One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y

Table 1 Optimized RT-PCR program using Quant Studio 3

Process	RT^c		PCR (40cycle)		Melting curve analysis
Subprocess	cDNA synthesis	Denature	Denature	Annealing-Elongation	Denature	Annealing	Melting^d	Hold
Tmp^a	50 °C	95 °C	95 °C	58 °C	95 °C	60 °C	0.03 °C/s	95 °C
Time	5 min	10 s	5 s	31 s	15 s	15 s		1 s
Measure^b				●			●

^aTemperature
^bMeasure fluorescent intensity in the steps marked with black dot (●)
^cReverse transcription
^dTemperature was increased 0.03 °C per second in this step to dissociate dsDNA gradually

ISSN: 1743-422X