title: Universal primers for the amplification and sequence analysis of actin-1 from diverse mosquito species.
abstract: We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5'-ATGGTCGGYATGGGNCAGAAGGACTC-3') and Act-8R (5'-GATTCCATACCCAGGAAGGADGG-3'), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report.
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iow State University, Ames, IA 50011, USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Staley
authors.forename: Karin S
authors.initials: KS
authors.lastname: Dorman
authors.forename: Lyric C
authors.initials: LC
authors.lastname: Bartholomay
authors.forename: Ildefonso
authors.initials: I
authors.lastname: Fernández-Salas
authors.forename: Jose A
authors.initials: JA
authors.lastname: Farfan-Ale
authors.forename: Maria A
authors.initials: MA
authors.lastname: Loroño-Pino
authors.forename: Julian E
authors.initials: JE
authors.lastname: Garcia-Rejon
authors.forename: Luis
authors.initials: L
authors.lastname: Ibarra-Juarez
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Blitvich
mesh_terms: D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D009033:Culicidae / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000199:Actins; D017931:DNA Primers