Fully Automated, Homogeneous Nucleic Acid Detection Technology Based on Dry-Reagent Assay Chemistry and Time-Resolved Fluorometry (Abstracts of Oak Ridge Posters‪)‬

Nucleic acid diagnostic techniques are increasingly used for bacterial detection (1). However, because of a lack of automation, DNA testing has thus far been mainly performed by laboratory personnel with a high level of expertise in molecular biology. Recently, homogeneous detection technologies (2) have greatly decreased the risk of cross-contamination. To further improve the reproducibility of PCR, manufacturers have introduced universal PCR master mixes containing all reagents at predetermined concentrations. Although these ready-to-use mixes have facilitated PCR setup, they still require cold storage and careful liquid handling for consistent performance. More recently, the automation of all assay steps including sample preparation has become feasible, and fully integrated, high-performance nucleic acid analyzers (3) have become commercially available. However, the high cost of analyzers and assays still limits their routine use. To facilitate the everyday use of PCR, we have developed a new instrument platform, GenomEra[TM], which combines rapid thermal cycling (4); low-cost plastic reaction vessels (4); a homogeneous, dual-label assay technology based on end-point time-resolved fluorescence (TRF) detection; software with an intuitive user interface; and ready-to-use dry-reagent reagent sets (5). The GenomEra assay chips are made of polypropylene and metal foil, allowing optimal optical characteristics and a high speed of thermal transfer. The foil acts as a mirror to enhance the intensity of the long-lifetime fluorescence measured from the closed reaction vessels. Thermal cycling in the GenomEra instrument is based on a conveyor that transfers the reaction chips cyclically between thermal blocks maintained at constant temperatures. Combined with the assay chips that are laminated with metal foil, the rate of temperature change inside the reaction chamber can be highly accelerated compared to conventional 1-block instruments. In addition to the denaturation, annealing/ extension, and measurement blocks, "hot" and "cold" blocks set to more extreme temperatures are used to further increase the speed of temperature change. The automated platform is intended for rapid, sensitive, small-scale analysis of any nucleic acid that can be detected by PCR. The target-specific reagents are predispensed and dried inside the assay vessels, and after the user has added the preprocessed sample (various sample preparation methods can readily be used), the analysis is performed automatically starting from the selection of an applicable temperature profile and ending with the reporting of unambiguous qualitative results.