Figure 4.

Detection of nonspecific amplification by T_m. For both A and B, the top amplification curves show real-time PCR data in relative fluorescence units (RFU) versus PCR cycle number, and the bottom panels show postamplification melt data as the negative first derivative of RFU with respect to temperature (−d(RFU)/dT) versus temperature in °C. A representative data set using a series of unrelated organisms along with 1 pg of Ames DNA control was amplified by the pagA:capB:IPC triplex primer set. A: Real-time and amplicon melt data from the pagA read in F1 channel. B: Data from capB read in F2 channel. The Salmonella choleraesius 9150 sample (*) amplified weakly in the F1 channel with a Ct of 42.2 compared with 34.4 for the Ames control (+). The F1 Melt Tm for the S. choleraesius amplicon was significantly different at 76.0°C compared with 79.3°C for the control (A, bottom), indicating that the S. choleraesius amplification product observed during real-time PCR was nonspecific. Triplicate re-testing of the S. choleraesius sample gave no detectable amplification (data not shown).