High-throughput qualitative multiplex 5′ nuclease assay using post-only PCR analysis

Homogenous fluorescence PCR assays offer distinct advantages for qualitative testing and are gaining immense popularity in fields like diagnostic microbiology. To meet the demand of high-volume laboratories, we developed a protocol for qualitative multiplex 5′ nuclease assays using post-only PCR analysis. This novel approach overcomes throughput problems encountered with the established methods for TaqMan™ detection on the ABI PRISM™ 7700 Sequence Detection system and permits off-site TaqMan PCR, which can be analyzed several days after the reactions are completed. We have validated this novel protocol using an assay for the identification of Bordetella pertussis against real-time and plate-read analysis methods and have shown that our proposed protocol produces no difference in qualitative calls.