Mechanistically Guided Combinatorial Coding Based on Strand Displacement Probes for Multiplex Pathogen Variant...

Area of Science:

• Molecular Diagnostics
• Genetics
• Bioengineering

Background:

• Accurate discrimination of closely related genetic variants at single-nucleotide resolution is challenging, especially in multiplexed assays.
• Existing methods often involve complex procedures and high costs for identifying multiple genetic variants simultaneously.

Purpose of the Study:

• To develop a novel, mechanistically guided strategy for combinatorial color-coded identification of genetic variants.
• To establish a scalable and cost-effective platform for molecular diagnostics and pathogen surveillance.

Main Methods:

• A Color-Coding Strategy for Multiple Variants Based on Single-Mutation-Responsive Strand-Displacement Padlock Probes was developed.
• The strategy integrates isothermal amplification reactions (n) with multichannel strand displacement probes (m) to create an n-dimensional coding framework.
• A programmable algorithm was used for automatic decoding of variant-specific 'n-color codons'.

Main Results:

• The system successfully identified up to 15 SARS-CoV-2 variants in a single assay using only three reaction tubes.
• The assay demonstrated high performance in clinical evaluation, achieving 100% positive agreement with RT-qPCR and correctly identifying variant identities in 94.7% of samples.
• The platform showed reduced reaction numbers, experimental complexity, and cost compared to conventional methods.

Conclusions:

• The developed color-coding strategy enables efficient and accurate identification of multiple genetic variants with minimal reactions.
• The modular and programmable design makes the platform adaptable to emerging variants, showing potential for pathogen surveillance and genetic analysis.
• This approach offers a significant advancement in scalable and cost-effective molecular diagnostics.