title: Simple, specific molecular typing of dengue virus isolates using one-step RT-PCR and restriction fragment length polymorphism.
abstract: A one-step RT-PCR and one-enzyme RFLP was used to detect and distinguish among flaviviruses, including the four serotypes of dengue and the St. Louis Encephalitis, West Nile and Yellow Fever viruses in cultured virus samples or acute-phase human serum. Using a previously described RT-PCR, but novel RFLP procedure, results are obtained in 24 h with basic PCR and electrophoresis equipment. There is 95% agreement between RT-PCR/RFLP results and those achieved by indirect immunofluorescence assays, and 100% agreement between RT-PCR/RFLP results and gene sequencing. This method is more rapid than tests of cytopathic effect based on virus isolation in tissue culture, and simpler than real-time PCR. It does not require specialized equipment, radioisotopes or computer analysis and is a method that can be applied widely in the developing world. It allows for prompt determination of whether a flavivirus is the cause of illness in a febrile patient, rapid identification of dengue serotypes in circulation, and improved patient management in cases where prior dengue exposure make dengue hemorrhagic fever or dengue shock syndrome a risk.
authors: Gorgas Memorial Institute for Health Studies, Panama City, Panama. ortiz.ay@gmail.com
authors: Alma
authors: A
authors: Ortiz
authors: Zeuz
authors: Z
authors: Capitan
authors: Yaxelis
authors: Y
authors: Mendoza
authors: Julio
authors: J
authors: Cisneros
authors: Brechla
authors: B
authors: Moreno
authors: Yamitzel
authors: Y
authors: Zaldivar
authors: Mariana
authors: M
authors: Garcia
authors: Rebecca E
authors: RE
authors: Smith
authors: Jorge
authors: J
authors: Motta
authors: Juan Miguel
authors: JM
authors: Pascale
mesh_terms: D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D058889:Molecular Typing / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*