title: A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses.
abstract: Mosquito-borne alphaviruses are widely distributed throughout the world, causing important human illnesses. Therefore, the development of methods to enable early diagnosis of infections by alphavirus is essential. We show here the development and evaluation of a quantitative real-time RT-PCR using genus-specific primers to the nsP1 viral gene of all mosquito-borne alphaviruses. The specificity and sensitivity of the assay were tested using seven alphaviruses and RNA transcribed from Venezuelan equine encephalitis virus. The detection limits of real-time RT-PCR ranged from 10 to 76 copies per ml. The melting temperature (TM) values for amplification of the alphavirus genomes were 83.05 °C and 85.28 °C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with TM peaks of 84.83 to 85.28 °C and encephalitic alphaviruses of 83.34 °C to 84.68 °C could be discriminated both diseases. Real-time RT-PCR may prove very useful for the screening and preliminary diagnosis in outbreaks and surveillance studies as well as for measuring the viral load in pathogenesis studies.
authors: Virology Research Center, School of Medicine of Ribeirao Preto of University of Sao Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil. mafarignoli@hotmail.com.
authors: Marilia Farignoli
authors: MF
authors: Romeiro
authors: Virology Research Center, School of Medicine of Ribeirao Preto of University of Sao Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors: William Marciel
authors: WM
authors: de Souza
authors: Virology Research Center, School of Medicine of Ribeirao Preto of University of Sao Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors: Aline Lavado
authors: AL
authors: Tolardo
authors: Virology Research Center, School of Medicine of Ribeirao Preto of University of Sao Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors: Luiz Carlos
authors: LC
authors: Vieira
authors: Laboratório de Virologia Clínica e Molecular, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors: Dyana Alves
authors: DA
authors: Henriques
authors: Laboratório de Virologia Clínica e Molecular, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors: Jansen
authors: J
authors: de Araujo
authors: Instituto de Ciências da Saúde, Centro Universitário de Sinop, Universidade Federal de Mato Grosso Mato Grosso, Sinop, Mato Grosso, Brazil.
authors: Carlos Eduardo Hassegawa
authors: CE
authors: Siqueira
authors: Laboratório de Pesquisa em Virologia, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, São Paulo, Brazil.
authors: Tatiana Elias
authors: TE
authors: Colombo
authors: Laboratório de Virologia, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors: Victor Hugo
authors: VH
authors: Aquino
authors: Virology Research Center, School of Medicine of Ribeirao Preto of University of Sao Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors: Benedito Antonio Lopes
authors: BA
authors: da Fonseca
authors: Instituto de Ciências da Saúde, Centro Universitário de Sinop, Universidade Federal de Mato Grosso Mato Grosso, Sinop, Mato Grosso, Brazil.
authors: Roberta Vieira
authors: RV
authors: de Morais Bronzoni
authors: Laboratório de Pesquisa em Virologia, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, São Paulo, Brazil.
authors: Maurício Lacerda
authors: ML
authors: Nogueira
authors: Laboratório de Virologia Clínica e Molecular, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors: Edison Luiz
authors: EL
authors: Durigon
authors: Virology Research Center, School of Medicine of Ribeirao Preto of University of Sao Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors: Luiz Tadeu Moraes
authors: LT
authors: Figueiredo
mesh_terms: D000524:Alphavirus / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D009033:Culicidae / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D019562:Viral Load / Q000379:methods*
chemical_list: D012367:RNA, Viral