Design of Molecular Diagnostic Assays for Detection of Dengue in Pakistan

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