31 Emerging Bacterial Foodborne Pathogens and Methods of Detection 723Figure 31.2.Use of fluorescence resonance energy transfer–based PCR to detect pathogens. A.This PCR-based
method has a number of components. Template DNA from the sample, containing a pathogen-specific target gene, is
isolated, and oligonucleotide primers specific to the target gene and used to amplify the target gene are added. A
probe specific for a central sequence in the target gene is labeled with a fluorogen and added to the mixture. A short
oligonucleotide probe, specific for the fluorescent probe, labeled with a quenching molecule, is also present. This
probe has a low melting temperature so that it will not bind to the fluorescent probe during PCR. Finally, the PCR
reaction must contain DNA polymerase that retains its 5’-exonuclease activity. B.In the first two steps of the PCR,
template DNA from the sample is heated to a temperature that will denature all its DNA. The temperature is then low-
ered to allow annealing of the primers for amplification and the fluorescently labeled probe. C.The pathogen-specific
target gene is amplified by Taq polymerase. Any fluorescent probe that has annealed to the DNA is digested by the
exonuclease activity of the Taq polymerase to release free fluorogen. D.After a number of rounds of amplification,
PCR products are cooled. At this point, the quencher-labeled probe will bind to any remaining fluorescent probe, pre-
venting fluorescence of that probe. The more pathogens are initially present in the sample, the more copies of the
amplified gene will be present. This leads to more of the fluorescent probe binding to the gene and being digested by
the DNA polymerase. Only the free fluorogen will fluoresce due to the presence of the quencher-labeled oligonu-
cleotide. At the end of the reaction, the number of pathogen cells in the original sample is estimated from the fluores-
cence signal; the more pathogen DNA present, the higher the fluorescence. F, fluorescent label; Q, quencher.