Antigen sandwich ELISA predicts RT-PCR detection of dengue virus genome in infected culture fluids of Aedes albopictus C6/36 cells.

Antigen detection by sandwich ELISA was evaluated to predict RT-PCR detection of dengue viral genome in infected culture fluid of Aedes albopictus clone C6/36 cells. Serum specimens collected from dengue patients within 5 days from onset of fever in 2 hospitals in Metro Manila, Philippines, were inoculated into C6/36 cells, and incubated at 28 degrees C. A total of 282 infected culture fluid specimens were harvested and examined by sandwich ELISA and RT-PCR to detect dengue viral antigen and genome, respectively. In the sandwich ELISA, the P/N ratio was calculated by dividing optical density (OD) of a given test specimen by the OD of the standard negative specimen. Samples with a P/N ratio > or = 4.001 were positive for viral genome detection by RT-PCR. The sensitivity and specificity of antigen sandwich ELISA with RT-PCR as the standard, were 90.4% and 100%, respectively. Although antigen sandwich ELISA is less sensitive than RT-PCR, its usefulness lies in its capability to screen a large number of samples at a minimum cost, especially during an outbreak. Samples that meet a set cutoff value can undergo confirmation by RT-PCR for further epidemiological studies.

A simple reverse transcription-polymerase chain reaction for dengue type 2 virus identification

We show here a simplified reverse transcription-polymerase reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reation vessel was carried out following a digestion of virus with 1 per cent Nondet P-40. The 50 µl assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37ºC for RT followed by a variable amount of cycles of two-step PCR amplification (92ºC for 60 sec, 53ºC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7 per cent agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 10².8 TCID 50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription: polymerase chain reaction (RT-PCR) method

Apresentamos neste trabalho uma tecnica simplificada de RT-PCR para identificacao de virus de dengue. Cinco estirpes brasileiras de virus de dengue dos tipos 1 e 2, isoladas de pacientes e o virus da vacina de febre amarela como controle negativo, foram utilizadas nos testes. Celulas C6/36 foram infectadas e os fluidos de cultura coletados apos 7 dias. A RT-PCR, era efetuada em um unico tubo contendo 1 ul de fluidos infectados e diluidos 1/10 em agua destilada ou em mistura detergente contendo Nonidet P40. A mistura de reacao, num volume de 50 ul, continha 50 pmol de primers especificos, que amplificam sequencia com 482 pares de bases para virus do dengue tipo 1 e 210 pares de bases para dengue tipo 2, ou, ainda, primers para grupo dengue que amplificam sequencia com 511 pares de bases...