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

Background: We report the development of an enzyme-linked immunosorbent ass ay-like single-tube assay for the detection of infectious agents in a micro titer tray format. Methods and Results: The method, sequential nucleic acid amplification and capture (SNAAC), combines amplification with hybridization of the product t o a surface/matrix-bound oligonucleotide probe. After amplification of the target sequence using species-specific primers, one of which contains a det ection tag such as fluorescein or biotin, a denaturation and hybridization cycle is performed. This allows capture by an oligonucleotide that is coval ently bound to the surface of a microtiter tray well or other support. Afte r washing to remove unincorporated solution-phase oligonucleotide bearing t he detection tag, the level of captured product is determined through a col orimetric reaction using an automated plate reader. We show the value and u tility of the SNAAC detection method using cloned sequences of the importan t human respiratory pathogen Chlamydia pneumoniae. Conclusions: SNAAC is a simple, rapid, and inexpensive method for the detec tion of low levels of infectious agents that is readily adaptable to curren t clinical laboratory equipment, thus avoiding the need to develop or purch ase new instrumentation.