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Chapter 14 : Fluorescence Resonance Energy Transfer
Category: Clinical Microbiology; Bacterial Pathogenesis
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.
FRET signal generation uses the overlap of donor dye fluorescence with acceptor dye absorption.
Two FRET Formats. In both formats the first dye (F1) is excited by λ1 which emits light at a longer wavelength (λ2). In the first format, fluorescence from the fluorescent dye (F1) is quenched by the second dye (Q) and signal is only produced by separating the two dyes. In the second format, the emitted light (λ2) from F1 is absorbed by F2 and re-emitted at a third wavelength (λ3), and signal is only produced when the two dyes are near each other.
Hydrolysis (TaqMan) probe detection. The dual-labeled, single-strand oligonucleotide probe is designed to anneal to sequences within the PCR product and contains two fluorochromes. The 5′ fluorochrome is called the “reporter” (R), whereas the 3′ fluorochrome functions as a quencher (Q). (A) During amplification, the probe binds to the template between the two PCR primers. (B) The Taq polymerase encounters the probe and starts chewing away at the end with its 5′-to-3′ exonuclease activity. (C) The reporter fluorophore is released into solution, where it is free to fluoresce. (D) Multiple reporter fluorochromes are available to permit multiplexing PCR reactions for controls.
Melting curve analysis. The decrease in FRET fluorescence signal due to the FRET probes disassociating with the amplification product is measured after the completion of all PCR cycles. The reaction vessel is slowly heated, and the signal is measured as a function of temperature. To aid determination of the middle of the melting curve (Tm , shown at the top), the negative derivative of the melting curve, divided by the derivative of the temperature (shown at the bottom), is plotted. The transformed data display the Tm (vertical line) as the apex of a peak.
(A) Effect of polymorphisms on hybridization FRET probes melting curve. A polymorphism under the probe will lower the annealing temperature and result in a lower melting curve Tm . (B) LightCycler data with the melting curve above and melting peak below. A positive sample without mutations under the probes (Tm , 63°C) and a sample with one base pair mutation (Tm , 61°C) are shown. The negative control has no peak.
Tm differences from polymorphisms in the 315 codon of katG in Mycobacterium tuberculosis associated with isoniazid resistance compared to the wild-type (WT) sequence. Reprinted from reference 45 with permission of the publisher.
Differentiation of HSV-1 and HSV-2 by T m using one set of primers and probes.
Tm differences of mycobacterial species 16S rRNA, M. tuberculosis (TB), M. kansasii (MK), and M. intracellulare (MI), obtained with one set of primers and probes. Reprinted from reference 40 with permission of the publisher.
Hybridization probe with SYBR Green. SYBR Green nonspecifically intercalates into double-stranded probe-amplification product, absorbs light (λ1), and fluoresces (λ2). The dye on the probe absorbs the emitted SYBR Green fluorescence and emits at a third wavelength (λ3).
Values of R0 with donor-acceptor pairs
Comparison of real-time thermocyclers a
Hybridization FRET probe assays