A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses

Area of Science:

• Molecular Diagnostics
• Virology
• Bioengineering

Background:

• RNA viruses, such as Newcastle disease virus (NDV), pose diagnostic challenges due to high mutation rates and genomic variability.
• Traditional methods for avian virus detection (e.g., RT-PCR) often lack the required specificity and sensitivity.
• Padlock probes (PLPs) combined with Rolling Circle Amplification (RCA) offer a promising platform for versatile nucleic acid detection.

Purpose of the Study:

• To develop a novel padlock probe (PLP) design strategy for detecting hypervariable RNA viruses, specifically Newcastle disease virus (NDV).
• To create a sensitive and specific assay capable of tolerating mutations in viral genomes.
• To enable multiplex detection of multiple poultry RNA viruses and facilitate digital quantification.

Main Methods:

• Designed nine padlock probes (PLPs) targeting conserved regions with degenerate bases, based on 335 NDV sequences (Class I and II).
• Evaluated PLP performance against eight out of ten Class II NDV genotypes.
• Extended the assay for simultaneous detection of NDV, infectious bronchitis virus (IBV), and avian influenza virus (AIV), coupled with paper-based microfluidic digital quantification.

Main Results:

• The novel PLP design demonstrated high coverage and specificity for detecting multiple NDV genotypes.
• Achieved a low detection limit of less than ten copies of viral RNA.
• Successfully developed a multiplex assay for simultaneous detection of three key poultry RNA viruses (NDV, IBV, AIV).

Conclusions:

• The developed PLP design strategy effectively addresses the challenge of detecting highly mutable emerging virus strains with enhanced sensitivity and specificity.
• This approach offers a robust platform for molecular diagnostics of variable RNA viruses.
• The integration with paper-based microfluidics enables sensitive digital quantification for improved pathogen surveillance.