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Related Experiment Video

Updated: Mar 28, 2026

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Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay.

Khanate Sayasit1, Chutikarn Chaimayo1, Warinya Nuwong1

  • 1Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Biorxiv : the Preprint Server for Biology
|March 27, 2026
PubMed
Summary

A new alignment-free method identifies conserved viral signatures for broad-spectrum diagnostics. This approach enables the development of a sensitive RT-qPCR assay for Orthoflaviviruses like dengue virus, Zika virus, and Japanese encephalitis virus.

Keywords:
Alignment-freeOrthoflavivirusPrimer-ProbeRT-qPCR Assayk-mer

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Area of Science:

  • Virology
  • Molecular Diagnostics
  • Bioinformatics

Background:

  • The genus Orthoflavivirus, encompassing dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV), poses significant global health threats due to co-circulation and rapid expansion.
  • High nucleotide divergence (>25%-30%) within this genus complicates the development of inclusive molecular diagnostics using traditional methods.
  • Computational limitations of multiple sequence alignment hinder the detection of conserved motifs across large viral datasets.

Purpose of the Study:

  • To develop a novel alignment-free computational pipeline for identifying conserved diagnostic targets in highly variable viral genera.
  • To design and validate a broad-spectrum molecular assay for the simultaneous detection of key Orthoflaviviruses.
  • To establish a scalable computational framework for enhanced pandemic preparedness.

Main Methods:

  • A systematic alignment-free design pipeline utilizing k-mer analysis and compacted De Bruijn graphs was developed.
  • Analysis of 11,846 RefSeq viral genomes to identify phylogenetically conserved signatures within the Orthoflavivirus genus.
  • Design and validation of a TaqMan RT-qPCR assay targeting a conserved 600-bp region in the non-structural protein 5 gene.

Main Results:

  • A conserved 600-bp region within the non-structural protein 5 gene was identified, enabling broad-spectrum assay design.
  • The developed RT-qPCR assay demonstrated a limit of detection of 1-10 copies/µL for DENV1-4, ZIKV, and JEV with no cross-reactivity.
  • Clinical evaluation showed 97.33% overall accuracy, with 100% sensitivity and specificity for DENV serotypes and 100% specificity for ZIKV.

Conclusions:

  • The alignment-free, k-mer guided approach effectively uncovers conserved diagnostic targets in highly variable viral genera.
  • The developed broad-spectrum RT-qPCR assay is a robust tool for frontline surveillance of Orthoflaviviruses.
  • The computational framework offers a scalable solution for future pandemic preparedness and pathogen detection.