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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

From: Absence of knockdown resistance suggests metabolic resistance in the main malaria vectors of the Mekong region

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