title: Development and evaluation of a fluorogenic real-time RT-PCR for the detection of dengue 3 virus.
abstract: A dengue-3-specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was developed using the novel Light Upon eXtension (LUX™) fluorogenic technology. A labeled forward primer and a standard reverse primer that target a conserved region within the non-structural 1 (NS1) gene of dengue 3 strains were designed. The dengue-3-specific assay did not recognize other dengue serotypes and related flaviviruses. Using a tenfold serial dilution of plasmid DNA containing the dengue 3 NS1 gene as standards, the range of dengue virus detection was determined to be from 10(3) to 10(9) copies/ml or from 80 to 8 × 10(7) copies/reaction with an average correlation coefficient of ≥ 0.99. The mean intra-assay coefficient of variation (CV) at 2.01% and the mean inter-assay CV at 2.68% suggest the repeatability of the procedure. Moreover, the fluorogenic assay was evaluated by using clinical specimens and comparing test results with historical data obtained from conventional RT-PCR, which served as the criterion standard. Using patient sera as test samples, the assay demonstrated 95.45% sensitivity and 100% specificity, respectively. These results reveal that the real-time RT-PCR assay may be utilized as a rapid, convenient, and sensitive tool for the detection of dengue 3 in clinical and laboratory specimens.
authors.affiliation: Division of Natural Sciences and Mathematics, University of the Philippines Visayas Tacloban College, Tacloban City, Leyte, Philippines.
authors.forename: Irene L
authors.initials: IL
authors.lastname: Tan
authors.forename: Mark Pierre S
authors.initials: MP
authors.lastname: Dimamay
authors.forename: Corazon C
authors.initials: CC
authors.lastname: Buerano
authors.forename: Jhoe Anthony R
authors.initials: JA
authors.lastname: Alfon
authors.forename: Carol Z
authors.initials: CZ
authors.lastname: Tanig
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Matias
authors.forename: Filipinas F
authors.initials: FF
authors.lastname: Natividad
mesh_terms: D003715:Dengue / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D006801:Humans; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics*
chemical_list: D005456:Fluorescent Dyes; C546113:NS1 protein, Dengue virus type 3; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins