Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens

Abstract

Background: More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed. Objective: To develop a semi-nested polymerase chain reaction (PCR) assay based on primers within the NS3 gene for the simultaneous detection and typing of dengue viruses in human sera. Study design: A first round of single step reverse transcription-polymerase chain reaction (RT-PCR) was carried out with a pair of consensus primers, followed by a second round of semi-nested amplification using the upstream consensus primer and four type-specific downstream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID50-titrated virus-infected tissue culture fluid, and total RNA extracted from C6/36 cells infected with dengue and other flaviviruses, respectively. A retrospective study was performed on acute sera from thirteen patients with dengue (confirmed by virus isolation) employing semi-nested PCR in parallel with virus re-isolation and a single-step RT-PCR method for the typing of dengue viruses in human sera. Results: The semi-nested PCR assay could detect up to 1 pfu of dengue virus, but not other flaviviruses. The semi-nested PCR and single-step RT-PCR assays correctly typed dengue viruses in twelve and five sera, respectively, whereas none of the sera was positive by virus re-isolation. Conclusions: This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.