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Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

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Abstract

Nucleic acid amplification test (NAAT)-based point-of-care (POC) devices are rapidly growing for use in low-resource settings. However, key challenges are the ability to store the enzyme-based reagents in dry form in the device and the long-term stability of those reagents at elevated temperatures, especially where ambient temperatures could be as high as 45 °C. Here, we describe a set of excipients including a combination of trehalose, polyethylene glycol and dextran, and a method for using them that allows long-term dry storage of enzyme-based reagents for an isothermal strand displacement amplification (iSDA) reaction in a porous matrix. Various porous materials, including nitrocellulose, cellulose, and glass fiber, were tested. Co-dried reagents for iSDA always included those that amplified the ldh1 gene in Staphylococcus aureus (a polymerase and a nicking enzyme, 4 primers, dNTPs and a buffer). Reagents also either included a capture probe and a streptavidin-Au label required for lateral flow (LF) detection after amplification, or a fluorescent probe used for real-time detection. The reagents showed the best stability in a glass fiber matrix when stored in the presence of 10% trehalose and 2.5% dextran. The reagents were stable for over a year at ∼22 °C as determined by lateral flow detection and gel electrophoresis. The reagents also exhibited excellent stability after 360 h at 45 °C; the assay still detected as few as 10 copies of ldh1 gene target by lateral flow detection, and 50 copies with real-time fluorescence detection. These results demonstrate the potential for incorporation of amplification reagents in dry form in point-of-care devices for use in a wide range of settings.

Graphical abstract: Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

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Supplementary files

Article information


Submitted
02 Jun 2020
Accepted
12 Aug 2020
First published
19 Aug 2020

Analyst, 2020, Advance Article
Article type
Paper

Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

S. Kumar, R. Gallagher, J. Bishop, E. Kline, J. Buser, L. Lafleur, K. Shah, B. Lutz and P. Yager, Analyst, 2020, Advance Article , DOI: 10.1039/D0AN01098G

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