RNA and DNA diagnostics on microspheres: current and emerging methods

Emerging infections and rapidly evolving pathogens are a challenge to conventional nucleic acid-based diagnostic tests. Current nucleic acid-based diagnosis is limited in the spectrum of analytes that can be simultaneously detected. This can potentially be addressed by single-tube multiplexing or multiple-well multiplexing (where multiplexing is defined as using a mixture of reagents, capable of detecting multiple diagnostic targets in a single tube or in highly parallel arrays). The number of organisms that can be targeted in a single tube by the current gold standard in clinical laboratories [quantitative real-time polymerase chain reaction (qPCR)] is limited by the number of resolvable fluorophores. The demand for multiplex technologies has led to the development of various high-throughput assays and broad-spectrum formats. In this article, current microsphere-mediated formats, their clinical applications and established microsphere-based diagnostics are reviewed. We then discuss some novel microsphere-integrated techniques and their future prospects in clinical diagnostics. We differentiate between innovations in the molecular reactions with microspheres and novel microsphere encoding strategies to increase multiplexing capacities.