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

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.