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Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Rolando, Justin C. and Jue, Erik and Barlow, Jacob T. and Ismagilov, Rustem F. (2020) Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification. Nucleic Acids Research, 48 (7). Art. No. e42. ISSN 0305-1048. PMCID PMC7144905. https://resolver.caltech.edu/CaltechAUTHORS:20200302-155937790

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Abstract

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 HRM 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1093/nar/gkaa099DOIArticle
https://github.com/IsmagilovLab/Digital_NAAT_2Ch_MeltCurve_AnalyzerRelated ItemData
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144905PubMed CentralArticle
ORCID:
AuthorORCID
Rolando, Justin C.0000-0001-8948-319X
Jue, Erik0000-0001-7585-3794
Barlow, Jacob T.0000-0002-1842-4835
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:© 2020 The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received October 04, 2019; Revised January 08, 2020; Editorial Decision February 04, 2020; Accepted February 06, 2020. Published: 27 February 2020. Data Availability: The complete sequencing data generated during this study are available in the National Center for Biotechnology Information Sequence Read Archive repository with the BioProject ID: PRJNA574638. The MATLAB script described here has been deposited in the open-access online repository GitHub and may be accessed using the following direct link: https://github.com/IsmagilovLab/Digital_NAAT_2Ch_MeltCurve_Analyzer. This project benefited from the use of instrumentation at the Jim Hall Design and Prototyping Lab and the Millard and Muriel Jacobs Genetics and Genomics Laboratory. We thank Daan Witters and Pedro Ojeda for initial selection of CT primers, Eric Liaw for helpful discussions of error propagation and Natasha Shelby for help with writing and editing this manuscript. 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 to J.T.B.]; National Science Foundation Graduate Research Fellowships [DGE-1144469 to E.J.]; and the Joseph J. Jacobs Institute for Molecular Engineering for Medicine (Caltech). Funding for open access charge: DTRA [W15QKN-16-9-1002]. Conflict of interest statement. The content of this manuscript is the subject of a patent application filed by Caltech.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
Defense Threat Reduction Agency (DTRA)MCDC-18-01-01-007
Defense Threat Reduction Agency (DTRA)W15QKN-16-9-1002
Burroughs Wellcome FundUNSPECIFIED
NIH Predoctoral FellowshipT32GM112592
NSF Graduate Research FellowshipDGE-1144469
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Issue or Number:7
PubMed Central ID:PMC7144905
Record Number:CaltechAUTHORS:20200302-155937790
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200302-155937790
Official Citation:Justin C Rolando, Erik Jue, Jacob T Barlow, Rustem F Ismagilov, Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification, Nucleic Acids Research, 48:7, e42, https://doi.org/10.1093/nar/gkaa099
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101668
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:03 Mar 2020 00:09
Last Modified:02 Feb 2021 23:48

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