A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification
We show here a simplified reverse transcription-polymerase chain 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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
1997-05-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
| Subjects: | |
| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02761997000300016 |
| _version_ | 1797757425678286848 |
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| author | Luiz Tadeu M Figueiredo Weber Chelli Batista Akira Igarashi |
| author_facet | Luiz Tadeu M Figueiredo Weber Chelli Batista Akira Igarashi |
| author_sort | Luiz Tadeu M Figueiredo |
| collection | DOAJ |
| description | We show here a simplified reverse transcription-polymerase chain 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 reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml 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% 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 TCID50/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 |
| first_indexed | 2024-03-12T18:15:41Z |
| format | Article |
| id | doaj.art-dbdb5b2bb68343f89affff50398f9fd3 |
| institution | Directory Open Access Journal |
| issn | 0074-0276 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T18:15:41Z |
| publishDate | 1997-05-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-dbdb5b2bb68343f89affff50398f9fd32023-08-02T09:10:45ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz0074-02761678-80601997-05-0192339510.1590/S0074-02761997000300016A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus IdentificationLuiz Tadeu M FigueiredoWeber Chelli BatistaAkira IgarashiWe show here a simplified reverse transcription-polymerase chain 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 reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml 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% 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 TCID50/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 techniquehttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02761997000300016dengue identificationdengue diagnosisreverse transcriptionpolymerase chain reaction |
| spellingShingle | Luiz Tadeu M Figueiredo Weber Chelli Batista Akira Igarashi A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification Memorias do Instituto Oswaldo Cruz dengue identification dengue diagnosis reverse transcription polymerase chain reaction |
| title | A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification |
| title_full | A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification |
| title_fullStr | A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification |
| title_full_unstemmed | A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification |
| title_short | A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification |
| title_sort | simple reverse transcription polymerase chain reaction for dengue type 2 virus identification |
| topic | dengue identification dengue diagnosis reverse transcription polymerase chain reaction |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02761997000300016 |
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