Methods in Anopheles Research - MR4

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8.5.1.5 Resistance to dieldrin (rdl) assayChapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.5 Resistance to dieldrin (rdl) assayPage 1 of 2Chris Bass, Martin Williamson, John Vontas, Hilary Ranson, Martin Donnelly and Lin FieldIntroductionTwo TaqMan assays have been developed, one for each of the two alternative mutations found in An.gambiae s.s. (A296G) and An. arabiensis (A296S). This assay is suitable for use on real-time PCRmachines that have two or more detection channels. An alternate PCR based assay can be found inChapter 5.3.3.A296G Assay conditions96 48 1 Reagent500 μl 250 μl 5.0 μl sterile H 2 O1.0 ml 500 μl 10.0 μl SensiMix DNA kit (Quantace)160 μl 80 μl 1.6 μl primer GlyRdlF (800nM) TCATATCGTGGGTATCATTTTGGCTAAAT160 μl 80 μl 1.6 μl primer GlyRdlR (800nM) CGACATCAGTGTTGTCATTGTCAAG40 μl 20 μl 0.4 µl TaqMan MGB probe (200 nM) WT1 VIC-ACGTGTTGCATTAGG40 μl 20 μl 0.4 µl TaqMan MGB probe (200 nM) Gly 6FAM-ACGTGTTGGATTAGG1.9 ml 950 μl 19 μl Total (To each 19 μl reaction add 1-2 μl genomic DNA)A296G Assay PCR cycle conditions95°C/10 min x 1 cycle; (95°C/10sec, 62°C/45sec) x 40 cycles. Measure fluorescence at the end of eachcycle.A296S Assay conditions96 48 1 Reagent500 μl 250 μl 5.0 μl sterile H 2 O1.0 ml 500 μl 10.0 μl SensiMix DNA kit (Quantace)160 μl 80 μl 1.6 μl primer SerRdlF (800nM) TCATATCGTGGGTATCATTTTGGCTAAAT160 μl 80 μl 1.6 μl primer SerRdlR (800nM) TCGTTGACGACATCAGTGTTGT40 μl 20 μl 0.4 µl TaqMan MGB probe (200 nM) WT2 VIC-TTACACCTAATGCAACACG40 μl 20 μl 0.4 µl TaqMan MGB probe (200 nM) Ser 6FAM-CACCTAATGAAACACG1.9 ml 950 μl 19 μl Total (To each 19 μl reaction add 1-2 μl genomic DNA)A296S Assay PCR cycle conditions95°C/10 min x 1 cycle; (95°C/10sec, 60°C/45sec) x 40 cycles. Measure fluorescence at the end of eachcycle.Interpreting Results and ExamplesBoth rdl assays use two probes, the first specific for the wild type allele is labeled with VIC and thesecond, specific for the mutant allele (S296 or G296), is labeled with FAM. In either assay, a substantialincrease in VIC fluorescence indicates a homozygous wild type, a substantial increase in FAMfluorescence indicates a homozygous mutant and a, usually intermediate, increase in both signalsindicates a heterozygote (Figures 8.5.1.5.1 and 8.5.1.5.2).
Chapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.5 Resistance to dieldrin (rdl) assayPage 2 of 225A) Cycling FAM probe (Glycine)R/RFluorescence2015S/R105S/S5 10 15 20 25 30 35 40Cycle1510B) Cycling VIC probe (Alanine)S/SFluorescence5S/RR/R5 10 15 20 25 30 35 40CycleFigure 8.5.1.5.1. Real-time TaqMan detection of A296G6050A) Cycling FAM probe (Serine)R/RFluorescence40302010S/RS/S5 10 15 20 25 30 35 40CycleFluorescence4540353025B) Cycling VIC probe (Alanine)S/SS/R201510R/R5 10 15 20 25 30 35 40CycleFigure 8.5.1.5.2. Real-time TaqMan detection of A296S
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\Methods inAnopheles ResearchSecond
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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INTRODUCTIONChapter 5 : Insecticide
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Chapter 5 : Insecticide Resistance5
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Methods in Anopheles Research - MR4

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