title: Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses.
abstract: A new approach was developed for the rapid detection and identification of Brazilian alphaviruses and flaviviruses. The methodology involves the genus-specific detection of Alphavirus and Flavivirus by a duplex reverse transcription-PCR (D-RT-PCR), followed by multiplex nested PCR (M-N-PCR) or nested PCR (N-PCR) assays for species-specific identification. By this protocol, 25 arboviruses were specifically detected and identified. Detection levels between 10(1.3) and 10(3.5) 50% tissue culture infective doses (TCID(50))/ml of Flavivirus and Alphavirus strains were achieved by D-RT-PCR, and levels of <1 TCID(50)/ml were achieved by M-N-PCR assays. To assess the suitability and clinical application of this methodology, a total of 101 human or animal stored samples were analyzed. Results obtained suggest that this technique could be applied as a rapid diagnostic tool in clinical samples in which arbovirus infection is suspected and differential diagnosis is required, avoiding the need to test specimens by separate PCR methods.
authors.affiliation: Virus Research Unit, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Sao Paulo, Brazil.
authors.forename: Roberta Vieira
authors.initials: RV
authors.lastname: de Morais Bronzoni
authors.forename: Flávia Graciela
authors.initials: FG
authors.lastname: Baleotti
authors.forename: Rita Maria
authors.initials: RM
authors.lastname: Ribeiro Nogueira
authors.forename: Márcio
authors.initials: M
authors.lastname: Nunes
authors.forename: Luiz Tadeu
authors.initials: LT
authors.lastname: Moraes Figueiredo
mesh_terms: D000524:Alphavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D018354:Alphavirus Infections / Q000821:virology; D001938:Brazil; D017931:DNA Primers; D005416:Flavivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D018177:Flavivirus Infections / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D017931:DNA Primers