The Fluorescence Characteristics of Enrichment Media in the Wavelength Range of Real-Time PCR Thermocycler Optical Path Assignments

The aim of this study was to investigate the fluorescence characteristics of common enrichment media with regard to disturbance of fluorescence readings of real-time polymerase chain reaction (PCR) reactions. Confirmation of effective amplification of target DNA during the real-time PCR process depends on measurement of the fluorescence emitted from the probe fluorophore used. Possible background fluorescence emitted from enrichment media, when applied directly to the sample, could lead to interference with this measurement. This inhibition of the detection process leads to false negative results, even although amplification of the target DNA was successful. Among the 11 enrichment media investigated, half-Fraser broth displayed significant fluorescent emission if excited in the wavelengths 492, 517 and 535 nm, according to the FAM, TET and HEX filter sets of the real-time PCR thermocycler. A shift to the excitation wavelengths of 585 (ROX) and 635 nm (CY-5) leads to no measureable emission caused by half-Fraser broth. A further investigation revealed that Fraser supplements containing acriflavine and nalidixic acid show strong emission if exited with the tested wavelengths. The related molecular structures of acriflavine and fluorescein, the basic molecular structures of FAM, HEX and TET, implicate similarity of the fluorescent properties of these molecules. In summary, consideration of the fluorescence characteristics of the applied enrichment media is important if subsequent direct quantification of food borne pathogens with real-time PCR from enrichment media is anticipated. This requirement was demonstrated as Fraser supplements contained in half-Fraser broth did raise the background fluorescence substantially.