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. 2013 Sep 11;8(9):e71832. doi: 10.1371/journal.pone.0071832

Figure 1. Establishment of a multiplex qPCR to differentiate Cpp. biotypes, biotype hybrids and Cx. Torrentium.

Figure 1

A) Signal intensities for single species and mixed species DNA samples. The DNA of single individuals of the three taxa was extracted and quantified. For the reaction mixtures, either 200 ng of single species DNA templates or a 1∶1 mix of two species DNA templates (100 ng DNA species 1 and 100 ng species 2) were subjected to PCR testing. The reaction mix was prepared including the sets of primers and probes specific for Cpp. pipiens, Cpp. molestus and Cx. torrentium in a total reaction volume of 20 µL (for individual concentration please refer to Figure S1C). The signal intensities of each species-specific probe were measured for single taxon samples and taxon mixtures and are expressed as crossing points (Ct-values). A sample that is not targeted by the respective species-specific probe, is indicated as non detected (ND). B) As pools of up to 25 mosquitoes were analysed, the detection of individual DNA from Cpp. biotype pipiens, Cpp. biotype molestus or Cx. torrentium in mixed samples were analysed by the multiplex qPCR test. DNA of single individuals from the three taxa was extracted and quantified. The reaction mixtures were prepared with either 1∶1 mixed DNA template of two species (100 ng of each single species DNA sample) or 1∶25 diluted DNA sample (8 ng DNA of species 1 and 192 ng DNA of species 2). Subsequently, the DNA templates were subjected to a reaction mix containing species-specific sets of primer/probe for all three taxa and tested for amplification signals. The signal intensity for the 1∶1 mixture was set to 1 and signal intensities of 1∶25 mixed samples are expressed as relative values. The signal intensities are color-coded as followed: Cpp. pipiens in white, Cpp. molestus in red and Cx. torrentium in black. In the graph, relative signal intensities for 1∶25 DNA mixes of Cpp. biotype pipiens and Cpp. biotype molestus are named pip∶mol when Cpp. pipiens DNA was diluted in Cpp. molestus DNA, or mol∶pip when Cpp. molestus DNA was diluted in Cpp. pipiens DNA. Accordingly 1∶25 DNA mixes of Cpp. pipiens with Cx. torrentium were named pip∶tor if Cpp. pipiens DNA was diluted in Cx. torrentium DNA and tor∶pip if Cx. torrentium DNA was diluted in Cpp. pipiens DNA. The same nomenclature was used for 1∶25 mixes of Cpp. biotype molestus DNA with Cx. torrentium DNA. Values presented are the mean and standard deviation of two independent experiments.