Methods in Anopheles Research - MR4

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8.5.1.1 An. gambiae s.l. species complex ID assayChapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.1 An. gambiae s.l. species complex ID assayPage 1 of 6Chris Bass, Martin Williamson, John Vontas, Hilary Ranson, Martin Donnelly and Lin FieldIntroductionTwo alternative assays have been developed. The first (Bass et al. 2007) (2-Plex Assay) uses two probesto distinguish between the main malaria vectors An. gambiae s.s. and An. arabiensis as one group andAn. quadriannulatus, An. melas or An. merus as a second group. This assay is suitable for use on realtimePCR machines that have two detection channels (such as Biorad’s Mini Opticon). This assay can beused alone to discriminate vector from non-vector (in regions where An. merus/melas/bwambae are notpresent) or in combination with an existing TaqMan assay (Walker et al. 2007) to further distinguish An.arabiensis from An. gambiae s.s..The second assay (Bass et al. 2008) (3-Plex Assay) is an enhancement of the first assay and uses threeprobes to distinguish between An. arabiensis, An. gambiae s.s. and An. quadriannulatus/merus/melas/bwambae as a group. This assay requires a real-time PCR machine that has a least threedetection channels. Traditional PCR assays can be found in Chapters 8.4.1 and 8.4.3.2-Plex Assay96 48 1 Reagent500 μl 250 μl 5.0μl sterile H 2 O1 ml 500 μl 10.0 μl SensiMix DNA kit (Quantace)160 μl 80 μl 1.6 μl primer ComF (800 nM) GCTTGGTGGTTTGTCCG160 μl 80 μl 1.6 μl primer ComR (800 nM) CTGTGTCGACGTGGTCCC40 μl 20 μl 0.4 µl probe AG/AA (200 nM) 6FAM- GACCAAGACGAGC40 μl 20 μl 0.4 µl probe AQ/AM (200 nM) VIC- GACCAAGACGCGC1.9 ml 950 μl 19 μl Total (To each 19 μl reaction add 1-2 μl genomic DNA)2-Plex Assay PCR cycle conditions95°C/10 min x 1 cycle; (95°C/15sec, 50°C/20sec, 72°C/20sec) x 45 cyclesMeasure fluorescence at the end of each cycle3-Plex Assay96 48 1 Reagent630 μl 315 μl 6.3μl sterile H 2 O1.25 ml 625 μl 12.5 μl SensiMix DNA kit (Quantace)200 μl 100 μl 2.0 μl primer Uni F (800nM) GTGAAGCTTGGTGCGTGCT200 μl 100 μl 2.0 μl primer Uni R (800nM) GCACGCCGACAAGCTCA50 μl 25 μl 0.5 µl LNA probe Aa+(200nM)[Cy5]AC+A+T+AG+GATGGA+G+A+AGG [BHQ2]20 μl 10 μl 0.2 µl TaqMan MGB probe (80 nM) Ag + VIC-TGGAGCGGaACAC50 μl 25 μl 0.5 µl TaqMan MGB probe (200nM) Aq + 6FAM-TGGAGCGGgACAC2.4 ml 1.2 ml 24 μl Total (To each 24 μl reaction add 1-2 μl genomic DNA)3-Plex Assay PCR cycle conditions95°C/10 min x 1 cycle(95°C/25sec, 66°C/60sec) x 40 cyclesMeasure fluorescence at the end of each cycle
Chapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.1 An. gambiae s.l. species complex ID assayPage 2 of 6Interpreting Results and Examples: 2-Plex AssayInterpreting results is carried out by examining the levels of FAM and VIC fluorescence during PCR. Asubstantial increase in FAM fluorescence during PCR indicates an An. gambiae s.s. or An. arabiensisspecimen while a substantial increase in VIC fluorescence indicates an An. quadriannulatus, An. melas orAn. merus specimen (Figure 8.5.1.1.1.). An increase in both dyes would indicate a hybrid or acontaminated sample.To help identify species the software that accompanies the real-time PCR machine may allow endpointfluorescence values for the two dyes to be automatically corrected for background and plotted againsteach other in bi-directional scatter plots (Figure 8.5.1.1.2.).3025Cycling of FAM probe (An.arabiensis/An. gambiae)Fluorescence20151055 10 15 20 25 30 35 40 45Cycle6050Cycling of VIC probe (An.quadriannulatus/An. melas/An. merus)Fluorescence403020105 10 15 20 25 30 35 40 45CycleFigure 8.5.1.1.1 Species identification using the 2-plex TaqMan assay designed to distinguish theprincipal vector species An. gambiae s.s. and An. arabiensis from the other members of the complex. Inthis example two or more specimens of An. gambiae s.s., (red trace) An. arabiensis, (blue trace) An.melas (green trace), An. merus (purple trace) and An. quadriannulatus (orange trace).
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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