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Arch Hellen Med, 27(5), September-October 2010, 818-821


Detection of dengue virus by simple RT-PCR using universal degenerate primers:
Observations from a preliminary study

1Clinical Microscopy Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok
2Wiwanitkit House, Bangkhae, Bangkok, Thailand

OBJECTIVE Dengue hemorrhagic fever (DHF) is caused by a mosquito-borne flavivirus with four distinct serotypes (dengue types 1–4: DEN1–4). All four serotypes are endemic in most of the countries in the tropical and subtropical regions. Because of the high sensitivity and specificity of PCR diagnostic techniques, several PCR-based strategies for the detection of dengue have been developed recently. In this study aimed to develop a one-step reverse transcription (RT)-PCR assay using universal primer for rapid detection of viral RNA of all dengue serotypes.

METHOD A reverse transcriptase polymerase chain reaction (RT-PCR) was developed by using a degenerate primer pair specific to the non-structural (NS1), which bears conserve region in the dengue genome of all serotypes. The primer was designed based on the genome sequence of dengue strains collected in Thailand. The RT-PCR assay using the newly designed primers was optimized and evaluated by the detection of dengue RNA in mosquito-cell culture and patient blood.

RESULTS The NS1 region of dengue (1–4) was successfully amplified giving an RT-PCR product of the expected size (637 bp). Since dengue virus is thought to replicate in mononuclear cells in vivo, the RNA extracted from peripheral blood mononuclear cells of patients suspected of being infected with DHF was used as template for RT-PCR. The results indicate that detection of viral RNA in mononuclear cells could be an effective alternative technique to the determination of immunoglobulin changes in plasma.

CONCLUSIONS According to this study, this technique is rapid, convenient and provides accurate diagnosis in suspected dengue.

Key words: Dengue, Mononuclear cells, Reverse-transcription PCR.

© Archives of Hellenic Medicine