Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics
The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription– polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | Spanish |
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Instituto Nacional de Salud
2016-05-01
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| Series: | Revista Peruana de Medicina Experimental y Salud Pública |
| Subjects: | |
| Online Access: | https://rpmesp.ins.gob.pe/index.php/rpmesp/article/view/2101 |
| _version_ | 1819014926712176640 |
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| author | Daría Camacho Jesús Reyes Leticia Franco Guillermo Comach Elizabeth Ferrer |
| author_facet | Daría Camacho Jesús Reyes Leticia Franco Guillermo Comach Elizabeth Ferrer |
| author_sort | Daría Camacho |
| collection | DOAJ |
| description | The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription– polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5′UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5′UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous. |
| first_indexed | 2024-12-21T02:23:36Z |
| format | Article |
| id | doaj.art-16bc1243425f4046b77166b0facf95eb |
| institution | Directory Open Access Journal |
| issn | 1726-4634 1726-4642 |
| language | Spanish |
| last_indexed | 2024-12-21T02:23:36Z |
| publishDate | 2016-05-01 |
| publisher | Instituto Nacional de Salud |
| record_format | Article |
| series | Revista Peruana de Medicina Experimental y Salud Pública |
| spelling | doaj.art-16bc1243425f4046b77166b0facf95eb2022-12-21T19:19:03ZspaInstituto Nacional de SaludRevista Peruana de Medicina Experimental y Salud Pública1726-46341726-46422016-05-0133226927310.17843/rpmesp.2016.332.21011739Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnosticsDaría Camacho0Jesús Reyes1Leticia Franco2Guillermo Comach3Elizabeth Ferrer4Instituto de Investigaciones Biomédicas (BIOMED-UC), Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, VenezuelaInstituto de Investigaciones Biomédicas (BIOMED-UC), Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, VenezuelaCentro Nacional de Microbiología. Instituto de Salud Carlos III. Majadahonda, Madrid, España.Instituto de Investigaciones Biomédicas (BIOMED-UC), Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, VenezuelaInstituto de Investigaciones Biomédicas (BIOMED-UC), Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, VenezuelaThe purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription– polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5′UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5′UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous.https://rpmesp.ins.gob.pe/index.php/rpmesp/article/view/2101alphavirusflavivirusclonación, moleculardiagnóstico |
| spellingShingle | Daría Camacho Jesús Reyes Leticia Franco Guillermo Comach Elizabeth Ferrer Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics Revista Peruana de Medicina Experimental y Salud Pública alphavirus flavivirus clonación, molecular diagnóstico |
| title | Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics |
| title_full | Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics |
| title_fullStr | Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics |
| title_full_unstemmed | Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics |
| title_short | Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics |
| title_sort | cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics |
| topic | alphavirus flavivirus clonación, molecular diagnóstico |
| url | https://rpmesp.ins.gob.pe/index.php/rpmesp/article/view/2101 |
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