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 3 of 6An. gambiae s.s. /An. arabiensisAn. quadriannulatus/An.merus/An. melasFailedreactionsFigure 8.5.1.1.2. Scatter plot analysis of TaqMan fluorescence data. In this example real time PCR wascarried out using the newly developed TaqMan assay designed to distinguish the principal vector speciesAn. gambiae s.s. and An. arabiensis from the other members of the complex on 96 samples.Fluorescence values of the FAM labeled probe specific for An. gambiae s.s. and An. arabiensis were thenplotted against the VIC labeled probe specific for An. quadriannulatus, An. melas and An. merus.Interpreting Results and Examples: 3-Plex AssayA substantial increase in Cy5 fluorescence (probe Aa) during PCR identifies an An. arabiensis specimen,an increase in VIC fluorescence (probe Ag) identifies an An. gambiae s.s. sample, and an increase in6FAM fluorescence (probe Aq) identifies An. quadriannulatus, An. melas or An. merus specimen (Figure8.5.1.1.3.). An increase in two or more of the dyes would indicate a hybrid or a contaminated sample.