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

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 down-stream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID(50)-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.