title: Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.
abstract: BACKGROUND
  Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis.
  
  
  OBJECTIVE
  To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples.
  
  
  METHODS
  RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction.
  
  
  RESULTS
  The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach.
  
  
  CONCLUSIONS
  This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.
authors.affiliation: Laboratorio de Virología, Instituto Nacional de Salud, Bogotá, D.C, Colombia.
authors.forename: María C
authors.initials: MC
authors.lastname: Méndez
authors.affiliation: Robert Koch Institut, Berlin, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Domingo
authors.affiliation: Laboratorio de Arbovirus y Enfermedades Víricas Importadas, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, España.
authors.forename: Antonio
authors.initials: A
authors.lastname: Tenorio
authors.affiliation: Laboratorio de Virología, Instituto Nacional de Salud, Bogotá, D.C, Colombia.
authors.forename: Lissethe C
authors.initials: LC
authors.lastname: Pardo
authors.affiliation: Laboratorio de Virología, Instituto Nacional de Salud, Bogotá, D.C, Colombia.
authors.forename: Gloria J
authors.initials: GJ
authors.lastname: Rey
authors.affiliation: Laboratorio de Virología, Instituto Nacional de Salud, Bogotá, D.C, Colombia.
authors.forename: Jairo A
authors.initials: JA
authors.lastname: Méndez
mesh_terms: D000818:Animals; D001921:Brain / Q000821:virology; D003105:Colombia; D017931:DNA Primers; D019353:Endemic Diseases; D005838:Genotype; D006801:Humans; D008099:Liver / Q000821:virology; D051379:Mice; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014766:Viremia / Q000821:virology; D015004:Yellow Fever / Q000175:diagnosis / Q000821:virology; D015005:Yellow fever virus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral