Polymerase Chain Reaction Species Diagnostic Assay for Anopheles quadrimaculatus Cryptic Species (Diptera: Culicidae) Based on Ribosomal DNA ITS2 Sequences

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Abstract
Species-specific differences in the nucleotide sequences of the 2nd internal transcribed spacer (ITS2) of nuclear ribosomal DNA (rDNA) were used to develop a diagnostic assay based on the polymerase chain reaction (PCR) that can distinguish 4 of the 5 cryptic sibling species in the common malaria mosquito, Anopheles quadrimaculatus Say, complex. The assay requires only a small amount of tissue from an individual mosquito and a mixture of 5 PCR primers. The plus strand universal primer is derived from a sequence in the 5.8S coding region that is identical in all members of the complex. The 4 minus strand primers were selected from species-unique sequences within the ITS2 region. PCR amplification produces a different sized fragment for each of the 4 species which can be visualized readily under ultraviolet light after electrophoresis through an ethidium bromide-containing agarose gel. The assay has been developed and tested only with An. quadrimaculatus complex specimens from Florida populations.

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