title: Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection.
abstract: BACKGROUND
  Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman(®) assay and conventional nested PCR assay.
  
  
  OBJECTIVE
  To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti).
  
  
  METHODS
  Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR(®) Green assay, real time Taqman(®) assay to compare the sensitivity and specificity.
  
  
  RESULTS
  Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR(®) green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay.
  
  
  CONCLUSIONS
  We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.
authors.affiliation: Department of Internal Medicine Nepal Police Hospital, Maharagjung, Kathmandu, Nepal.
authors.forename: Damodar
authors.initials: D
authors.lastname: Paudel
authors.forename: Richard
authors.initials: R
authors.lastname: Jarman
authors.forename: Kriengsak
authors.initials: K
authors.lastname: Limkittikul
authors.forename: Chonticha
authors.initials: C
authors.lastname: Klungthong
authors.forename: Supat
authors.initials: S
authors.lastname: Chamnanchanunt
authors.forename: Ananda
authors.initials: A
authors.lastname: Nisalak
authors.forename: Robert
authors.initials: R
authors.lastname: Gibbons
authors.forename: Watcharee
authors.initials: W
authors.lastname: Chokejindachai