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.
abstract: BACKGROUND
  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.
authors.affiliation: Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
authors.forename: Marilia Farignoli
authors.initials: MF
authors.lastname: Romeiro
authors.affiliation: Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
authors.forename: William Marciel de
authors.initials: WM
authors.lastname: Souza
authors.affiliation: Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
authors.forename: Aline Lavado
authors.initials: AL
authors.lastname: Tolardo
authors.affiliation: Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
authors.forename: Luiz Carlos
authors.initials: LC
authors.lastname: Vieira
authors.affiliation: Laboratório de Pesquisa em Virologia, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, São Paulo, Brasil.
authors.forename: Tatiana Elias
authors.initials: TE
authors.lastname: Colombo
authors.affiliation: Laboratório de Virologia, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
authors.forename: Victor Hugo
authors.initials: VH
authors.lastname: Aquino
authors.affiliation: Laboratório de Pesquisa em Virologia, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, São Paulo, Brasil.
authors.forename: Maurício Lacerda
authors.initials: ML
authors.lastname: Nogueira
authors.affiliation: Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
authors.forename: Luiz Tadeu Moraes
authors.initials: LT
authors.lastname: Figueiredo
mesh_terms: D052160:Benzothiazoles; D001938:Brazil; D017931:DNA Primers; D003959:Diamines; D005416:Flavivirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D018177:Flavivirus Infections / Q000175:diagnosis* / Q000821:virology; D005456:Fluorescent Dyes; D006801:Humans; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I