Download a simple integrated diagnostic platform for dna testing of chlamydia

A SIMPLE INTEGRATED DIAGNOSTIC PLATFORM FOR DNA TESTING
OF CHLAMYDIA TRACHOMATIS INFECTION
D. J. Shina, L. Chena and T. H. Wang*a,b,c,d
Department of Biomedical Engineering, Johns Hopkins University, UNITED STATES
b
Department of Mechanical Engineering, Johns Hopkins University, UNITED STATES
c
Institute for NanoBioTechnology, Johns Hopkins University, UNITED STATES
d
Center for POC Tests for Sexual Transmitted Diseases, Johns Hopkins University, UNITED STATES
a
ABSTRACT
This paper presents an integrated nucleic acid amplification platform for detection of Chlamydia trachomatis (Ct)
infections from self-collected vaginal swab samples. The assay combines magnetic bead-based DNA purification with
loop-mediated isothermal amplification (LAMP) to enable high-sensitivity nucleic acids testing with low-complexity
instrumentation. The reagents are processed on a droplet cartridge utilizing a novel lamination-based fabrication
technique. The platform utilizes a simple rotary mechanism for a completely USB-powered actuation of magnetic beads.
We demonstrate the performance of our prototype by correctly identifying blinded vaginal swab samples collected from a
high-risk population for transmission and acquisition of Ct.
KEYWORDS: Sexually-transmitted infections, loop-mediated isothermal amplification, colorimetric detection, surface
patterning, solid-phase DNA extraction
INTRODUCTION
Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen and a common sexually-transmitted disease
(STD). Ct is very widespread, with an estimated 2.86 million infections annually in the United States alone. Most infected
people do not present with symptoms, which makes it difficult to monitor the prevalence of Ct in a population. However,
in symptomatic patients, Ct infection can result in permanent damage to reproductive organs in women. In order to assist
with the control of Ct infection in a population, it is necessary to develop a diagnostic platform which is both accurate and
sufficiently convenient and accessible to the general public .
Current technologies for Ct screening are broadly found in two formats: cartridge-based nucleic-acids amplification
tests (NAAT) [1] and lateral flow device-based immunoassays (LFD) [2]. While NAAT provides superior sensitivity, the
sophisticated instrumentation for cartridge processing makes these solutions costly. The LFD tests can be found in a more
affordable format applicable to operation in limited-resource settings, although sensitivity is generally considered
inadequate [3]. We propose an intermediate solution which further simplifies the instrument requirements for NAAT
formats by developing a single-stream colorimetric LAMP assay which is automated by a low-power magnetic bead
actuation mechanism.
THEORY
The foundation of the presented work rests upon three key concepts: 1) solid-phase nucleic acid isolation; 2)
colorimetric loop-mediated isothermal amplification; 3) surface patterning. Solid-phase DNA extraction is a widely used
technique in molecular biology. While the underlying mechanisms of action is contingent upon the surface coating of the
solid phase material, the general idea is to adsorb nucleic acid from a lysate mixture and release them in elution buffer. In
particular, magnetic colloid micro/nanoparticles have gained favor due to their inherently superior surface-to-volume
ratio. In current work, beads coated with polyhistidine residues are used for DNA extraction. Bead surface charge is
positive at acidic pH and negative at alkaline pH, allowing elution of captured DNA directly into LAMP mixture.
Colorimetric detection of DNA amplification obviates the need for sophisticated detection instrument, thereby
reducing the cost of each screening test. LAMP has recently emerged as a promising candidate for colorimetric detection,
due to its exceptionally high total DNA synthesis [4]. Amplification of product results in generation of pyrophosphates
during synthesis, which chelates and precipitates magnesium ion in solution. The decrease in magnesium ion
concentration in solution can be monitored with a metal indicator dye, in the form of a color shift from violet to blue [5].
Surface patterning plays an important role in two ways. Hydrophilic spots enable the formation of compartments of
aqueous reagents on the device, while smooth hydrophobic surfaces facilitate the transfer of solid particles between
aqueous compartments. In this work, smooth hydrophobic surfaces are generated by simply laminating the hydrophilic
PMMA with PTFE tape (contact angle ~105°). Hydrophilic PMMA spots (contact angle ~68°) are revealed by etching
the spot perimeter with CO2 laser cutter and peeling away the PTFE film.
EXPERIMENTAL
Schematic of the assay is presented in Figure 1A. The automated DNA processing cartridge accepts 100 µL of sample
lysate and produces a colorimetric result within 1 hour. DNA extraction is facilitated by charge-based interaction of
nucleic acids with magnetic beads (Figure 1B). The lysate is transferred into a sample loading slot and mixed with
binding solution. This lowers the mixture pH to 5 and favors adsorption of DNA to bead surface. After washing, the
978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001
1350
17th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
27-31 October 2013, Freiburg, Germany
beads are transported to the LAMP mixture for direct elution of DNA. The LAMP mixture has a pH of 8.8, which favors
release of bound DNA from bead surface. Incubation at 65˚C initiates isothermal DNA amplification. Upon target
amplification via LAMP, color change from violet to blue is observed using a metal ion indicator dye.
Automation of the assay is achieved with instrumentation described in Figure 1C. Rotary actuation is an economical
alternative to linear translation and well-suited to point-of-care applications. The prototype utilizes a USB-powered servo
motor with a magnet mounted on its rotor arm. A sequence of angular positions is programmed to guide the magnet
across various reagents.
A
B
C
Figure 1: A) Overview of assay platform. Sample lysate is loaded directly to the input chamber containing a mixture of
binding buffer and paramagnetic beads. Sample preparation is automated by a USB-controlled rotary manipulator. B)
Overview of single-stream Chlamydia trachomatis assay. C) Overview of the automated cartridge processing instrument.
A USB-powered and controlled servomotor enables programmable movement of permanent magnet mounted on its arm.
The assay is realized on PMMA-based cartridges fabricated as illustrated in Figure 2A. The cartridge consists of two
functional layers separated by a spacer. The upper layer is laminated with PTFE sealing tape to generate a smooth
hydrophobic surface, followed by laser-assisted etching to expose the PMMA surface below. This enables anchoring of
multiple aqueous reagent pendants sealed in a continuous fluorinated oil phase. The continuous PTFE surface on the
lower layer forms contact with the reagents and facilitates transportation of magnetic beads across reagents (Figure 2B).
As shown in Figure 2C, efficient splitting of magnetic beads from reagents can be achieved. The fabrication process has a
rapid turnaround time and does not require access to clean room facility.
B
A
C
Figure 2: A) Cartridge fabrication overview. Each cartridge is assembled from 2 functional layers and a spacer frame
and bonded with acrylic pressure-sensitive adhesive tape. B) Schematic of cartridge side profile after assembly. The
PTFE surface on the lower layer compresses reagent droplets, forming cylindrical columns of aqueous reagents isolated
in a continuous fluorinated oil phase. Magnetically actuated beads can freely traverse through reagents on a continuous
PTFE surface. C) Photograph of magnetic bead plug dissociation sequence.
1351
ladder
RESULTS AND DISCUSSION
Assay sensitivity was initially evaluated in a bench top process using Ct cell culture dilutions and verified using 2%
agarose gel electrophoresis. Successful LAMP amplicons are elongated as a growing chain of repeating sequences, which
is observed on the gel image as a set of growing ladders (Figure 3A). Amplification was observed for up to 101
CFU/reaction. Afterwards, readout sensitivity was evaluated by testing dilutions of Ct DNA extract as the input. Assay
was performed in 25 µL reaction volume. Magenta/violet indicates negative amplification while light blue indicates
positive amplification. Color changes were observed up to 10 copies per reaction (Figure 3B). Gel electrophoresis
verification confirmed successful amplification up to same input concentration (data not shown).
6
5
4
3
2
1
10 10 10 10 10 10 1
NTC +
10
4
10
3
10
2
10
1
1
NTC
Figure 3: A) Sensitivity results obtained with Chlamydia trachomatis cell culture dilutions verified using 2% agarose gel
electrophoresis. Amplification is observed for up to 101 CFU/reaction. B) Sensitivity results for colorimetric readout.
Afterwards, our prototype was tested using self-collected vaginal swabs from a high-risk population for Ct
transmission and acquisition as described previously [6]. The swabs were initially expressed in 1x Tris-EDTA buffer and
tested with the gold standard Gen-Probe Aptima Combo 2 assay. Six remaining aliquots of identified samples were
blinded and tested on our platform. At the end of LAMP reaction, magnetic beads were cleared to the periphery of the
reaction to reveal the color of the completed reaction. Figure 4 shows colorimetric readout correctly identifying infected
samples.
Sample ID
1
2
3
4
5
6
Gen-Probe
−
+
−
+
−
+
Prototype
−
+
−
+
−
+
Chip Positive
Chip Negative
Post-reaction cartridge
Magnetic bead
Figure 4: On-chip testing of six vaginal swab samples. Infected samples (2, 4 and 6) turned blue while negative samples
(1, 3 and 5) remained violet or magenta, as shown in the photograph.
CONCLUSION
This study demonstrates an integrated Ct screening platform capable of performing NAAT using a simple, compact
instrumentation. Our design focuses on reduced complexity, as evidenced by features such as colorimetric detection,
bead-based sample preparation and lamination-based device fabrication. Our approach suggests that the sensitivity of
DNA amplification-based tests in the laboratory could be retained in a format that is more accessible to the general public.
ACKNOWLEDGEMENTS
The authors would like to thank financial support from NIH (R01CA155305, U54CA151838 and R21CA173390) and
NSF (1159771 and 0967375)
REFERENCES
[1] Gaydos C. et al., 2013. J Clin Microbiol. 51(6): 1666-1672.
[2] Sabido M. et al., 2009. J Clin Microbiol. 47(2): 475.
[3] van Dommelen et al., 2010. Sex Transm Infect. 86: 355-359.
[4] Notomi T. et al., 2000. Nucl. Acids Res. 28(12): E63.
[5] Goto M. et al., 2009. BioTechniques. 46(3):167-172.
[6] Masek B. et al., 2009. J Clin Microbiol. 47(6): 1663-1667.
CONTACT
*T.H. Wang, tel: +1-410-5167086; [email protected]
1352