Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Revista da Sociedade Brasileira de Medicina Tropical |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822016000300279 |
Resumo: | Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies. |
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Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in BrazilFlavivirusArbovirusReal-time RT-PCRDiagnosisAbstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.Sociedade Brasileira de Medicina Tropical - SBMT2016-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822016000300279Revista da Sociedade Brasileira de Medicina Tropical v.49 n.3 2016reponame:Revista da Sociedade Brasileira de Medicina Tropicalinstname:Sociedade Brasileira de Medicina Tropical (SBMT)instacron:SBMT10.1590/0037-8682-0444-2015info:eu-repo/semantics/openAccessRomeiro,Marilia FarignoliSouza,William Marciel deTolardo,Aline LavadoVieira,Luiz CarlosColombo,Tatiana EliasAquino,Victor HugoNogueira,Maurício LacerdaFigueiredo,Luiz Tadeu Moraeseng2016-09-19T00:00:00Zoai:scielo:S0037-86822016000300279Revistahttps://www.sbmt.org.br/portal/revista/ONGhttps://old.scielo.br/oai/scielo-oai.php||dalmo@rsbmt.uftm.edu.br|| rsbmt@rsbmt.uftm.edu.br1678-98490037-8682opendoar:2016-09-19T00:00Revista da Sociedade Brasileira de Medicina Tropical - Sociedade Brasileira de Medicina Tropical (SBMT)false |
dc.title.none.fl_str_mv |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
title |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
spellingShingle |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil Romeiro,Marilia Farignoli Flavivirus Arbovirus Real-time RT-PCR Diagnosis |
title_short |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
title_full |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
title_fullStr |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
title_full_unstemmed |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
title_sort |
Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil |
author |
Romeiro,Marilia Farignoli |
author_facet |
Romeiro,Marilia Farignoli Souza,William Marciel de Tolardo,Aline Lavado Vieira,Luiz Carlos Colombo,Tatiana Elias Aquino,Victor Hugo Nogueira,Maurício Lacerda Figueiredo,Luiz Tadeu Moraes |
author_role |
author |
author2 |
Souza,William Marciel de Tolardo,Aline Lavado Vieira,Luiz Carlos Colombo,Tatiana Elias Aquino,Victor Hugo Nogueira,Maurício Lacerda Figueiredo,Luiz Tadeu Moraes |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Romeiro,Marilia Farignoli Souza,William Marciel de Tolardo,Aline Lavado Vieira,Luiz Carlos Colombo,Tatiana Elias Aquino,Victor Hugo Nogueira,Maurício Lacerda Figueiredo,Luiz Tadeu Moraes |
dc.subject.por.fl_str_mv |
Flavivirus Arbovirus Real-time RT-PCR Diagnosis |
topic |
Flavivirus Arbovirus Real-time RT-PCR Diagnosis |
description |
Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-06-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822016000300279 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822016000300279 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/0037-8682-0444-2015 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Sociedade Brasileira de Medicina Tropical - SBMT |
publisher.none.fl_str_mv |
Sociedade Brasileira de Medicina Tropical - SBMT |
dc.source.none.fl_str_mv |
Revista da Sociedade Brasileira de Medicina Tropical v.49 n.3 2016 reponame:Revista da Sociedade Brasileira de Medicina Tropical instname:Sociedade Brasileira de Medicina Tropical (SBMT) instacron:SBMT |
instname_str |
Sociedade Brasileira de Medicina Tropical (SBMT) |
instacron_str |
SBMT |
institution |
SBMT |
reponame_str |
Revista da Sociedade Brasileira de Medicina Tropical |
collection |
Revista da Sociedade Brasileira de Medicina Tropical |
repository.name.fl_str_mv |
Revista da Sociedade Brasileira de Medicina Tropical - Sociedade Brasileira de Medicina Tropical (SBMT) |
repository.mail.fl_str_mv |
||dalmo@rsbmt.uftm.edu.br|| rsbmt@rsbmt.uftm.edu.br |
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