title: One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples.
abstract: BACKGROUND
  Dengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.
  
  
  METHODS
  The DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.
  
  
  RESULTS
  The RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10(2) to 10(6) copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.
  
  
  CONCLUSIONS
  Our results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.
authors.affiliation: Department of Microbiology, The Public Health Agency of Sweden, SE-171 82, Solna, Sweden. erik.alm@folkhalsomyndigheten.se.
authors.forename: Erik
authors.initials: E
authors.lastname: Alm
authors.affiliation: Department of Microbiology, The Public Health Agency of Sweden, SE-171 82, Solna, Sweden. gunnel.lindegren@folkhalsomyndigheten.se.
authors.forename: Gunnel
authors.initials: G
authors.lastname: Lindegren
authors.affiliation: Department of Microbiology, The Public Health Agency of Sweden, SE-171 82, Solna, Sweden. kerstin.falk@folkhalsomyndigheten.se.|Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77, Stockholm, Sweden. kerstin.falk@folkhalsomyndigheten.se.
authors.forename: Kerstin Ingrid
authors.initials: KI
authors.lastname: Falk
authors.affiliation: Department of Microbiology, The Public Health Agency of Sweden, SE-171 82, Solna, Sweden. nina.lagerqvist@folkhalsomyndigheten.se.
authors.forename: Nina
authors.initials: N
authors.lastname: Lagerqvist
mesh_terms: D003429:Cross Reactions; D017931:DNA Primers; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods; D044967:Serum / Q000821:virology
chemical_list: D017931:DNA Primers