Abstract: A real-time PCR melting curve assay that unambiguously distinguishes Culex pipiens complex mosquitoes from the morphologically similar species Culex restuans has been developed. Differentiating between the two species is important because Cx. pipiens complex mosquitoes are more important disease vectors than Cx. restuans. Aligned mitochondrial DNA sequences of each species were examined for suitable priming sites. A 60 bp region of the cytochrome c oxidase subunit II gene was chosen, and designed primers consisted of a common forward primer, and species-specific reverse primers. Additionally, a 20 bp long “tail” was added to the Cx. pipiens complex-specific reverse primer to increase its melting temperature. The pipiens complex-specific amplicon melts at 78°C, while the restuans-specific amplicon melts at 72°C. We tested the assay with pipiens complex colony specimens, and Cx. restuans collected as egg rafts from the Chicago area. Specimens from rafts were raised to adults for genomic DNA extraction, with species determination confirmed morphologically at the 4th instar larval stage. Mixed pools of females were constructed to test the assay’s ability to detect small numbers of one species in pools of 50 individuals. The assay was capable of detecting a single individual of one species in a pool with 49 individuals of the other species; however, the assay is not quantitative because efforts to associate the height of the melt curve with the number of individuals of each species in a sample were unsuccessful. Nevertheless, the assay determined the presence/absence of each species, and worked on individual specimens. Unlike other assays that require a separate visualization step, the assay described herein consists of one straightforward PCR, does not require the purchase of fluorescent probes, and produces unambiguous results.
