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High Fidelity Machine-Learning-Assisted False Positive Discrimination in Loop-Mediated Isothermal Amplification Using
Ming Dong1, Aneesh Kshirsagar1, Anthony J Politza2
1Department of Electrical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802, United States.
ACS Nano
|February 23, 2024
Summary
This study presents a nanopore-based method for accurate nucleic acid detection using loop-mediated isothermal amplification (LAMP). The probe-free system enhances LAMP reliability by distinguishing true positives from false positives through amplicon sizing and counting.
Area of Science:
- Biotechnology
- Molecular Biology
- Nanotechnology
Background:
- Loop-mediated isothermal amplification (LAMP) is a valuable nucleic acid detection method for point-of-care applications.
- LAMP assays can be compromised by false positives arising from contamination, nonspecific amplification, or signal reporting.
- Existing methods to improve specificity, such as dye-labeled probes, risk inhibiting the reaction.
Purpose of the Study:
- To develop a probe-free nanopore-based system for LAMP readout.
- To differentiate true LAMP amplicons from false positives using amplicon sizing and counting.
- To enhance the specificity and reliability of LAMP assays.
Main Methods:
- Developed a kinetic model to analyze LAMP reaction patterns.
- Utilized gel electrophoresis to distinguish true from false positive amplicon profiles.
- Implemented nanopore sensing for amplicon sizing and counting, analyzing event charge deficit (ECD) and frequencies.
- Integrated machine learning algorithms for false positive discrimination.
Main Results:
- Distinct kinetic and amplicon size patterns were identified between true and false LAMP positives.
- The nanopore-based sizing and counting method, coupled with machine learning, achieved 91.67% accuracy in discriminating false positives.
- This approach offers a reliable, probe-free readout for LAMP assays.
Conclusions:
- Nanopore-based amplicon sizing and counting provide a robust method for enhancing LAMP specificity.
- This probe-free system overcomes the limitations of dye-based detection, avoiding reaction inhibition.
- The developed system significantly improves the reliability of nucleic acid detection using LAMP technology.
