Abstract

Opening New Dimensions in Diagnostics with Hybridization Probes

Kenneth Pierce, Senior Research Scientist, Brandeis University

Probes that signal upon hybridization offer advantages over hydrolysable probes for PCR multiplexing. Single-stranded products generated by non-symmetric PCR can hybridize with multiple probes to identify any of a large number of specific mutations per probe. We have previously reported the use of dual-labeled fluorescent (“Lights On”) probes with quencher-labeled (“Lights Off”) probes to detect and identify clinically important mutations in bacterial genes that cause increased antibiotic resistance (Pierce et al, 2013, J Mol Diagn 15: 291-8). Combining Lights-On-Lights-Off detection with digital PCR offers the potential to detect low percentages of these mutant strains in mixtures containing drug-sensitive strains, thereby providing important information for antibiotic choice. But, many high through-put digital systems offer endpoint detection only at ambient temperature. We are exploring a new design in which low-melting-temperature Lights Off probes keep fluorescence at minimal levels in the absence of mutations causing resistance. Mutations at several “hot spots” of the genes can be targeted by using multiple probes and detected by a large increase in fluorescence at 25oC. In some cases those mutations can be distinguished from one another by the level of fluorescence. The combined technologies also offer new opportunities for detecting resistance mutations affecting anti-viral or cancer drug therapies.