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

Area of Science:

• Molecular Diagnostics
• Genetics
• Biotechnology

Background:

• Distinguishing closely related genetic variants at single-nucleotide resolution is difficult in molecular diagnostics, especially in multiplexed assays.
• Existing methods often require complex and numerous parallel reactions.

Purpose of the Study:

• To develop a Color-Coding Strategy for Multiple Variants Based on Single-Mutation-Responsive Strand-Displacement Padlock Probes (CCMVS) for combinatorial genetic variant identification.
• To enable accurate and scalable discrimination of genetic variants with minimal parallel reactions.

Main Methods:

• Integrated multiple isothermal amplification reactions (n) with multichannel strand displacement probes (m) to create an n-dimensional coding framework.
• Utilized "n-color codons" for unique variant encoding and a programmable algorithm for automatic decoding.
• Applied the CCMVS strategy to identify SARS-CoV-2 variants in various sample types, including clinical nasopharyngeal swabs.

Main Results:

• Successfully identified up to 15 SARS-CoV-2 variants using only three reaction tubes.
• Achieved 100% positive agreement with RT-qPCR for SARS-CoV-2 positive specimens.
• Assigned variant identities to 94.7% of clinical samples (72 out of 76).

Conclusions:

• The CCMVS platform offers reduced reaction numbers, experimental complexity, and cost compared to conventional methods.
• Its modular and programmable design allows for adaptability to emerging variants.
• Demonstrates significant potential for pathogen surveillance and broad genetic variant analysis.