Identification of methicillin-resistant Staphylococcus aureus using an integrated and modular microfluidic system

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired (HA-MRSA) infection worldwide. As a result, the rapid and specific detection of MRSA is crucial not only for early prevention of disease spread, but also for the effective treatment of these infections. We report here an integrated modular-based microfluidic system for MRSA identification, which can carry out the multi-step assay used for MRSA identification in a single disposable fluidic cartridge. The multi-step assay included PCR amplification of the mecA gene harboring methicillin resistance loci that can provide information on drug susceptibility, ligase detection reaction (LDR) to generate fluorescent ligation products appended with a zip-code complement that directs the ligation product to a particular address on a universal array containing zip-code probes and a universal DNA array, which consisted of a planar waveguide for evanescent excitation. The fluidic cartridge design was based on a modular format, in which certain steps of the molecular processing pipeline were poised on a module made from a thermoplastic. The cartridge was comprised of a module interconnected to a fluidic motherboard configured in a 3-dimensional network; the motherboard was made from polycarbonate, PC, and was used for PCR and LDR, while the module was made from poly(methylmethacrylate), PMMA, and contained an air-embedded waveguide serving as the support for the universal array. Fluid handling, thermal management and optical readout hardware were situated off-chip and configured into a small footprint instrument. In this work, the cartridge was used to carry out a multiplexed PCR/LDR coupled with the universal array allowed for simultaneous detection of five genes that encode for 16S ribosomal RNA (SG16S), protein A (spa), the femA protein of S. epidermidis (femA), the virulence factor of Panton-Valentine leukocidin (PVL) and the gene that confers methicillin resistance (mecA). Results indicated that this modular system could differentiate community-acquired MRSA (CA-MRSA) from hospital-acquired MRSA (HA-MRSA) based on the presence/absence of the PVL gene as well as S. aureus from other Staphylococcal species using the sequence content in the femA gene. This system can identify strains in <40 min and detect MRSA directly from a mixture of Staphylococci.