Rapid Detection of Methicillin-Resistant Staphylococcus Aureus using Bubble-Free Microfluidic PCR

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common hospital-acquired infection and resistant to certain drugs; therefore, reliable PCR detection of MRSA is critical for early prevention of disease spread and the effective treatment of infections. The major problems of microfluidic PCR are water evaporation, loss of reagents, and inconsistent optical path-length due to random bubble generations during the thermal cycling. We report a bubble-free microfluidic PCR for the detection of MRSA. In order to avoid the bubble generation, we utilized polyethylene-based microfluidic devices since it has low gas permeability. The proposed polyethylene microfluidic PCR also offers a uniform heat distribution without the amplification inhibition by a temperature drop in the aqueous nucleic acid sample. The polyethylene layer was spin-coated over PDMS channel for effective hybrid integration of two different functions of polymeric materials (i.e. degasing pump for PDMS and polyethylene for uniform PCR and optical path-length). Consequently overall sample loss such as PCR reagent and nucleic acid was almost eliminated by bubble-free condition. The detection limit of mecA which is methicillin-resistance gene was found to be 6.4 x 102 copies of MRSA genomic DNA by the end-point fluorescence analysis for approximately 30 min reaction. The bubble-free multiplexed microfluidic MRSA PCR array will benefit for rapid detection and drug resistance determination effectively.