Suppressing Primer-Driven Nonspecific Amplification in LAMP Using TrueLAMP.

Area of Science:

• Molecular Biology
• Nucleic Acid Amplification
• Diagnostic Assays

Background:

• Loop-mediated isothermal amplification (LAMP) is a rapid nucleic acid detection method.
• Nonspecific amplification in LAMP can lead to false-positive results, limiting its diagnostic accuracy.
• There is a need for improved LAMP formulations that enhance specificity without compromising sensitivity.

Purpose of the Study:

• To develop and evaluate TrueLAMP, a novel colorimetric LAMP formulation.
• To assess TrueLAMP's ability to suppress nonspecific amplification while maintaining target-specific detection.
• To validate TrueLAMP's performance using SARS-CoV-2 as a model system.

Main Methods:

• TrueLAMP formulation incorporates a nonspecific amplification inhibitor.
• Colorimetric LAMP reactions targeting SARS-CoV-2 were performed using TrueLAMP polymerase and buffer.
• A two-step incubation protocol (60 °C for 10 min followed by 65 °C) was employed.
• Amplification was monitored colorimetrically and validated by agarose gel electrophoresis.

Main Results:

• TrueLAMP completely eliminated amplification in no-template controls across multiple primer sets, achieving 100% specificity.
• A limit of detection of 250 copies per reaction with 95% detection probability was achieved using the Lamb primer set.
• Stable endpoint colorimetric signals allowed for flexible readout without time-critical monitoring.

Conclusions:

• TrueLAMP effectively suppresses primer-driven nonspecific amplification, significantly improving the specificity of LAMP assays.
• The method demonstrates high sensitivity and specificity, making it suitable for accurate molecular diagnostics.
• TrueLAMP's compatibility with simple equipment and stable colorimetric readout supports decentralized and routine molecular testing.