Development and Characterization of a Solid Phase for Single-Step Enrichment of Pathogenic Targets

Abstract

The identification of low abundance target nucleic acids in a complex matrix can be challenging due to the abundance background material. Current methods use two-step processes which are time consuming, prone to contamina- tion and usually limited to one pathogen. In this study we describe a single-step target-capture approach using magnetic microbeads with capture probes covalently attached through a phosphorus dendrimer linker. This approach was also used successfully for simultaneous capturing of two low abundance pathogenic nucleic acids present in a complex matrix (800- fold excess of background nucleic acids) by using a multi-pathogen solid phase. The thermal stability of the solid phase allows denaturation and capture to proceed sequentially and the recovery of the targets to be performed by heat denatura- tion without the risk of probe shedding. The critical variables involved in the development of the solid phase and the steps required for further optimization are discussed.