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Real-Time, Digital LAMP with Commercial Microfluidic Chips Reveals the Interplay of Efficiency, Speed, and Background Amplification as a Function of Reaction Temperature and Time

Rolando, Justin C. and Jue, Erik and Schoepp, Nathan G. and Ismagilov, Rustem F. (2019) Real-Time, Digital LAMP with Commercial Microfluidic Chips Reveals the Interplay of Efficiency, Speed, and Background Amplification as a Function of Reaction Temperature and Time. Analytical Chemistry, 91 (1). pp. 1034-1042. ISSN 0003-2700. PMCID PMC6322147. https://resolver.caltech.edu/CaltechAUTHORS:20181221-105356677

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Abstract

Real-time, isothermal, digital nucleic acid amplification is emerging as an attractive approach for a multitude of applications including diagnostics, mechanistic studies, and assay optimization. Unfortunately, there is no commercially available and affordable real-time, digital instrument validated for isothermal amplification; thus, most researchers have not been able to apply digital, real-time approaches to isothermal amplification. Here, we generate an approach to real-time digital loop-mediated isothermal amplification (LAMP) using commercially available microfluidic chips and reagents and open-source components. We demonstrate this approach by testing variables that influence LAMP reaction speed and the probability of detection. By analyzing the interplay of amplification efficiency, background, and speed of amplification, this real-time digital method enabled us to test enzymatic performance over a range of temperatures, generating high-precision kinetic and end-point measurements. We were able to identify the unique optimal temperature for two polymerase enzymes while accounting for amplification efficiency, nonspecific background, and time to threshold. We validated this digital LAMP assay and pipeline by performing a phenotypic antibiotic susceptibility test on 17 archived clinical urine samples from patients diagnosed with urinary tract infections. We provide all the necessary workflows to perform digital LAMP using standard laboratory equipment and commercially available materials. This real-time digital approach will be useful to others in the future to understand the fundamentals of isothermal chemistries, including which components determine amplification fate, reaction speed, and enzymatic performance. Researchers can also adapt this pipeline, which uses only standard equipment and commercial components, to quickly study and optimize assays using precise, real-time digital quantification, accelerating development of critically needed diagnostics.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acs.analchem.8b04324DOIArticle
https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.8b04324PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322147PubMed CentralArticle
ORCID:
AuthorORCID
Rolando, Justin C.0000-0001-8948-319X
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:© 2018 American Chemical Society. ACS AuthorChoice - This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Received: September 21, 2018; Accepted: December 5, 2018; Published: December 19, 2018. This research was supported in part by the Burroughs Wellcome Fund Innovation in Regulatory Science Award (to R.F.I.) and a grant from the Jacobs Institute for Molecular Engineering for Medicine. Research reported in this publication was also supported by the Department of Health and Human Services (HHS) Office of the Assistant Secretary for Preparedness and Response (ASPR) and the Wellcome Trust under the CARB-X program (federal award no. IDSEP160030-02); the content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of HHS Office of the ASPR. This work is funded in part by CARB-X as a collaboration between Talis Biomedical Corporation and Caltech. This project benefited from the use of instrumentation at the Jim Hall Design and Prototyping Lab at the California Institute of Technology. We thank Travis Schlappi for performing the extractions of the archived clinical samples and Natasha Shelby for contributions to writing and editing this manuscript. Author Contributions: All authors have given approval to the final version of the manuscript. The authors declare the following competing financial interest(s): The technology described in this publication is the subject of a patent application filed by Caltech. R.F.I. has a financial interest in Talis Biomedical Corporation.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
Burroughs Wellcome FundUNSPECIFIED
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Department of Health and Human ServicesIDSEP160030-02
Wellcome TrustUNSPECIFIED
Talis Biomedical CorporationUNSPECIFIED
Issue or Number:1
PubMed Central ID:PMC6322147
Record Number:CaltechAUTHORS:20181221-105356677
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20181221-105356677
Official Citation:Real-Time, Digital LAMP with Commercial Microfluidic Chips Reveals the Interplay of Efficiency, Speed, and Background Amplification as a Function of Reaction Temperature and Time. Justin C. Rolando, Erik Jue, Nathan G. Schoepp, and Rustem F. Ismagilov. Analytical Chemistry 2019 91 (1), 1034-1042. DOI: 10.1021/acs.analchem.8b04324
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91953
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:21 Dec 2018 19:05
Last Modified:03 Oct 2019 20:39

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