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.
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.
Crabtree
M B
MB
Arbovirus Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80522, USA.
Savage
H M
HM
Miller
B R
BR
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003465:Culex / Q000145:classification* / Q000235:genetics; D017344:Genes, Insect; D008969:Molecular Sequence Data; D009656:North America; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity