Development of a Multiplexed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Identify Common Members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala

Rebekah J. Kent Centers for Disease Control and Prevention, Division of Vector-borne Disease, Arbovirus Diseases Branch, Fort Collins, Colorado

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Stephen Deus Centers for Disease Control and Prevention, Division of Vector-borne Disease, Arbovirus Diseases Branch, Fort Collins, Colorado

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Martin Williams Centers for Disease Control and Prevention, Division of Vector-borne Disease, Arbovirus Diseases Branch, Fort Collins, Colorado

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Harry M. Savage Centers for Disease Control and Prevention, Division of Vector-borne Disease, Arbovirus Diseases Branch, Fort Collins, Colorado

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Morphological differentiation of mosquitoes in the subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala is difficult, with reliable identification ensured only through examination of larval skins from individually reared specimens and associated male genitalia. We developed a multiplexed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify common Cx. (Cux.) and Cx. (Phc.). Culex (Cux.) chidesteri, Cx. (Cux.) coronator, Cx. (Cux.) interrogator, Cx. (Cux.) quinquefasciatus, Cx. (Cux.) nigripalpus/Cx. (Cux.) thriambus, and Cx. (Phc.) lactator were identified directly with a multiplexed primer cocktail comprising a conserved forward primer and specific reverse primers targeting ribosomal DNA (rDNA). Culex nigripalpus and Cx. thriambus were differentiated by restriction digest of homologous amplicons. The assay was developed and optimized using well-characterized specimens from Guatemala and the United States and field tested with unknown material from Guatemala. This assay will be a valuable tool for mosquito identification in entomological and arbovirus ecology studies in Guatemala.

Author Notes

*Address correspondence to Rebekah J. Kent, Centers for Disease Control and Prevention, Division of Vector-borne Disease, Arbovirus Diseases Branch, 3150 Rampart Road, Fort Collins, CO 80521. E-mail: fxk7@cdc.gov

Financial support: This research was funded by the Centers for Disease Control and Prevention, the Universidad de Valle del Guatemala, and a Robert E. Shope International Fellowship in Infectious Diseases award to RJK.

Authors' addresses: Rebekah J. Kent, Stephen Deus, Martin Williams, and Harry M. Savage, Centers for Disease Control and Prevention, Division of Vector-borne Disease, Arbovirus Diseases Branch, Fort Collins, CO, E-mails: fxk7@cdc.gov, sdeus@ridgeviewclassical.com, zoq9@cdc.gov, and hms1@cdc.gov.

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