Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Virology

Background:

• Current COVID-19 variant detection methods like sequencing and PCR have limitations.
• Sequencing is costly and time-consuming.
• PCR methods can suffer from low sensitivity and background interference.

Purpose of the Study:

• To develop a novel, highly sensitive, and specific method for detecting single-base mutations in viral variants.
• To address the limitations of existing diagnostic techniques for rapid and accurate viral variant identification.

Main Methods:

• Development of a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy.
• Integration of nicking endonuclease, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES).
• Establishment of a luminescence platform for specific mutation detection.

Main Results:

• Achieved a detection limit of 2.01 × 10^5 copies/μL for COVID-19 D796Y substitution.
• Demonstrated excellent specificity with a variant/wild-type ratio < 0.0625%.
• Successfully detected mutations in biological samples (throat, blood) and identified multiple mutations (D614G, H69/V70del) without interference.

Conclusions:

• The LM-IR-INA strategy provides a highly sensitive and specific approach for viral variant detection.
• The developed TRES-based method is superior to steady-state techniques and applicable to complex biological samples.
• This strategy shows potential as a universal platform for detecting various viral mutations.