrophoresis on a 3% (w/v) agarose
gel stained with GelRed nucleic acid stain (VWR, Spain) and imaged
with UV lamp (l 254 nm).
2.6. Lateral flow assay
Gold nanoparticles functionalised with streptavidin (SA-AuNPs)
with an average diameter of 40 nm and optical density (OD) 10
were purchased from BBI solutions (Cardiff, UK). In order to in-
crease the signal obtained in the lateral flow strips, this conjugate
was concentrated around 6-fold by centrifugation.
The membrane used was FF170HP nitrocellulose (GE Healthcare
Europe GmbH, Germany), and the absorbent pad was composed of
cotton litter fibbers grade 320 (Ahlstrom, Sweden). The test and
control lines were prepared by drawing a line with a pipette tip. On
the test line a pre-incubated (15 min at 22  C) mixture of
1.7 mg mL1 of streptavidin and 67 mM of the biotinylated capture
probe in PBS buffer was immobilised, whilst for the control line,
biotinylated goat anti-rabbit IgG antibody, 0.8 mg mL1, (Sigma
Aldrich, Spain) in PBS buffer was immobilised. Subsequently, the
membrane was allowed to dry at 22  C for 1 h, followed by blocking
with 1% w/v skimmed milk powder and 0.1% v/v Empigen detergent
for 15 min, under shaking conditions. The membrane was left to
dry, again at 22  C for approximately 2 h and then stored at 4  C
 S. Kortli et al. / Analytica Chimica Acta 1112 (2020) 54e61 57

Fig. 1. Schematic representation of ELONA assay.

Fig. 2. Calibration curves obtained from the amplicons using different ratios of
modified dNTPs: natural dNTPs and a reverse primer modified with one biotin in Fig. 4. Calibration curves comparing PCR vs. RPA incorporating 60% of biotinylated
50 end. dCTP and detecting with SA-polyHRP.

until use. The test strips were cut in strips of 4 mm width (M70,
Advanced Sensor Systems P.Ltd.).
Six microliters of streptavidin linked to AuNPs were mixed with
6 mL of RPA product, 2 mL of 10 x PBS-tween buffer and 6 mL Milli Q
water. The mixture was incubated for 3 min at 22  C before being
wicked on to the test strip. In order to explore the sensitivity of the
assay, RPA was carried out with a wide range of starting concen-
trations of ssDNA (109 M, 1010 M, 1011 M, 1012 M, 1013 M,
1014 M, 1015 M, 0 M) or genomic DNA (80 pg, 8 pg, 0.8 pg, 0.08 pg,
0.008 pg, 0.0008 pg, 0.00008 pg, 0 pg). A Smartphone camera was
used to take an image of the strip and the intensity of the bands
were analysed using Image J software. These values were plotted in
GraphPad Prism software using a sigmoidal 4 PL model in order to
obtain the LOD of the assay. Triplicate measurements were per-
formed for each concentration and the LOD was calculated inter-
polating the resulting value from the formula zero concentration
(blank) value ± 3 x standard deviation of this value.

3. Results and discussion

Fig. 3. Calibration curves incorporating 60% of biotinylated dCTP and comparing use of
SA-HRP (one HRP molecule per molecule of SA) vs. SA-polyHRP (more than one HRP Biotinylated dNTPs were explored as a means to achieve ultra-
per molecule of SA).
sensitive molecular detection, using the potential biowarfare agent,
Yersinia pestis as a model. ELONA was used to optimise assay pa-
rameters, which were then translated to a lateral flow assay, and
58 S. Kortli et al. / Analytica Chimica Acta 1112 (2020) 54e61
Fig. 5. Schematic representation of lateral flow assay.
both assays were applied to the analysis of synthetic and genomic
DNA.
The assay took advantage of our previously reported tailed
primer approach, which avoids the need for the generation of single
stranded DNA prior to detection via hybridisation. Primers are
specifically designed with the primer recognition sequence, fol-
lowed by a carbon spacer, which acts to prevent further elongation,
and a single stranded tail. The use of these primers results in a
dsDNA amplicon tethered by two ssDNA tails [25,26,28]. In the
assays described herein, only the forward primer was modified,
achieving a dsDNA amplicon with one ssDNA tail on the 5’ end, as
the signal was obtained from biotinylated dNTPs incorporated
during the amplification. For a better understanding of the result-
ing amplicon see Fig. S1.
3.1. Primers design
The primers for this work were specifically designed for the pla
gene, according to Riehm et al., whom reported that the best real-
time PCR assay was the 50 -nuclease targeting pla gene. The authors
claimed that it can be recommended as diagnostic tool for
establishing a presumptive diagnosis when bubonic plague is
clinically suspected [5]. Once the primers were designed, they were
tested using Primer Blast software to check its specificity against
other related species (Table S3).
3.2. Optimisation of biotinylated dNTPs
In order to achieve the maximum incorporation of the bio-
tinylated dNTPs during amplification, different ratios of two
different commercial types of biotinylated dNTPs were evaluated
(biotin-dCTP and biotin-dUTP) with three different polymerases.
(Tfi polymerase, Therminator polymerase and Kapa2G Robust po-
lymerase). The Tfi polymerase can be directly used in the same
protocols normally carried out with Taq DNA polymerase, and is
comparable to that of Taq in terms of yield, specificity, fidelity and
robustness [29]. The Therminator polymerase is a 9 N™ DNA Po-
lymerase variant with an enhanced ability to incorporate modified
substrates such as dideoxynucleotides, ribonucleotides and acy-
clonucleotides [30] whilst, the Kapa2G Robust polymerase is ideally
suited for challenging PCR applications and difficult samples [31].
Only the Kapa2G Robust polymerase was observed to achieve good
 S. Kortli et al. / Analytica Chimica Acta 1112 (2020) 54e61
Fig. 6. Calibration curve for the determination of LOD in lateral flow assay: (a) Yersinia pestis synthetic DNA; (b) Yersinia pestis genomic DNA.
levels of amplification with both of the biotinylated dNTPs (Fig. S2)
and was thus used to evaluate the best ratios of the biotinylated
dNTPs. Increasing the ratio of biotinylated dNTPs: natural dNTPs an
increase in size of the amplicon was observed by the shift of the
band in the electrophoresis gel, demonstrating the successful
incorporation of these modified dNTPs. However, when higher
amount of biotinylated dNTP was used, an inhibitory effect was
observed, resulting in lower amplification efficiency (Fig. S2c).
In order to confirm that the biotinylated dNTPs were incorpo-
rated during amplification, the amplicon products were detected
using ELONA. In this assay, the plate was functionalised with a
thiolated capture DNA probe complementary to the ssDNA tail of
the amplicon, and as detector probe SA-HRP was added (Fig. 1).
The maximum signal was achieved when 60% and 40% of bio-
tinylated dCTP and dUTP, was used respectively (Figs. S3aeb). The
effect of incorporating both biotinylated dNTPs together was also
explored with the aim of further enhancing the final signal ach-
ieved. When both dNTPs were used, the highest signal was
observed using a combination of 60% biotinylated dCTP and 20%
biotinylated dUTP (Fig. S3c). Calibration curves were performed
with these optimum ratios and the detection limits (LOD) obtained
were compared to that achieved using two different controls. One
was based on the use of biotinylated reverse primer, where only
one biotin is incorporated on the 5’ end of the amplicon, as this
format is frequently exploited for subsequent binding to strepta-
vidin linked reporter molecules. The other control was performed
with the commercial Biotin PCR labelling core kit (Jena Bioscience),
which is reported to have already prepared 50% of biotinylated
dUTP in the master mix.
Detection limits of 45 pM, 52 pM and 55 pM, were obtained for
biotinylated dCTP alone (60%), biotinylated dUTP alone (40%) and
the Biotin PCR labelling core kit, respectively. A higher detection
limit was obtained with a combination of both biotinylated dNTPs,
which can be attributed to an inhibitory effect when too high level
of modified: natural dNTPs is used (Fig. 2).
The use of a commercially available SA-polyHRP80 conjugate
consisting of five identical covalent HRP homopolymer blocks
59
providing 400 HRP molecules was explored as a means to further
improve the detection limit incorporating 60% of biotinylated dCTP.
A LOD 941 fM, two orders of magnitude lower, was obtained (Fig. 3).
3.3. Recombinase polymerase amplification (RPA) and lateral flow
assay (LFA) detection
Having achieved an ultrasensitive detection limit via the use of
biotinylated dNTPs, the possibility of exploiting these modified
dNTPs using isothermal amplification and lateral flow assay
detection was pursued.
The Recombinase Polymerase Amplification (RPA) method of
isothermal amplification was chosen as it, operates at 37-42  C with
minimal sample preparation and is capable of amplifying as low as
1e10 DNA copies in less than 20 min [32]. RPA has been used to
amplify diverse targets, including RNA, miRNA, ssDNA and dsRNA
from a wide variety of organisms and samples and applied to a
range of assay formats [33], suitable for deployment to the point-
of-need. The amplification time and different ratios of bio-
tinylated: natural dCTPs were evaluated (Figs. S4 and S5) obtaining
similar amplification level (314 fM) as that achieved using PCR with
incorporating 60% of biotinylated dCTP and in just 15 min of reac-
tion (Fig. 4).
3.4. Isothermal amplification with the tailed forward primer was
then combined with LFA detection
As previously reported, the test line consisted of an immobilised
biotinylated oligonucleotide probe complementary to the single
stranded tail of the RPA amplicon [25], whilst biotinylated antibody
was immobilised on the control line to bind with the SA-AuNPs. In
the presence of analyte, the amplicon binds to the capture probe
immobilised on the test line and the red colour of the SA-AuNP
conjugate is observed both at the test line and the control line
(Fig. 5).
The concentration of the SA-AuNP conjugate, the amount of
sample added to the LFA and the buffer used was optimised. A six-
60 S. Kortli et al. / Analytica Chimica Acta 1112 (2020) 54e61
fold concentration of the SA-AuNP conjugate and 6 mL of RPA
amplicon in a final volume of 20 mL, gave the sharpest and most
intense red bands (Fig. S6). Whilst PBS is the most commonly used
buffer for LFAs [34], the use of PBS-tween acts as a block to avoid
non-specific binding [35], as can be seen in Fig. S6c.
To establish the sensitivity achievable with LFA detection, a
range of concentrations of synthetic DNA were isothermally
amplified using the tailed forward primer and the biotinylated
dCTP (Fig. S7a). The test line was visible to the naked eye at con-
centrations as low as 10 fM for Y. pestis (Fig. 6a), and using a
Smartphone camera and Image J software, the LOD was improved
to 7 fM.
Having established the sensitivity attainable, genomic DNA was
isothermally amplified (Fig. S7b), and captured on the developed
LFA, achieving a detection limit of 0.63 fg (31.42 fg uL1 or 0.13
genomic equivalent) using Image J software (Fig. 6b). These
detection limits are around 16-fold better compared with the
detection using real-time PCR [5] and 6-fold better than our pre-
vious results using tailed primers [27]. Comparing with lateral flow
assays describing the detection of genomic DNA from other mi-
croorganisms, including Mycobacterium tuberculosis (25 fg) [36],
Skeletonema costatum (0.94 pg mL1) [37], Coxiella burnetti (7
copies/reaction) [38] and Pseudomonas aeruginosa (10 fg) [39], a
lower detection limit is reported here.
4. Conclusions
We have reported the first example of a nucleic acid based
lateral flow assay, exploiting a tailed primer and biotinylated dNTPs
to facilitate ultrasensitive detection limits for use at the point-of-
need, with a complete assay time of less than 30 min. To demon-
strate the functionality of the developed assay, Y. pestis genomic
DNA was isothermally amplified and detected in a lateral flow
assay, achieving a LOD of 0.63 fg. Future work will focus on the
implementation of an integrated lateral flow assay, where the
isothermal amplification and detection of the amplicon will be
carried out in the same platform, with the only required end-user
intervention being sample addition.
Funding
The authors are grateful to King Abdulaziz University, under the
financing of the collaborative project “Selection and application of
aptamers against anabolic steroids” for funding.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Salah Kortli: Data curation, Formal analysis, Investigation,
Writing - review & editing. Miriam Jauset-Rubio: Conceptualiza-
tion, Data curation, Formal analysis, Investigation, Writing - orig-
inal draft, Writing - review & editing. Herbert Tomaso: Validation.
Mohammed Nooredeen Abbas: Project administration, Supervi-
sion, Investigation, Writing - review & editing. Abdulaziz S.
Bashammakh: Conceptualization, Funding acquisition, Writing -
review & editing. Mohammad S. El-Shahawi: Conceptualization,
Funding acquisition, Writing - review & editing. Abdulrahman O.
Alyoubi: Conceptualization, Funding acquisition, Writing - review
& editing. Mounir Ben-Ali: Project administration, Supervision,
Investigation. Ciara K. O’Sullivan: Conceptualization, Project
administration, Supervision, Investigation, Writing - original draft,
Funding acquisition, Writing - review & editing.
Acknowledgements
The genomic DNA from Yersinia pestis was kindly provided by
The Friedrich-Loeffler-Institut (FLI), Institute of Bacterial Infections
and Zoonoses (Germany).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.aca.2020.03.059.
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