Journal
NUCLEIC ACIDS RESEARCH
Volume 48, Issue 7, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkaa099
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Funding
- Defense Threat Reduction Agency (DTRA) Award [MCDC-18-01-01-007, W15QKN-16-9-1002]
- Burroughs Wellcome Fund Innovation in Regulatory Science Award
- National Institutes of Health Biotechnology Leadership Pre-doctoral Training Program (BLP) Fellowship [T32GM112592]
- National Science Foundation Graduate Research Fellowships [DGE-1144469]
- Joseph J. Jacobs Institute for Molecular Engineering for Medicine (Caltech)
- DTRA [W15QKN-16-9-1002]
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Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HAM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.
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