A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva.
A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Emily A
EA
Oblath
W Hampton
WH
Henley
Jean Pierre
JP
Alarie
J Michael
JM
Ramsey
D059018:DNA Contamination; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012463:Saliva / Q000382:microbiology*; D013295:Streptococcus mutans / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors
D017931:DNA Primers; D004269:DNA, Bacterial