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Duplex Reverse Transcription-PCR Followed by Nested PCR Assays for Detection and Identification of Brazilian Alphaviruses and Flaviviruses

2005; American Society for Microbiology; Volume: 43; Issue: 2 Linguagem: Inglês

10.1128/jcm.43.2.696-702.2005

1098-660X

Roberta Vieira de Morais Bronzoni, Flávia Graciela Baleotti, Rita Maria Ribeiro Nogueira, Márcio Roberto Teixeira Nunes, Luíz Tadeu Moraes Figueiredo,

Viral Infections and Outbreaks Research

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.