Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses - PubMed (original) (raw)

Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses

Roberta Vieira de Morais Bronzoni et al. J Clin Microbiol. 2005 Feb.

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.

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Figures

FIG. 1.

Agarose gel electrophoresis of amplicons from D-RT-PCR (A) and M-N-PCR (B) for Alphavirus. Lanes 1 to 3, negative controls (RNA extract from uninfected mouse brain tissue, BUJV, and water); lane 4, molecular size marker (DNA ladder, 100 or 50 bp); lanes 5 to 12, Alphavirus VEEV BeAr 40403, VEEV 78V 3531, MUCV BeAn 8, PIXV BeAr 35645, EEEV SPAn 14723, WEEV Rio 1257, AURAV BeAr 10315, and MAYV BeAr 20290.

FIG. 2.

Agarose gel electrophoresis of amplicons from D-RT-PCR (A), M-N-PCR (B), and N-PCR (C) for Flavivirus. Lanes 1 to 4, negative controls (RNA extract from uninfected mouse brain tissue, uninfected cell culture supernatant, BUJV, and water); lane 5, molecular size marker (DNA ladder, 100 bp); lanes 6 to 22, Flavivirus DENV 1 RibH 830, DENV 1 RioH 289731, DENV 2 Cea 24622, DENV 2 SpH 125367, DENV 2 Toc 213, DENV 3 Rio, DENV 3 RibH 1, YFV 17D, YFV BeAnm131, DENV 4 Boa Vista, SLEV BeH 355964, SLEV BeAn 421498, SLEV SpAn 11916, SLEV BeAr 417704, BSQV BeAn 4073, ILHV BeH 7445, and ROCV SpH 34675.

FIG. 3.

D-RT-PCR detection, followed by M-N-PCR identification of flaviviruses and alphaviruses in clinical samples. Lanes 1 to 4, Flavivirus genus; lanes 5 to 8, Alphavirus genus; lane 9, molecular size marker (DNA ladder, 100 bp); lanes 10 to 13, M-N-PCR flavivirus assay of DENV 1, DENV 2, DENV 3, and YFV; lanes 14 to 17, M-N-PCR alphavirus assay of MUCV, EEEV, WEEV, and MAYV; lane 18, water.

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Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses - PubMed (original) (raw)