Optimization of fluorescence measurement in duplex real-time PCR with TaqMan® probes labeled with VIC and quenched by TAMRA

Real-time PCR with 5′-nuclease TaqMan® probes (1,2) is a relatively robust method for specific DNA sequence detection and quantification. The method is often used in duplex format, where the first set of primers and the probe target the specific DNA sequence of interest, and the second set is used as a reference. In the original duplex TaqMan system, the first probe is labeled with FAM (6carboxyfluorescein) and quenched by TAMRA (6-carboxytetramethylrhoda mine) and the second probe is labeled with VIC (4,7,2′-trichloro-7′-phenyl-6carboxyfluorescein) and quenched by TAMRA (3). This system is widely used both in commercial kits and in original systems, mainly because of an affordable price. However, it produces fluorescence spectra with overlapping peaks that can be correctly measured only using advanced instruments equipped with a spectrograph on the emission side and supported with a powerful software to process the spectra. Real-time PCR cyclers equipped with filter-based optics are unable to correctly monitor fluorescence of the original duplex TaqMan system because of interference between optical channels. For example, amplification of the primers/probe system with a FAM label, which is reflected by an increase in the recorded fluorescence in the FAM channel, leads also to a certain increase in the recorded fluorescence in the VIC channel. The instruments offer an option to eliminate such interference between optical channels by data transformation using some undisclosed calculation. However, this option is imprecise, and an increase in the fluorescence in the FAM channel leads to a decrease in the calculated fluorescence in the VIC channel. Another source of problems is the emission of TAMRA, which is recorded in the same channel as VIC by several intsruments. The reason for the described problems is that standard filters used in real-time PCR cyclers, with filter-based optics, are not optimal for work with a combination of dyes FAM, VIC, and TAMRA. As a basis for optimization of optical channels, we measured a threedimensional (3-D) fluorescence spectrum of the PCR system. The previously described real-time PCR for the detection of Salmonella enterica (4) with a FAM-labeled, TAMRAquenched probe was used in duplex with the previously described real-time PCR for the detection of Escherichia coli (5) with a VIC-labeled, TAMRAquenched probe. The ratio of the template DNA from S. enterica and E. coli was 1:1 (200:200 ng). Reactions were performed in TopYieldTM flatbottom 8-strips (Nunc, Roskilde, Denmark) in a GeneAmp® 9700 Optimization of fluorescence measurement in duplex real-time PCR with TaqMan® probes labeled with VIC and quenched by TAMRA