Methods in Anopheles Research - MR4

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 5 of 6PCR from 200 to 50 nM showed a degree of improvement but did not completely eliminate thebackground signal. In practice this background does not inhibit scoring as an An. quadriannulatus, An.melas or An. merus specimen shows a strong signal in the Cy5 channel while a true Anopheles gambiaes.s. individual only shows signal in the VIC channel.10080Cycling of VIC probe (An.gambiae s.s.)Fluorescence604020605005 10 15 20 25 30 35 40CycleCycling of Cy5 probe (An.arabiensis)Fluorescence40302010155 10 15 20 25 30 35 40CycleCycling of FAM probe (An.quadriannulatus/merus/melas)Fluorescence1055 10 15 20 25 30 35 40CycleFigure 8.5.1.1.4. Species identification using the multiplex real-time PCR assay. When the assay is run at60°C a background signal from the VIC probe is observed when using An. quadriannulatus, An. melas orAn. merus DNAs. An. gambiae s.s. (red trace), An. arabiensis (blue trace) and An. quadriannulatus/An.melas/An. merus (green, pink and light blue traces).ReferencesBass C, Williamson MS, Field LM (2008) Development of a multiplex real-time PCR assay foridentification of members of the Anopheles gambiae species complex. Acta Trop 107:50-53Bass C, Williamson MS, Wilding CS, Donnelly MJ, Field LM (2007) Identification of the main malariavectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay. Malar J 6:155Walker ED et al. (2007) Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensisby TaqMan single nucleotide polymorphism genotyping. Malar J 6:23