TABLE 5.

Amplification programs for monoplex serovar-specific PCR assays

SerovarAmplification programMelting range (°C)
195°C, 1 s; 55°C, 10 s, single acquisition; 72°C; 4 s; 45 cycles50-70
295°C, 0 s; 57°C; 4 s; 72°C, 2 s, single acquisition; 45 cycles65-95
3Amplification I, 95°C, 0 s; 57°C, 5 s; 15 cycles; amplification II, 95°C, 0 s; 57°C, 3 s; 2nd target, 53°C, 1°C/step, delay 1 cycle; single acquisition; 45 cycles65-85
495°C, 10 s; 55°C, 6 s, single acquisition; 72°C, 5 s; 45 cycles50-80
5Amplification I, 95°C, 2 s; 57°C, 6 s; 72°C, 2 s; 10 cycles; amplification II, 95°C, 5 s; 57°C, 8 s; ramp rate, 5°C/s; 2nd target, 54°C, 1°C/step, delay 1 cycle; single acquisition; 72°C, 3 s; 45 cycles50-70
695°C, 0 s; 58°C, 2 s; 72°C, 1 s, single acquisition; 40 cycles63-90
795°C, 0 s; 55°C, 1 s, single acquisition; 40 cycles65-95
8Amplification I, 95°C, 0 s; 66°C, 5 s; 15 cycles; amplification II, 95°C, 0 s; 66°C, 2 s; 2nd target, 58°C, 1°C/step, delay 1 cycle; 72°C, 1 s, single acquisition; 40 cycles66-95
995°C, 0 s; 62°C, 1 s, single acquisition; 33 cycles70-95
1095°C, 2 s; 56°C, 5 s, single acquisition; 72°C, 2 s; 45 cycles55-75
1195°C, 2 s; 53°C, 10 s; ramp rate, 5°C/s, single acquisition; 72°C; 4 s; 50 cycles54-75
1295°C, 10 s; 59°C, 5 s, single acquisition; 72°C, 6 s; 45 cycles50-80
1395°C, 0 s; 57°C, 2 s; 72°C, 2 s, single acquisition; 40 cycles63-90
1495°C, 0 s; 55°C; 4 s; 72°C, 2 s, single acquisition; 45 cycles70-90