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

Abstract

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.

Keywords: Isothermal; KFD; Magnetic bead; PSR; Virus.

Fig 1

Reagent setup of simplified magnetic bead viral RNA extraction assay. This includes Lysis, washing and elution reagents in presence of magnetic field viral RNA is eluted

Fig 2

Schematics of simplified KFD detection protocol. The KFD samples processed for viral RNA isolation using simplified magnetic bead-based assay coupled with an isothermal RT-PSR assay with a visual color detection.

Fig 3

Reverse Transcription Polymerase Spiral Reaction. A) Lyophilized reagents B) Dry Bath C) Visual interpretation of results by adding SYBR Green Dye D) Visual interpretation of results by adding HNB dye

Fig 4

Sensitivity studies of Reverse Transcription Polymerase Spiral Reaction. Comparison of sensitivity of RT-PSR and visual detection (A) real-time kinetics of KFDV RT-PSR amplification of the env gene showing the amplification curve with serial 10-fold dilutions of the KFDV IVT RNA (10⁶ to 10 RNA copies/rxn), (B) naked eye visualization through HNB dye

Fig 5

Comparative evaluation of RT-PSR reagent stability in dry down formats with clinical samples.

Fig 6

Summary of comparative evaluation of whole methodology with clinical samples.