Simultaneous detection of Dengue virus serotypes by nested-multiplex PCR

Dengue virus is a single positive RNA virus causing Dengue fever (break-bone fever), belonging to the Flavivirus in the family of Flaviviridae. Four antigenically distinct serotypes have been reported from this virus developing infection with asymptomatic or clinical signs of high fever, headache, body aches, nausea and rash in human.  This virus is mainly transmitted by mosquitoes Aedes aegypti or Aedes albopictus and in the last decade it has gained importance in the public health in Iran. It was reported that non-human primates, bovids, horses, pigs, birds, and rodents can be infected with this virus. Therefore, early detection of the virus in mosquitoes and humans can be important in controlling the disease. In the present study, we introduce a primer pair detecting Dengue virus (all four serotypes) and a nested-Multiplex PCR detecting each serotype simultaneously. For this aim we used recombinant plasmid constructs (D۱-, D۲-, D۳- and D۴- pEX-A۱۲۸ plasmid) having ca. ۶۶۰ nucleotides from ۵` end of the corresponding serotype and larvae of Aedes mosquito. Our results confirmed that the ۵` end of the virus is suitable for simultaneously detection of Dengue virus serotypes by RT-PCR and discrimination of serotypes from each other by multiplex PCR. In conclusion, we propose that to lower the cost, the Dengue virus should be preferably detected first by serotype common RT-PCR. In the case of positive result, the serotype determination can be performed by nested-multiplex PCR. Since for the management of public health, monitoring of the circulating Dengue virus is of great importance, the presented method is a suitable assay for this aim.

کلیدواژه ها:

DDengue virus RT ، PCR Multiplex PCR Serotypes RNA virus