Solid-phase amplification and detection: a single-tube diagnostic assay for infectious agents.

Abstract

BACKGROUND: We report the development of an enzyme-linked immunosorbent assay-like single-tube assay for the detection of infectious agents in a microtiter tray format. METHODS AND RESULTS: The method, sequential nucleic acid amplification and capture (SNAAC), combines amplification with hybridization of the product to a surface/matrix-bound oligonucleotide probe. After amplification of the target sequence using species-specific primers, one of which contains a detection tag such as fluorescein or biotin, a denaturation and hybridization cycle is performed. This allows capture by an oligonucleotide that is covalently bound to the surface of a microtiter tray well or other support. After washing to remove unincorporated solution-phase oligonucleotide bearing the detection tag, the level of captured product is determined through a colorimetric reaction using an automated plate reader. We show the value and utility of the SNAAC detection method using cloned sequences of the important human respiratory pathogen Chlamydia pneumoniae. CONCLUSIONS: SNAAC is a simple, rapid, and inexpensive method for the detection of low levels of infectious agents that is readily adaptable to current clinical laboratory equipment, thus avoiding the need to develop or purchase new instrumentation.