Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)
Autor(a) principal: | |
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Data de Publicação: | 2008 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Revista do Instituto de Medicina Tropical de São Paulo |
Texto Completo: | https://www.revistas.usp.br/rimtsp/article/view/31151 |
Resumo: | HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination. |
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Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) Otimização da PCR em tempo real - Sybr Green para detecção do Herpes Vírus Humano tipo 6 (HHV-6) Human herpes virus 6 (HHV-6)PCRReal-time PCRSybr Green HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination. HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL) até 10-9 (equivalente a 2,46 moleculas/µL). As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com primers HHV3 5' TTG TGC GGG TCC GTT CCC ATC ATA 3' e HHV4 5' TCG GGA TAG AAA AAC CTA ATC CCT 3' e pelo método convencional, PCR nested usando primers HHV1 (externo): 5' CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (externo): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3', HHV3 (interno) e HHV4 (interno): 5' TCG GGA TAG AAA AAC CTA ATC CCT 3'. O limite de detecção foi determinado pelas diluições seriadas do plasmídio contendo um fragmento de HHV6: para o ensaio com Sybr Green, foi de 24,6 moleculas/µL e para a PCR nested, 2,46 moleculas/µL. Elegemos o PCR em Tempo Real - Sybr Green como método diagnóstico e quantitativo do HHV-6 devido a sua boa sensibilidade e menor risco de contaminação. Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo2008-02-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://www.revistas.usp.br/rimtsp/article/view/31151Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 No. 1 (2008); 61-63 Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 Núm. 1 (2008); 61-63 Revista do Instituto de Medicina Tropical de São Paulo; v. 50 n. 1 (2008); 61-63 1678-99460036-4665reponame:Revista do Instituto de Medicina Tropical de São Pauloinstname:Instituto de Medicina Tropical (IMT)instacron:IMTenghttps://www.revistas.usp.br/rimtsp/article/view/31151/33035Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Pauloinfo:eu-repo/semantics/openAccessCanto, Cynthia Liliane Motta doSumita, Laura MassamiMachado, Adriana FreireTateno, AdrianaCunha, Eveline Vieira daMachado, Clarisse Martins2012-07-07T19:07:28Zoai:revistas.usp.br:article/31151Revistahttp://www.revistas.usp.br/rimtsp/indexPUBhttps://www.revistas.usp.br/rimtsp/oai||revimtsp@usp.br1678-99460036-4665opendoar:2022-12-13T16:51:50.655029Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT)true |
dc.title.none.fl_str_mv |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) Otimização da PCR em tempo real - Sybr Green para detecção do Herpes Vírus Humano tipo 6 (HHV-6) |
title |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) |
spellingShingle |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) Canto, Cynthia Liliane Motta do Human herpes virus 6 (HHV-6) PCR Real-time PCR Sybr Green |
title_short |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) |
title_full |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) |
title_fullStr |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) |
title_full_unstemmed |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) |
title_sort |
Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6) |
author |
Canto, Cynthia Liliane Motta do |
author_facet |
Canto, Cynthia Liliane Motta do Sumita, Laura Massami Machado, Adriana Freire Tateno, Adriana Cunha, Eveline Vieira da Machado, Clarisse Martins |
author_role |
author |
author2 |
Sumita, Laura Massami Machado, Adriana Freire Tateno, Adriana Cunha, Eveline Vieira da Machado, Clarisse Martins |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Canto, Cynthia Liliane Motta do Sumita, Laura Massami Machado, Adriana Freire Tateno, Adriana Cunha, Eveline Vieira da Machado, Clarisse Martins |
dc.subject.por.fl_str_mv |
Human herpes virus 6 (HHV-6) PCR Real-time PCR Sybr Green |
topic |
Human herpes virus 6 (HHV-6) PCR Real-time PCR Sybr Green |
description |
HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination. |
publishDate |
2008 |
dc.date.none.fl_str_mv |
2008-02-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://www.revistas.usp.br/rimtsp/article/view/31151 |
url |
https://www.revistas.usp.br/rimtsp/article/view/31151 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
https://www.revistas.usp.br/rimtsp/article/view/31151/33035 |
dc.rights.driver.fl_str_mv |
Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Paulo info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Paulo |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo |
publisher.none.fl_str_mv |
Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo |
dc.source.none.fl_str_mv |
Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 No. 1 (2008); 61-63 Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 Núm. 1 (2008); 61-63 Revista do Instituto de Medicina Tropical de São Paulo; v. 50 n. 1 (2008); 61-63 1678-9946 0036-4665 reponame:Revista do Instituto de Medicina Tropical de São Paulo instname:Instituto de Medicina Tropical (IMT) instacron:IMT |
instname_str |
Instituto de Medicina Tropical (IMT) |
instacron_str |
IMT |
institution |
IMT |
reponame_str |
Revista do Instituto de Medicina Tropical de São Paulo |
collection |
Revista do Instituto de Medicina Tropical de São Paulo |
repository.name.fl_str_mv |
Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT) |
repository.mail.fl_str_mv |
||revimtsp@usp.br |
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1798951646816894976 |