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 contamination 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 denaturation 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.