We demonstrate a multiplexed loop mediated isothermal amplification (LAMP) assay for infectious disease diagnostics, where the analytical process flow of target pathogens genomic DNA is performed manually by moving magnetic beads through a series of plugs in a capillary. Heat is provided by a water bath and the results read by the naked eye, enabling applications in low resource settings

Cooper, Jonathan M.

Reboud, Julien

Xu, Gaolian

Zhao, Hang

Chemical Communications

English

PubMed

A sample-to-answer nucleic-acid based assay, processed magnetically inside capillaries, enables multiplexing analysis in low resource settings.</p

Gaolian Xu

Hang Zhao

Jonathan M. Cooper

Julien Reboud

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A capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients

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Cite this:Chem. Commun., 2016,
52, 12187
A capillary-based multiplexed isothermal nucleic
acid-based test for sexually transmitted diseases
in patients†
Gaolian Xu,‡ Hang Zhao,‡ Jonathan M. Cooper and Julien Reboud*
We demonstrate a multiplexed loopmediated isothermal amplification
(LAMP) assay for infectious disease diagnostics, where the analytical
process flow of target pathogens genomic DNA is performedmanually
by moving magnetic beads through a series of plugs in a capillary.
Heat is provided by a water bath and the results are read by the
naked eye, enabling applications in low resource settings.
Recently, rapid, high performance nucleic acid testing (NAT)
based diagnostic technologies have shown the ability to detect
infections early.1 However, the existing technologies for the
diagnosis of sexually transmitted diseases, such as chlamydia
and gonorrhoea, suffer from limitations in speed, cost, and the
significant user expertise required.1 The most widely used
technology is based on the polymerase chain reaction (PCR),
which attains high performance, but is difficult to carry out in
resource limited areas.1 As an alternative, low cost nucleic acid
based assays using isothermal amplification have the ability to
provide platforms for point-of-care (POC) diagnostics in both
low and high resource settings, due to their high sensitivities
and specificities.2
Isothermal amplifications, such as the loop-mediated
systems (LAMP), also offer the capability of multiplexing,3,4
which in turn can be used to inform treatment in e.g. infectious
diseases (where the relative sensitivities to specific drug
regimes are often determined genetically). Here we focus on
detecting two sexually-transmitted pathogens, Chlamydia
trachomatis and Neisseria gonorrhoea, which are prevalent in both
high as well as low and middle income countries (LMICs).5 The
LAMP reaction generally requires six DNA primers for each
target to be detected, limiting the possibilities of simulta-
neously amplifying multiple targets in single reactions, due to
the high level of potential cross-talk between the reactions. This
constraint has restricted the development of multiplexing POC
devices, since adding independent reactions to a single device
in turn increases practical challenges linked to reagent mixing,
analysis and temperature control.
Here we show that by exploiting capillary-based platforms
for multiplexing and in line sample processing,6–8 through the
use of plugs separated by an immiscible phase, we are able
to demonstrate a powerless, low-cost, easy-to-use sample-to-
answer multiplexed LAMP assay. Our new platform circumvents
the requirements for external fluid actuation and electronic
thermal control systems, potentially facilitating its deployment
as a POC device in resource-limited settings.
The advantages of using capillary-based platforms, such as
ease of use and multiplexing DNA amplification reactions of
extracted samples, have been realised previously,6 while single
target DNA extraction was successfully demonstrated,7 and DNA
lysis incorporated only recently within a similar format using
a FTA card (Flinders Technology Associates), but requiring
multiple pipetting steps.8 In this paper, we now go beyond
the current state of the art and show the complete nucleic-acid
processing (from crude samples to the answer) in a multiplexed
and easy-to-use format.
To achieve this, we integrated three different capabilities into
the capillary-based system, namely: manual sample manipulation
with magnetic beads to extract DNA from complex samples;
tolerance to temperature variability (enabling the use of heated
water to drive the reaction); and a visual detection of a calcein
fluorescent probe on the amplified product, illuminated by a
UV flashlight.9
Previously, magnetic beads have been integrated within
microfluidic devices for DNA separation and purification,10,11
providing a simple manual method for collection of bound
probes or targets without the need for centrifugation or filtra-
tion and with no requirement for external power.12 They also
advantageously replace previously used FTA cards,8 which are
difficult to handle and limit the applicability of previously
developed tip-based systems to trained experts. In the assay
configuration that we describe, the magnetic beads served as
Division of Biomedical Engineering, School of Engineering, University of Glasgow,
Oakfield Avenue, Rankine Building, G12 8LT Glasgow, UK.
E-mail: Julien.Reboud@glasgow.ac.uk
† Electronic supplementary information (ESI) available: Additional methods and
experimental sections. See DOI: 10.1039/c6cc05679b
‡ These authors contributed equally to this work.
Received 10th July 2016,
Accepted 8th September 2016
DOI: 10.1039/c6cc05679b
www.rsc.org/chemcomm
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12188 | Chem. Commun., 2016, 52, 12187--12190 This journal is©The Royal Society of Chemistry 2016
solid carriers for DNA (Fig. 1). By moving a handheld magnet,
the DNA passed through a series of discrete process steps,
defined by a series of plugs within the capillary, which enabled
us to purify and enrich the sample, removing any inhibitors
prior to the amplification reaction. The series of plugs simply
comprised different aqueous phases separated by mineral oil,13
which also served to prevent evaporation during the amplifica-
tion step at 60 1C. Multiplexing was enabled by sequentially
adding separate plugs containing different reaction mixtures
specific to each of the targets.
The whole process was completed within 60 min. 1 ml of
sample (in this case a model target DNA, at different concentra-
tions in PBS) was manually pipetted into the pre-filled capillary
(Fig. 1 and Experimental methods in the ESI†), where it was
mixed with lysis buffer, which also contained the magnetic
beads. Epoxy was used to seal the ends of the capillary to prevent
spillage and the capillary was left at room temperature for
3 minutes to lyse the cells and capture the DNA on the beads
(in a low pH and high ionic strength solution). The beads
(carrying the DNA) were then subsequently moved through the
mineral oil separator into the washing section (Fig. 1B(3–4) and
2A), where they were held for 1 min to remove residues and
contaminants from the beads, while retaining the DNA.14
A single wash step was sufficient to purify the DNA to a level
that enabled the LAMP reactions to proceed efficiently. The
magnet was moved further along the capillary, through an oil
separator into the LAMP reaction mixture, where the amplifica-
tion reaction was performed at 60 1C for 50minutes, using a water
bath as the heat source. This ‘‘plug’’ also served as the elution
section, where the DNA was released from the beads and detected
using a hand-held UV-flashlight to reveal green fluorescence.15
The results of the LAMP amplification were directly read out
visually, and we established a limit of detection of the capillary-
based assay by processing ten-fold serially diluted cultured
E. coli (Promega), which contained the conserved fragment of
C. trachomatis at 2.5  103 bacteria per ml (Fig. 2C and D),
corresponding to 2.5 copies of the plasmid in 1 ml. This approach
was validated using a real-time (RT) amplification approach, where
the signal was quantified using fluorescence microscopy4 (Fig. 2D).
As the target concentration increased, the exponential phase of
Fig. 1 (A) The integrated capillary LAMP system with preloaded reagents
after the addition of the sample. (1) Epoxy glue sealant; (2) lysis buffer and
magnetic beads (black spots) to release and capture DNA; (3) mineral oil;
(4) washing buffer; (5) LAMP reaction mix. (B) Process sequence: (1) pre-
filled capillary (2) sample (red) injection, lysis and DNA binding; (3) sealing
with epoxy glue (black) and aggregation of magnetic beads; (4–6) passing
throughmineral oil into the washing buffer to remove impurities; (7) elution
of DNA in the LAMP reaction mix and LAMP amplification (by heating to
60–64 1C); (8) illumination using a UV flashlight to reveal the assay results.
Fig. 2 Continuous images showing, under the influence of magnetic
field, the movement of MBs (red arrow) between solutions through
immiscible mineral oil. (A) The movement of MBs from water (blue) to
mineral oil (white); (B) the movement of MBs from oil (white) to water
(blue). Pictures were taken using a smartphone camera and an LED ring
lighting from above. (C) Examples of fluorescence images of the LAMP mix
plug containing different concentrations of E. coli carrying the target
DNA as a plasmid, at the end of capillaries illuminated with a UV flashlight
(from the bottom) and captured using a smartphone camera. (1) 2.5 
106 bacteria per ml; (2) 2.5 105 bacteria perml; (3) 2.5 104 bacteria per ml;
(4) 2.5 103 bacteria per ml; (5) 2.5 102 bacteria per ml. (6) ddH2O negative
control; (D) real-time amplification curves (1–4 normalised, left axis),
and ddH2O as a negative control (5 and 6, right axis, not normalised
to ease readability): (1) 2.5  106 bacteria per ml (black square); (2) 2.5 
105 bacteria per ml (red circle); (3) 2.5  104 bacteria per ml (blue lozenge);
(4) 2.5  103 bacteria per ml (green triangle); (5) 2.5  102 bacteria per ml
(black line); (6) negative control (red line). (E) Threshold time (defined
as the time corresponding to 50% of the maximum fluorescence
intensity, Tt) as a function of target concentration. Data are the average
of at least 3 replicates and error bars represent the standard deviation.
The data were fitted with linear regression (R2 = 0.999).
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signal enhancement started earlier, from ca. 18 minutes for 2.5 
106 bacteria per ml to 33 minutes for 2.5  103 bacteria per ml.
To assess the efficiency of the capillary system, we used the
threshold time (Tt) (Fig. 2E), as the reaction time for the
fluorescence signal to reach 50% of the maximum,4 which
shows a linear decrease with increasing target concentration
(on a logarithmic scale). The value of Tt is important as it
enables us to quantify the original amount of target DNA
present in the reaction after calibration, opening up applica-
tions in optimising medical treatment such as those based on
viral load quantification.16
Contrary to more complex DNA amplification techniques,
such as PCR, LAMP does not require precise thermal control or
cycling of the reaction (previously LAMP has been implemented
using pocket warmers6 and thermos containers17). Here we
simply poured hot water (ca. 300 ml at 64 1C) into a thermally-
insulated polystyrene foam container providing the required
stability over 1 h, with the temperature only cooling down
to 60 1C over this period (Fig. 3A). The only external energy
required for the LAMP was the warming of the water, which can
be easily performed even in remote locations. The functionality
of the approach was compared with a controlled incubation
at 63 1C, using gel electrophoresis analysis of the amplified
products (see the ESI† for Experimental details), and showed no
appreciable difference (Fig. 3B).
To demonstrate the multiplexed capability of our capillary
platform, an additional LAMP reaction mixture plug targeted at
the detection of N. gonorrhoeae (see Methods in the ESI† for its
composition) was added after the plug targeting C. trachomatis, into
a single capillary, separated by mineral oil. Due to the sequential
nature of the elution in the two plugs dividing the DNA contents,
the volume of the second plug (N. gonorrhoeae) was reduced to 3 ml,
compared to that of the first one (C. trachomatis – 5 ml) (Fig. 4A).
After amplification, the fluorescence signal in the two sections
of each capillary indicated the presence of the targeted
sequence of each pathogen simultaneously (Fig. 4B).
Finally, we further demonstrated the applicability of the
technique in 6 patient samples, obtained as swabs and
extracted as part of routine clinical diagnostics at the NHS
West of Scotland Specialist Virology Centre. The results (yes/no
indication of the presence of the pathogens) were consistent
with those obtained using the benchmark assay real-time PCR
(Table S1 and ESI†).
Using the ability of the real-time LAMP reaction to quantify
the initial DNA concentrations (the amount of DNA present
after extraction, before amplification – see Fig. 2), we also
explored the influence of the retention time on the quantity
of eluted DNA. When the beads were left in the first plug
(4 in Fig. 4A), without moving the beads to the second LAMP
plug (through the oil plug into 5 in Fig. 4A), the value of Tt was
30.5  1.0 minutes. When the beads were moved through
the first plug into the mineral oil, the Tt value for the LAMP
mix of the first plug was 35.5  0.5 minute. Using the equation
derived from Fig. 2C, we calculated that the recovery of DNA
when the beads only pass through the plug of LAMP reaction
mix (4 in Fig. 4A) was 15%  5% of that when the beads are left
in the plug.
In conclusion, in this study, we have reported a capillary-
based, magnetically-actuated, multiplexed LAMP assay platform
for the rapid diagnosis of C. trachomatis and N. gonorrhoea.
Its simplicity, low-cost and low energy requirement (only a tea
cup volume of hot water required) holds significant promise for
its application in low resource settings.
This work was supported by a College of Science and
Engineering Studentship (GX, Glasgow, UK), a Lord Kelvin
and Adam Smith Research Fellowship (JR, Glasgow, UK), an
EPSRC fellowship (JC, EP/K027611/1), and an ERC Advanced
Investigator Award (JC, 340117). All the original data related to
this article are within the depository of the University of Glasgow
with http://dx.doi.org/10.5525/gla.researchdata.349. Additional
data related to this paper may be requested from the authors.
Fig. 3 (A) Temperature variability within the insulated chamber. ca. 300 ml
of water at 64 1C was poured into the chamber and monitored for 60 min.
Data are the average of at least 3 replicates and error bars represent the
standard deviation. (B) Agarose gel results of N. gonorrhoeae within the
capillary platform. Lane L 100 bp ladder; lanes 1 and 3 were positive and
lanes 2 and 4 were negative. Lanes 1/2 were the reactions kept at 63 1C
constantly, while lanes 3/4 were incubated in the chamber. No appreciable
difference between the two systems is visible.
Fig. 4 Multiplex LAMP assay. (A) Multiplex LAMP system with preloaded
reagents. (1) Lysis buffer with MBs; (2) mineral oil; (3) washing buffer;
(4) LAMP reaction reagent 1 (3 ml); (5) LAMP reaction reagent 2 (5 ml);
(B) simultaneous detection of C. trachomatis and N. gonorrhoea:
(1) N. gonorrhoea LAMP mix; (2) mineral oil; (3) C. trachomatis LAMP
mix. Three capillaries were measured with different samples (from the top
to bottom): N. gonorrhoea, C. trachomatis, and C. trachomatis and
N. gonorrhoea sample simultaneously. The pictures were obtained using
a smartphone camera and illumination with a UV flashlight (from the
bottom). (C) Real-time LAMP amplification curves of the reactions with
an initial concentration of 2.5  105 bacteria per ml, when (1) the beads are
moved into the first plug (4 in A) and left there and (2) when the beads
traverse the first plug and are stored in the mineral oil.
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12190 | Chem. Commun., 2016, 52, 12187--12190 This journal is©The Royal Society of Chemistry 2016
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Capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients

Capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients

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