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.

FIG. 1.

Alignment of the 3′ untranslated region of dengue viruses and binding sites for RT-PCR primers and probes. Except for Dengue-2, each serotype is represented by a Singaporean and two foreign isolates. Primer 1, pan-dengue forward primer; 2, pan-dengue reverse primer-reverse complementary; 3, D1 fluorescein-labeled probe-reverse complementary; primer 4, D1 LC Red-labeled probe-reverse complementary; primer 5, D2 fluorescein-labeled probe; 6, D2 LC Red-labeled probe; 7, D3 fluorescein-labeled probe; 8, D3 LC Red-labeled probe; 9, D4 fluorescein-labeled probe; and 10, D4 LC Red-labeled probe.