Figure 3

Melting temperature analysis and quantitative standard curves of SYBR^® Green Real-Time PCR assays.(A) primer set Psv RT-F/Psv RT-R on strain Psv ITM317; (B) primer set Psn RT-F/Psn RT-R on strain Psn ITM519; (C) primer set Psf RT-F/Psf RT-R on strain Psf NCPPB1464. Quantitative thermal dissociation curves were represented plotting fluorescence derivative values [-d (fluorescence units)/d (time)] versus temperature, obtained with DNA from the target P. savastanoi pathovar, extracted by thermal lysis from 10³ to 10⁷ CFU per reaction (red, orange, yellow, green and blue lines, respectively) and with no target DNAs (blue diamond), extracted from the two other P. savastanoi pathovars, from olive (A), oleander (B) and ash (C) and from a pool of bacterial unidentified epiphytes isolated from the same plants (from olive, oleander and ash in A, B and C, respectively). Standard curves were generated by plotting the Ct values versus the log of genomic DNA concentration of each tenfold dilution series in the range of linearity (from 50 ng to 5 pg per reaction). The Ct data obtained with target DNA from 10³ to 10⁷ CFU per reaction were reported (+). (See online for a colour version of this figure).