title: Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  RESULTS
  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.
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.|Universidade Federal do Paraná, Departamento de Bioprocessos e Biotecnologia, Curitiba, PR, Brasil.
authors.forename: Miriam Ribas
authors.initials: MR
authors.lastname: Zambenedetti
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.
authors.forename: Daniela Parada
authors.initials: DP
authors.lastname: Pavoni
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.
authors.forename: Andreia Cristine
authors.initials: AC
authors.lastname: Dallabona
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.
authors.forename: Alejandro Correa
authors.initials: AC
authors.lastname: Dominguez
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.
authors.forename: Celina de Oliveira
authors.initials: CO
authors.lastname: Poersch
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.
authors.forename: Stenio Perdigão
authors.initials: SP
authors.lastname: Fragoso
authors.affiliation: Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Genômica, Curitiba, PR, Brasil.|Fundação Oswaldo Cruz-Fiocruz, Instituto de Biologia Molecular do Paraná, Curitiba, PR, Brasil.
authors.forename: Marco Aurélio
authors.initials: MA
authors.lastname: Krieger
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics; D006526:Hepatitis C / Q000175:diagnosis*; D017909:Levivirus / Q000235:genetics*; D008954:Models, Biological; D012328:RNA Viruses / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards