Development and Utilization of Multiplex pcr - high Resolution Melting (hrm) assay for Detection of Arboviral Pathogens

Abstract

Arthropod borne viruses (arboviruses) are transmitted by blood sucking arthropods. Disease outbreaks caused by arboviruses are sporadic and unpredictable. Arboviral disease outbreaks in Kenya have confirmed the presence of a number of arboviral diseases within the human and animal populace. Currently, there are no approved or specific therapies for arboviral infections; treatment is limited to palliative care. Early detection and vector control are important strategies in control of arboviruses. However, there is not yet a single detection system that is capable of detecting all arboviruses simultaneously. The development of realtime thermocyclers capable of high resolution melting (HRM) analysis has enabled development of rapid, sensitive and low-cost surveillance assays. Multiplex-RT-PCR with HRM analysis offers a comprehensive means for broad range arboviral screening. I optimized a multiplex-PCR-HRM assay using sets of primers that universally amplify arboviruses within genera and utilized the assay for detection of arboviral pathogens in biological samples. The assay was able to differentiate 16 distinct arbovirus positive controls and identify Bunyamwera, Sindbis, Usutu, Wesselsbron, and Mosquito Flaviviruses from field collected mosquitoes. Moreover, the assay identified co-infections and discovered novel arboviruses. The combination of this assay with the current molecular and culture Cmethodologies will provide powerful, rapid and cost effective diagnostic methodologies that are essential in surveillance and outbreak investigation programs in vectors, hosts and the reservoirs of arboviruses