title: Cost-effective real-time reverse transcriptase PCR (RT-PCR) to screen for Dengue virus followed by rapid single-tube multiplex RT-PCR for serotyping of the virus.
abstract: Virus detection methodology provides detection of dengue virus in the early phase of the disease. PCR, targeting cDNA derived from viral RNA, has been used as a laboratory-based molecular tool for the detection of Dengue virus. We report the development and use of three real-time one-step reverse transcriptase PCR (RT-PCR) assays to detect dengue cases and serotype the virus involved. The first RT-PCR assay uses SYBR green I as the reporting dye for the purpose of cost-effective screening for dengue virus. The detection limit of the SYBR green I assay was 10 PFU/ml (0.01 equivalent PFU per assay) for all four dengue virus serotypes. The second RT-PCR assay is a duplex fluorogenic probe-based real-time RT-PCR for serotyping clinical samples for dengue viruses. The detection threshold of the probe-based RT-PCR format was 0.1 PFU for serotypes Dengue-1 and Dengue-2, 1 PFU for serotype Dengue-3, and 0.01 PFU for serotype Dengue-4. The third is a fourplex assay that detects any of the four serotypes in a single closed tube with comparable sensitivity. Validation of the assays with local clinical samples collected from 2004 to 2006 revealed that there was an 88% positive correlation between virus isolation and RT-PCR with regard to dengue virus detection and a 100% correlation with seroconversion in subsequent samples. The serotyping results derived from duplex and fourplex assays agree fully with each other and with that derived from immunofluorescence assays.
authors.affiliation: National Environment Agency, Environmental Health Institute, 11 Biopolis Way, 06-05/08 Helios Block, Singapore 138667.
authors.forename: Yee-Ling
authors.initials: YL
authors.lastname: Lai
authors.forename: Youne-Kow
authors.initials: YK
authors.lastname: Chung
authors.forename: Hwee-Cheng
authors.initials: HC
authors.lastname: Tan
authors.forename: Hoon-Fang
authors.initials: HF
authors.lastname: Yap
authors.forename: Grace
authors.initials: G
authors.lastname: Yap
authors.forename: Eng-Eong
authors.initials: EE
authors.lastname: Ooi
authors.forename: Lee-Ching
authors.initials: LC
authors.lastname: Ng
mesh_terms: D000330:Aedes; D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D002478:Cells, Cultured; D015342:DNA Probes; D003715:Dengue / Q000175:diagnosis* / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics* / Q000295:instrumentation / Q000379:methods; D012703:Serotyping; D013045:Species Specificity; D013997:Time Factors; D014776:Virus Cultivation
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I