Development of a polymerase chain reaction assay for differentiation between Culex pipiens pipiens and Cx. p. quinquefasciatus (Diptera: Culicidae) in North America based on genomic differences identified by subtractive hybridization

Abstract

Culex pipiens is a complex of mosquitoes that are involved in the transmission of pathogens, including St. Louis encephalitis virus in North America. The 2 major taxa in the complex, Cx. p. pipiens and Cx. p. quinquefasciatus, are nearly identical morphologically, making identification of field-collected specimens difficult, and attempts at differentiation based on biochemical and molecular techniques have been unsuccessful. We report here the use of genomic subtractive hybridization to identify a region of nucleic acid heterology between the genomes of Cx. p. pipiens and Cx. p. quinquefasciatus and the development of a polymerase chain reaction (PCR) assay to discriminate between them. PCR primers based on the nucleic acid sequence of a Cx. p. pipiens-unique DNA fragment were used to differentiate Cx. p. pipiens and Cx. p. pipiens/quinquefasciatus hybrids from Cx. p. quinquefasciatus by using extracted individual mosquito genomic DNA, crude DNA preparations from a mosquito head or legs, and DNA from triturated mosquito pools.