Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Memórias do Instituto Oswaldo Cruz |
Texto Completo: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762017000500339 |
Resumo: | BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. |
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Memórias do Instituto Oswaldo Cruz |
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Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnosticsdiagnosticsReal-time PCRinternal controlHCVRNA viruses BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.Instituto Oswaldo Cruz, Ministério da Saúde2017-05-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762017000500339Memórias do Instituto Oswaldo Cruz v.112 n.5 2017reponame:Memórias do Instituto Oswaldo Cruzinstname:Fundação Oswaldo Cruzinstacron:FIOCRUZ10.1590/0074-02760160380info:eu-repo/semantics/openAccessZambenedetti,Miriam RibasPavoni,Daniela ParadaDallabona,Andreia CristineDominguez,Alejandro CorreaPoersch,Celina de OliveiraFragoso,Stenio PerdigãoKrieger,Marco Aurélioeng2020-04-25T17:52:43Zhttp://www.scielo.br/oai/scielo-oai.php0074-02761678-8060opendoar:null2020-04-26 02:22:00.323Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruztrue |
dc.title.none.fl_str_mv |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
title |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
spellingShingle |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics Zambenedetti,Miriam Ribas diagnostics Real-time PCR internal control HCV RNA viruses |
title_short |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
title_full |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
title_fullStr |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
title_full_unstemmed |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
title_sort |
Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics |
author |
Zambenedetti,Miriam Ribas |
author_facet |
Zambenedetti,Miriam Ribas Pavoni,Daniela Parada Dallabona,Andreia Cristine Dominguez,Alejandro Correa Poersch,Celina de Oliveira Fragoso,Stenio Perdigão Krieger,Marco Aurélio |
author_role |
author |
author2 |
Pavoni,Daniela Parada Dallabona,Andreia Cristine Dominguez,Alejandro Correa Poersch,Celina de Oliveira Fragoso,Stenio Perdigão Krieger,Marco Aurélio |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Zambenedetti,Miriam Ribas Pavoni,Daniela Parada Dallabona,Andreia Cristine Dominguez,Alejandro Correa Poersch,Celina de Oliveira Fragoso,Stenio Perdigão Krieger,Marco Aurélio |
dc.subject.por.fl_str_mv |
diagnostics Real-time PCR internal control HCV RNA viruses |
topic |
diagnostics Real-time PCR internal control HCV RNA viruses |
dc.description.none.fl_txt_mv |
BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. |
description |
BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-05-01 |
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://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762017000500339 |
url |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762017000500339 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/0074-02760160380 |
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 |
Instituto Oswaldo Cruz, Ministério da Saúde |
publisher.none.fl_str_mv |
Instituto Oswaldo Cruz, Ministério da Saúde |
dc.source.none.fl_str_mv |
Memórias do Instituto Oswaldo Cruz v.112 n.5 2017 reponame:Memórias do Instituto Oswaldo Cruz instname:Fundação Oswaldo Cruz instacron:FIOCRUZ |
reponame_str |
Memórias do Instituto Oswaldo Cruz |
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Memórias do Instituto Oswaldo Cruz |
instname_str |
Fundação Oswaldo Cruz |
instacron_str |
FIOCRUZ |
institution |
FIOCRUZ |
repository.name.fl_str_mv |
Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruz |
repository.mail.fl_str_mv |
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