Singleplex Reactions in a Compact Array (SiRCA)

PCR amplification provides outstanding target identification and quantification over a large range of analyte concentrations. Multiplex PCR where two or more analytes are quantified is highly desirable in terms of improving throughput and reducing sample and reagent consumption. A significant problem with multiplex PCR however is the competition between primers for the same reagents and the possibility of primer-dimer formation. Additionally, fluorescent probes are the typical signaling method for determining a positive outcome. This increases the stringency needed for primer design and temperature optimization to ensure compatibility with other primer/probe sets. Due to spectral overlap between dyes and instrumentation limitations, most multiplex PCR reactions have been limited to 3-12 targets.

We are developing strategies for performing multi-well singleplex real-time PCR for the identification of multiple genetic targets with the simplicity of single-target PCR. We use a library of encoded microbeads to deliver specific primers to individual microwells and subsequently perform PCR after reagent delivery and microwell isolation. We are developing the necessary microfluidic architecture such as microfabrication techniques, valving, temperature control, and chip material. In addition, PCR assay conditions in small volumes along with primer sequences are being optimized.

Example of a three-plex SiRCA assay targeting methicillin-resistant staphylococcus aureus (MRSA) and a control, s. mutan. The red circles represent primers specific for s. aureus and show amplification as the gene is present in MRSA. The yellow circles represent primers specific for MRSA only and again show amplification. The blue circles represent the primers for s mutan with no amplification seen. The bright spots in the blue wells are the auto-fluorescence of the beads.