Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses.
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.
de Morais Bronzoni
Roberta Vieira
RV
Virus Research Unit, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Sao Paulo, Brazil.
Baleotti
Flávia Graciela
FG
Ribeiro Nogueira
Rita Maria
RM
Nunes
Márcio
M
Moraes Figueiredo
Luiz Tadeu
LT
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
D017931:DNA Primers