Table 1.

Overview of the two FRET/MCA assays developed for detection of knockdown resistance in An. epiroticus, An. subpictus, An. minimus s.s. and An. harrisoni: sequences of oligonucleotides and cycling conditions

	PRIMARY PCR	SECONDARY PCR	MELT CURVE ANALYSIS
FRET/MCA I	PRIMERS	PRIMERS	PROBE
An. epiroticus, An. Subpictus	Agd1: 5'-atagattccccgaccatg-3' Agd2: 5'-agacaaggatgatgaacc-3'	sundF-ROX: 5'-tagctacggtagtgaTagg-3'* Agd2: 5'-agacaaggatgatgaacc-3'	sundF-FAM: 5'-cataccactaaatttcctaTc-3'**
	CYCLING CONDITIONS	CYCLING CONDITIONS	MCA CONDITIONS
	3 min denaturation at 94°C 40 cycles: 1 min denaturation at 94°C, 30 s annealing at 47°C, 30 s extension at 72°C. 10 min extension at 72°C	4 min denaturation at 95°C 30 cycles: 1 min denaturation at 95°C, 30 s annealing at 53°C,30 s extension at 72°C. 8 min extension at 72°C	1 min at 95°C 1 min at 48°C 90 repeats heating for 20 s, starting at 48°C and with 0.3°C increments.
FRET/MCA II	PRIMERS	PRIMERS	PROBE
An. minimus s.l	Agd1Mi: 5'-ctgccaagatggaattttcac-3' Agd2H: 5'-gacaaaagcaaggctaag-3'	MinF-ROX: 5'-ggctacagtagtgaTagg-3'* FunminR: 5'-gacaaaagcaaggctaagaaaag-3'	Min-FAM: 5'-cttacgactaaatttcctaTcac-3'**
	CYCLING CONDITIONS	CYCLING CONDITIONS	MCA CONDITIONS
	3 min denaturation at 94°C 40 cycles: 1 min denaturation at 94°C, 30 s annealing at 47°C, 30 s extension at 72°C. 10 min extension at 72°C	4 min denaturation at 95°C 30 cycles: 1 min denaturation at 95°C, 45 s annealing at 51°C, 30 s extension at 72°C. 8 min extension at 72°C	1 min at 95°C 1 min at 48°C 80 repeats heating for 15 s, starting at 48°C and with 0.3°C increments.

* ROX fluorophore on capital T, ** FAM fluorophore on capital T