Ultrasensitive, amplification-free assays for detecting pathogens.

Hybridization of nucleic acid probes has the potential to directly detect pathogens without requiring resource-intensive amplification steps, but conventional approaches to direct hybridization are typically slow or suffer from low sensitivity. In this work, we have characterized a novel approach fo r rapid detection of low-abundance nucleic acids by direct hybridizati on in solution, with sensitive analysis enabled by electrophoretic preconcentration of nucleic acids at a nanoporous m embrane. We performed proof-of-concept testing of the assay using a m odel DNA virus, showing direct detection of as little as 400 amol (~240 m illion copies) with current (unoptimized) hardware. We extensively char acterized the preconcentration proces s for DNA, and determ ined rates of preconcentrat ion and efficiency of recovery as a function of preconcentration conditions, membrane formulation, and DNA size. The membrane preconcentration device also en ables rapid, ultra-sensitive size-based separation of nucleic acids, and has been demonstrated for multiplex PCR analysis of drug resistance genes in bacteria.