Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.2 Plasmodium detection assayPage 3 of 4P. falciparumP. vivax/P.ovale/P. malariaenegativeFigure 8.5.1.2.2. Scatter plot analysis of TaqMan fluorescence data. In this example the TaqMan assaywas carried out on ~30 Plasmodium genomic DNA samples and five no template negative controls.Fluorescence values of the FAM labeled probe specific for P. falciparum were plotted against those of theVIC labeled probe specific for P. vivax, P. ovale and P. malariae.Notes and TroubleshootingThe Plasmodium detection assay was originally optimized using purified Plasmodium genomic DNA astemplate and carrying out 45 cycles of PCR. When tests were carried out on blood fed mosquitoes usinga standard ‘quick and dirty’ DNA extraction, a small amount of non-specific fluorescence was sometimesseen after 40 cycles (see figure 7 below). For this reason we recommend restricting the number of cyclesin PCR to 40.70Fluorescence60504030Non-specific productsappearing after 40cycles of PCR20105 10 15 20 25 30 35 40 45CycleFigure 8.5.1.2.3. Cycling of VIC labelled probe specific for P. ovale, P. vivax, and P. malariae.