title: Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses.
abstract: BACKGROUND
  Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.
  
  
  RESULTS
  An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log₁₀ cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.
  
  
  CONCLUSIONS
  This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.
authors: Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
authors: Jesse J
authors: JJ
authors: Waggoner
authors: Janaki
authors: J
authors: Abeynayake
authors: Malaya K
authors: MK
authors: Sahoo
authors: Lionel
authors: L
authors: Gresh
authors: Yolanda
authors: Y
authors: Tellez
authors: Karla
authors: K
authors: Gonzalez
authors: Gabriela
authors: G
authors: Ballesteros
authors: Anna M
authors: AM
authors: Pierro
authors: Paolo
authors: P
authors: Gaibani
authors: Frances P
authors: FP
authors: Guo
authors: Vittorio
authors: V
authors: Sambri
authors: Angel
authors: A
authors: Balmaseda
authors: Kumudu
authors: K
authors: Karunaratne
authors: Eva
authors: E
authors: Harris
authors: Benjamin A
authors: BA
authors: Pinsky
mesh_terms: D000914:Antibodies, Viral / Q000235:genetics; D003716:Dengue Virus / Q000145:classification / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012703:Serotyping / Q000379:methods*
chemical_list: D000914:Antibodies, Viral; D012367:RNA, Viral
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