Download A MICROFLUIDIC CHIP COMBINING DNA EXTRACTION AND

A MICROFLUIDIC CHIP COMBINING DNA EXTRACTION AND REAL-TIME
PCR FOR IDENTIFYING BACTERIA IN SALIVA
E.A. Oblath, W.H. Henley, J.P. Alarie, J.M. Ramsey*
University of North Carolina at Chapel Hill, USA
ABSTRACT
The capability to quickly diagnose bacterial infections from saliva could greatly improve the ability of doctors to provide
appropriate treatment. Saliva is easily obtained and contains many of the same analytes as more traditional biological
samples. We have developed an aluminum oxide membrane (AOM)-PDMS-glass hybrid microfluidic chip that integrates
extraction, amplification, and detection of genomic DNA (gDNA) from bacteria found in saliva. Multiple species of bacteria,
including Streptococcus mutans, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA), can be simultaneously
identified through the use of parallel single-plex real-time polymerase-chain-reactions (rt-PCR).
KEYWORDS: Real-time PCR, Aluminum Oxide Membrane, DNA Extraction, Saliva
INTRODUCTION
Saliva has many advantages over blood as a diagnostic sample. Collection of saliva is non-invasive, painless, and does
not require a trained phlebotomist. Needles do not need to be used, reducing the possibility of accidental blood-borne
pathogen transmission. Saliva contains analytes from multiple biological sources, both systemic and local, and the levels of
analytes in saliva correlate well with those found in blood or urine [1]. Bacteria that cause respiratory infections have been
found in saliva and it also contains many organisms that are not infectious and can be used as controls [2, 3].
Traditional methods of identifying infectious bacteria rely on culturing the organisms. These methods require days to
provide results, and samples must be sent to a specialized laboratory. This is unsuitable when treatment decisions for
successful outcomes depend on rapid diagnoses. Rapid identification of bacteria from their genetic sequences can be
performed in a few hours via PCR amplification. While typical PCR methods are much faster than traditional methods, they
require labor-intensive nucleic acid extraction and purification that must be performed using specialized equipment in a
centralized lab. A point-of-care device integrating the DNA extraction and purification, PCR amplification, and detection of
PCR products would increase the accessibility of PCR as a diagnostic tool.
EXPERIMENTAL
AOM-PDMS-glass hybrid microfluidic devices are prepared using standard PDMS soft lithography and plasma bonding
techniques. Figure 1 shows an image of a chip and a schematic of its cross-section. An AOM is sandwiched between an
array of 7 parallel reaction wells and a microfluidic layer used to flow fluid through the membrane. Kim et al. have described
a PCR reactor that also incorporates an AOM for DNA extraction [4], but the design was limited to one reaction chamber and
off-chip gel electrophoresis was required for detection.
PDMS
Reaction well
Reaction wells
Waste
Reaction wells
AOM
Reaction wells
Waste
Glass slide
Figure 1: Image of chip (left) and schematic of chip cross-section (right)
DNA extraction from cell lysate is accomplished by filling the reaction wells with sample and applying vacuum to the
waste end of the channel until the entire sample has been pulled through the AOM. PCR reagents, including supermix, Taq
polymerase, bovine serum albumin to prevent adsorption of Taq polymerase to the AOM, and primers and probes, are then
added to the reaction wells and overlaid with mineral oil to prevent evaporation. The outlet of the channel is sealed with a
drop of uncured PDMS and the chip is placed on a Peltier stage for thermocycling. The chip is thermocycled with the
following program: 2 min at 50°C, 2 min at 95°C, 60 cycles of 30 s at 95°C followed by 65 s at 66°C, and finally, 2 min at
66°C. Using a fluorescence microscope and CCD camera, an image is taken at the end of each 65 s at 66°C step.
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001
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15th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
The presence of target bacteria is determined by monitoring the fluorescence signal from each reaction well due to
species-specific primers and probes. The probes utilize TaqMan chemistry. A quencher on the 3’ end of the probe
suppresses emission from a fluorescent dye on the 5’ end while the probe is intact. When the probe is cleaved during the
annealing/extension step in the cycle, the fluorescence is no longer quenched and a signal can be seen. By adding unique
primer and probe sets to different wells, multiple target species or strains of bacteria could be identified simultaneously while
using a single fluorescent dye. Streptococcus mutans, Staphylococcus aureus, and (MRSA) were used as model organisms to
design and test the chip. Three primer and probe sets were designed to use with the model organisms: S. mutans 16S rRNA,
for S. mutans, S. aureus nuc, for methicillin-susceptible S. aureus (MSSA) and MRSA, and S. aureus mecA, for MRSA.
RESULTS AND DISCUSSION
Purified gDNA was used to test for the simultaneous detection of two species of bacteria. 10 µL of a sample solution
containing 0.1 ng/mL S. mutans gDNA and 0.1 ng/mL MSSA gDNA was used for DNA extraction in each of the seven
wells. For this sample, there are 300-400 copies of gDNA from each species. After the entire sample was pulled through the
AOM, master mix and primer/probe sets were added to each well. Two wells contained the S. mutans 16S rRNA set, two
wells contained the S. aureus nuc set, and two wells contained the S. aureus mecA set. The center well was used as a
negative control and did not contain any primers or probes. As shown in Figure 2A, amplification was seen for the wells
containing the S. mutans 16S rRNA set and the S. aureus nuc set, correctly identifying the two types of gDNA used as the
sample. The same experiment was repeated with MRSA gDNA used in place of the MSSA gDNA. As shown in Figure 2B,
amplification is seen from all wells except the negative control, correctly identifying the sample as containing both S. mutans
gDNA and MRSA gDNA.
B
A
A
Figure 2: Real-time amplification plots for S. mutans 16S rRNA, S. aureus nuc, and S. aureus mecA genes. (A) Each well
contained 1pg of both S. mutans and MSSA gDNA as the sample while the primers were varied between the wells. (B) Each
well contained 1 pg of both S. mutans and MRSA gDNA as the sample while the primers were varied between the wells.
Cell lysate from cultured MSSA was used as a sample in another experiment. 10 µL of cell lysate was pulled through the
AOM from each well, and then master mix and primer/probe sets were added to each well. In this experiment six wells
contained the S. aureus nuc set and the center well did not contain any primers or probes and was used as a negative control.
As shown in Figure 3, amplification was seen in all six wells containing the S. aureus nuc primers and probe set. DNA from
the cell lysate was successfully captured on the AOM and separated from PCR inhibitors in the lysate.
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Figure 3: Real-time amplification plot for the S. aureus nuc gene. 10 µL of MSSA cell lysate was used as the sample for each
well, while the presence of primers and probes was varied between wells.
CONCLUSION
Multiplexed PCR is usually done with all primer sets combined in a single reaction. It is considered challenging to design
primers and probe sets that can be multiplexed without interfering with one another. Overlapping primers can produce
dimers, and other non-specific interactions between extraneous DNA or the amplicons with the primers and probes can
prevent amplification. An inherent bias in which short sequences are amplified faster than longer sequences can lead to
asymmetric amplification. The number of dye colors that can be concurrently analyzed by the instrumentation additionally
limits multiplexed rt-PCR. By multiplexing in space, our chip design eliminates non-specific interactions between primer
sets and requires only a single dye for target detection. Since interactions between primer sets do not need to be considered,
multiplexing in space also simplifies the addition of new primers sets for different target species. Our chip integrates
extraction, amplification, and detection of gDNA and can be used as a point-of-care device to rapidly detect many species of
infectious bacteria in saliva.
ACKNOWLEDGEMENTS
This work is supported by the National Institutes of Health (NIH) and National Institute of Dental and Craniofacial
Research (NIDCR) grant number U01DE017788-2.
REFERENCES
[1] L.F. Hofman, “Human Saliva as a Diagnostic Specimen,” Journal of Nutrition, vol. 131, pp. 1621S-1625S, 2001.
[2] O. Greiner, P.J.R. Day, P.P. Bosshard, F. Imeri, M. Altwegg, and D. Nadal, “Quantitatie Detection of Streptococcus
pneumoniae in Nasopharyngeal Secretions by Real-Time PCR,” Journal of Clinical Microbiology, vol. 39, pp. 31293134, 2001.
[3] S.B. Roth, J. Jalava, O. Ruuskanen, A. Ruohola, and S. Nikkari, “Use of an Oligonucleotide Array for Laboratory
Diagnosis of Bacteria Responsible for Acute Upper Respiratory Infections,” Journal of Clinical Microbiology, vol. 42,
pp. 4268-4274, 2004.
[4] J. Kim, M. Mauk, D. Chen, X. Qiu, J. Kim, B. Gale, and H.H. Bau, “A PCR Reactor With an Integrated Alumina
Membrane for Nucleic Acid Isolation,” Analyst, vol. 135, pp. 2408-2414, 2010.
CONTACT
*J.M. Ramsey, tel: +1-919-962-7492; [email protected]
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