Miniature on-chip detection of unpurified methicillin-resistant Staphylococcus aureus (MRSA) DNA using real-time PCR.
This paper evaluates the ability to detect three forms of methicillin-resistant Staphylococcus aureus (MRSA) in a microfluidic system. The MRSA was prepared off-chip by varying levels of sample preparation: one containing purified genomic DNA, another containing the supernatant of a crude preparation using simple reagents, and a third through boiled culture preparation without any additional reagents. Polydimethylsiloxane (PDMS) microfluidic chips were fabricated using soft lithography and then bonded to a 22mmx22mmx0.1mm glass cover slip. A lid fabricated in a similar manner was used during compression to prevent bubble formation and evaporation in the stationary well-based chip. A miniature thermal cycler based on a resistive heater and a small fan were used to cycle through desired temperatures for polymerase chain reaction and fluorescent intensity measurements were taken at each cycle. Each form of template provided positive results utilizing the developed micro-PCR system (verified with gel electrophoresis). A serial dilution of the purified genomic DNA provided a standard curve with an efficiency of 1.77. The lowest concentration that provided clear positive results came from a 3microL sample containing 11.2pg of DNA. The ability to detect MRSA in a sample having undergone minimal sample preparation is a necessary step in the development of a point-of-care detection system capable of identifying infectious organisms.
Mechanical Engineering Department, Vanderbilt University, 2301 Vanderbilt Place, VU Station B 351592, Nashville, TN 37235, United States. dustin.house@Vanderbilt.Edu <dustin.house@Vanderbilt.Edu>
Dustin L
DL
House
Chan Hee
CH
Chon
C Buddy
CB
Creech
Eric P
EP
Skaar
Dongqing
D
Li
D002874:Chromosome Mapping / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006361:Heating / Q000295:instrumentation*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008904:Miniaturization; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*