Fluorescence Resonance Energy Transfer

Abstract

This chapter discusses general fluorescence resonance energy transfer (FRET) probe technology and the use of FRET with single and dual hybridization probes in microbiology. It focuses on hydrolysis and hybridization FRET probes and the many variations used for detection of amplification products in PCR amplification. True multiplex assays simultaneously amplify different nucleic acid targets and result in multiple unique PCR products. These types of assays often have two or more sets of primers and probes and are most commonly used with genomic targets in which the amount of target nucleic acid is normally large and the ratio of the two target nucleic acids is close to unity. Recent applications of FRET probes in bacteriology include assays for Vibrio parahaemolyticus, Clostridium difficile, Brucella, Francisella tularensis, Clostridium perfringens, Listeria monocytogenes, Burkholderia cepacia complex, Campylobacter jejuni and Campylobacter coli, and penA and ponA genotypes in Neisseria gonorrhoeae. The use of homogeneous FRET probe technology for detection of PCR products provides an opportunity for microbiologists to use molecular detection in a closed system. The necessary specificity and sensitivity of many microbiology tests are achievable using PCR with FRET detection. The technology has become widely available, and configurations of instrumentation and FRET design are available for many applications. Hybridization FRET probes provide great sensitivity and specificity to real-time PCR with the benefit of sensitive detection of nucleic acid sequences with unexpected polymorphisms. The hybridization FRET probes also enable multiple formats for robust multiplexing reactions often with just a single set of primers and probes.