Diagnosis and serotyping of dengue using cost-effective high-resolution melting curve based real time PCR

Abstract

Dengue virus (DENV) is classified into four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 which are genetically-related but antigenically distinct types. Although primary infection with a serotype of DENV gives lifelong immunity to that serotype, the secondary infection with other serotype leads to the development of dengue hemorrhagic fever and dengue shock syndrome. Serological tests that can detect NS1 antigen on the day one of illness is nonspecific and less sensitive to diagnose dengue in the first week of illness. IgM and IgG antibody detection may not be positive until the later part of first week. Hence it is necessary to develop a nucleic acid-based test kit for detection of DENV in the early phase of the disease such that the complications can be prevented. Simultaneous detection of serotype of DENV helps in understanding the epidemiology of the disease. In the present study, a single primer pair was designed to 3′ untranslated region of DENV genome. After amplification, the melting temperature (Tm) for DENV-1, DENV-2, DENV-3 and DENV-4 was found to be 83.96 ± 0.58 ˚C, 81.99± 0.26 ˚C, 84.54± 0.15 ˚C, and 83.28 ± 0.12 ˚C, respectively.