Abstract:
Dengue disease is endemic in more than a hundred countries worldwide. The prevalent
situation in Pakistan is a similar challenge, with two serotypes predominantly occurring
in Karachi, Khyber Pakhtunkhwa, Punjab and Sindh. Assays for the amplification and
detection of dengue genome are based on the subtypes found globally, and in Pakistan,
kits imported from abroad are costly and non-specific to the particular strains prevalent
in the population. This study involved the retrieval of pol and NS3 sequences from
DENV2 serotype, and env sequences from DENV3 serotype using consensus sequences
occurring in Pakistan. Degenerate primers were designed and optimized to allow the
detection and diagnosis of these components in infected sera samples. Viral RNA was
extracted from the patient’s blood and converted to cDNA prior to amplification. The
primers were optimized to determine the most effective working temperatures and to
establish the assay’s validity. Degeneracy allowed for a wider scope of detection of
uncommon dengue sequences by providing multiple potential sites of complementarity.
The results of this study provide a means for the effective, inexpensive and specific
detection of dengue in Pakistan to help provide accurate clinical diagnosis and to
distinguish between dengue and other Flaviviridae with similar clinical presentations
circulating in the are
