Development of a PCR Test System for the Genetic Diagnosis of Hemorrhagic Fever With Renal Syndrome

Hemorrhagic fever with renal syndrome (HFRS) is an acute zoonotic disease caused by orthohantaviruses. Reliable molecular diagnostics are essential for early detection and epizootiological surveillance. This study aimed to develop and validate a reverse transcription quantitative real-time PCR (RT-qPCR) assay for detection of HFRS-associated orthohantavirus RNA.Species-specific primers (HV P1-F and HV P1-R) and a TaqMan probe (HV P1) targeting the nucleocapsid protein (N) gene within the S segment were designed. A plasmid positive control containing a 242-bp target fragment was constructed. Analytical sensitivity, specificity, reproducibility, and repeatability were evaluated. Field testing included samples from 343 small mammals collected in endemic areas of the West Kazakhstan Region. The assay consistently detected the plasmid control from 1 ng/µL to 1 fg/µL, with Ct values of 37.35 and 37.73 at 1 fg/µL. No specific amplification was observed at 100 ag/µL. A strong linear relationship between Ct values and template concentration was obtained (R² = 0.998). No false-positive reactions occurred with tick-borne encephalitis virus or Crimean-Congo hemorrhagic fever virus RNA. Reproducibility and repeatability showed 100% concordance of qualitative results. HFRS-associated orthohantavirus RNA was detected in 45 of 123 pooled field samples. The minimum infection prevalence was 13.1%, reaching 15.3% in Myodes glareolus and 13.3% in Apodemus uralensis and Microtus arvalis. The developed one-step RT-qPCR assay demonstrated high sensitivity, specificity, and reproducibility and is suitable for routine diagnostics and surveillance of natural HFRS foci.