Solid phase DNA extraction with a flexible bead-packed microfluidic device to detect methicillin-resistant Staphylococcus aureus in nasal swabs.
We have developed a bead-packed microfluidic device with a built-in flexible wall to automate extraction of nucleic acids from methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs. The flexible polydimethylsiloxane (PDMS) membrane was designed to manipulate the surface-to-volume ratio (SVR) of bead-packed chambers in the range of 0.05 to 0.15 (μm(-1)) for a typical solid phase extraction protocol composed of binding, washing, and eluting. In particular, the pneumatically assisted close packing of beads led to an invariant SVR (0.15 μm(-1)) even with different bead amounts (10-16 mg), which allowed for consistent operation of the device and improved capture efficiency for bacteria cells. Furthermore, vigorous mixing by asynchronous membrane vibration enabled ca. 90% DNA recovery with ca. 10 μL of liquid solution from the captured cells on the bead surfaces. The full processes to detect MRSA in nasal swabs, i.e., nasal swab collection, prefiltration, on-chip DNA extraction, and real-time polymerase chain reaction (PCR) amplification, were successfully constructed and carried out to validate the capability to detect MRSA in nasal swab samples. This flexible microdevice provided an excellent analytical PCR detection sensitivity of ca. 61 CFU/swab with 95% confidence interval, which turned out to be higher than or similar to that of the commercial DNA-based MRSA detection techniques. This excellent performance would be attributed to the capability of the flexible bead-packed microdevice to enrich the analyte from a large initial sample (e.g., 1 mL) into a microscale volume of eluate (e.g., 10 μL). The proposed microdevice will find many applications as a solid phase extraction method toward various sample-to-answer systems.
In-Vitro Diagnostics Lab, Samsung Advanced Institute of Technology, Samsung Electronics Co., Ltd., Republic of Korea.
Kyu-Youn
KY
Hwang
Sung Hong
SH
Kwon
Sun-Ok
SO
Jung
Kak
K
Namkoong
Won-Jong
WJ
Jung
Joon-Ho
JH
Kim
Kahp-Yang
KY
Suh
Nam
N
Huh
D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D017817:Nasal Lavage Fluid / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction; D052616:Solid Phase Extraction; D013203:Staphylococcal Infections / Q000382:microbiology; D013499:Surface Properties
D004269:DNA, Bacterial; D004129:Dimethylpolysiloxanes; C013830:baysilon