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Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics
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 im...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Instituto Oswaldo Cruz, Ministério da Saúde
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398160/ https://www.ncbi.nlm.nih.gov/pubmed/28403327 http://dx.doi.org/10.1590/0074-02760160380 |
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author | Zambenedetti, Miriam Ribas Pavoni, Daniela Parada Dallabona, Andreia Cristine Dominguez, Alejandro Correa Poersch, Celina de Oliveira Fragoso, Stenio Perdigão Krieger, Marco Aurélio |
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_sort | Zambenedetti, Miriam Ribas |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-5398160 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Instituto Oswaldo Cruz, Ministério da Saúde |
record_format | MEDLINE/PubMed |
spelling | pubmed-53981602017-05-01 Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics Zambenedetti, Miriam Ribas Pavoni, Daniela Parada Dallabona, Andreia Cristine Dominguez, Alejandro Correa Poersch, Celina de Oliveira Fragoso, Stenio Perdigão Krieger, Marco Aurélio Mem Inst Oswaldo Cruz Articles 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 Saúde 2017-04-06 2017-05 /pmc/articles/PMC5398160/ /pubmed/28403327 http://dx.doi.org/10.1590/0074-02760160380 Text en http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Articles Zambenedetti, Miriam Ribas Pavoni, Daniela Parada Dallabona, Andreia Cristine Dominguez, Alejandro Correa Poersch, Celina de Oliveira Fragoso, Stenio Perdigão Krieger, Marco Aurélio 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 |
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_short | 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 |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398160/ https://www.ncbi.nlm.nih.gov/pubmed/28403327 http://dx.doi.org/10.1590/0074-02760160380 |
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