Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction

We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

[1]  D. Gajdusek,et al.  Detection of flaviviruses by reverse‐transcriptase polymerase chain reaction , 1991, Journal of medical virology.

[2]  R. Schlesinger,et al.  Dengue Viruses , 1977, Virology Monographs Die Virusforschung in Einzeldarstellungen.

[3]  R. Chanock,et al.  Cloning full-length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins. , 1986, Virology.

[4]  G. Kuno,et al.  Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. , 1984, The American journal of tropical medicine and hygiene.

[5]  J. Guesdon,et al.  Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood. , 1990, Journal of virological methods.

[6]  S. Halstead,et al.  Pathogenesis of dengue: challenges to molecular biology. , 1988, Science.

[7]  G. Sarkar,et al.  Shedding light on PCR contamination , 1990, Nature.

[8]  P. Mason,et al.  Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. , 1987, Virology.

[9]  K. Osatomi,et al.  Complete nucleotide sequence of dengue type 3 virus genome RNA. , 1990, Virology.

[10]  V. Deubel,et al.  Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. , 1986, Virology.

[11]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[12]  G. Fox,et al.  The molecular epidemiology of dengue viruses: genetic variation and microevolution. , 1990 .

[13]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.