Simplified visual detection of Kyasanur Forest Disease virus employing Reverse Transcriptase-Polymerase Spiral Reaction (RT-PSR)

Among recently prevalent tick-borne infections in India, Kyasanur Forest Virus Disease (KFD) is an important public health concern. During last decade the emergence of cases apart from endemic zone raised concern about case positivity. Early diagnosis is therefore very important in disease management and primary containment. This study, aimed to develop a simplified viral RNA extraction in combination to dry down format of novel isothermal assay for (Reverse Transcription- Polymerase Spiral reaction) specific and rapid identification of Kyasanur Forest Disease Virus targeting viral envelope gene. The one step method was optimized by magnetic bead based viral RNA extraction followed by isothermal RT-PSR assay in heat bath at 63⁰C for 60 minutes. Further, visual results interpretation was done by color change of Hydroxy Naphthol Blue dye. The detection limit of the assay was found 10 RNA copies/rxn with comparable to silica column based viral RNA combined real time qPCR. No cross reactivity was observed with other closely related flaviviruses. The assay was evaluated with clinical samples has shown >99% concordance between two methods. This is the first report of sample extraction coupled isothermal detection of KFD in a simplified manner without a need of any hi-end equipment. The assay developed here has potential to use as an alternate for field-based detection in resource limited settings for KFD.