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Journal of Clinical Microbiology, February 2005, p. 696-702, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.696-702.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Roberta Vieira de Morais Bronzoni,1* Flávia Graciela Baleotti,1 Rita Maria Ribeiro Nogueira,2 Márcio Nunes,3 and Luiz Tadeu Moraes Figueiredo1

Virus Research Unit, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Sao Paulo,1 Flavivirus Laboratory, Oswaldo Cruz Foundation, Rio de Janeiro, Rio de Janeiro,2 Arbovirus Laboratory, Evandro Chagas Institute, Belém, Para, Brazil3

Received 5 February 2004/ Returned for modification 2 May 2004/ Accepted 18 September 2004

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 101.3 and 103.5 50% tissue culture infective doses (TCID50)/ml of Flavivirus and Alphavirus strains were achieved by D-RT-PCR, and levels of <1 TCID50/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.

* Corresponding author. Mailing address: Unidade Multidepartamental de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida dos Bandeirantes 3900, Ribeirão Preto, CEP 14049-900, São Paulo, Brazil. Phone: 55-16-602-3271. Fax: 55-16-633-6631. E-mail: rbronzoni{at}aol.com.br.

Journal of Clinical Microbiology, February 2005, p. 696-702, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.696-702.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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