The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.