A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray

Area of Science:

• Virology
• Molecular Biology
• Public Health

Background:

• The genus *Flavivirus* comprises significant arthropod-borne viruses impacting human and animal health.
• Existing specific RT-PCR assays necessitate a broad-range method for comprehensive flavivirus detection.
• Balancing genus-level specificity with species-level sensitivity is a key challenge in flavivirus diagnostics.

Purpose of the Study:

• To develop a novel, broad-range assay for detecting all *Flavivirus* species.
• To validate the assay's sensitivity and specificity across diverse flaviviruses.
• To establish a reliable tool for screening and confirmation of flavivirus infections.

Main Methods:

• Development of a SYBR Green-based reverse transcription quantitative PCR (RT-qPCR) assay.
• Integration of RT-qPCR with a low-density DNA microarray containing 84 oligonucleotide probes for all known *Flavivirus* species.
• Validation using 26 different *Flavivirus* strains and application in External Quality Assessment studies.

Main Results:

• The RT-qPCR demonstrated roughly equal sensitivity for detecting and quantifying all tested flaviviruses.
• The combined assay successfully screened and confirmed *Flavivirus* presence in laboratory and field samples.
• Performance was equivalent or superior to existing species-specific RT-PCRs, as shown in External Quality Assessment studies.

Conclusions:

• The novel RT-qPCR-microarray assay provides a sensitive and specific tool for broad-range flavivirus detection.
• This assay is suitable for screening and confirmation of flaviviruses in various sample types.
• The method represents an advancement in flavivirus diagnostics, addressing the need for comprehensive surveillance.