Development of single step RT-PCR for detection of Kyasanur forest disease virus from clinical samples

BACKGROUND: Kyasanur Forest Disease (KFD), a tick borne flavivirus, which was earlier endemic to Karnataka state, India, has been confirmed and detected from neighboring states of Tamil Nadu, Maharashtra, Goa and Kerala states in India. Increased human and vector surveillance therefore becomes essential for the identification of KFD affected regions and control of further spread of the disease. Currently, available KFD detection assays include realtime RT-PCR and nested RT-PCR assays. Here we describe the development of a sensitive single step RT-PCR assay for the detection of KFD viral RNA. This can be easily used in any BSL-2 laboratory for screening of KFD suspected cases or for differential diagnosis of viral hemorrhagic fever panel. METHOD: Three primer sets were designed and checked for sensitivity using known dilutions of KFD viral RNA (Ranging from 10(6) copies to 10 copies). The primer set (2) was found to be most sensitive was selected and tested for specificity for Kyasanur forest disease virus (KFDV) by testing against zika, dengue, chikungunya, crimean congo hemorrhagic fever (CCHF), yellow fever, japanese encephalitis (JE) and west nile viruses. A total of 104 samples (human, monkey and tick positive and negative samples) were tested using this assay. RESULT: No false positive or false negative results were seen for human, monkey or tick samples. The assay was specific for KFD and could detect upto 100 copies of KFD viral RNA. DISCUSSION AND CONCLUSION: The previously published sensitive real time RT-PCR assay requires higher cost in terms of reagents and machine setup and technical expertise has been the primary reason for development of this assay. A single step RT-PCR is relatively easy to perform and more cost effective than real time RT-PCR in smaller setups in the absence of Biosafety Level-3 facility. This study reports the development and optimization of single step RT-PCR assay which is more sensitive and less time-consuming than nested RT-PCR and cost effective for rapid diagnosis of KFD viral RNA.