Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics

Abstract

As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18–25 °C) for 1–2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q 10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.

Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19

“…However, a recent report introduced a point-of-care PCR mix designed for room temperature use, displayed in a microfluidic format with a more complex formulation comprising trehalose, mannitol, and PEG 20,000. This formulation demonstrated stability for 1–2 years at room temperature 21 . The optimal lyophilization time emerged as a crucial variable, underscored as a pivotal component within the protocol.…”

Development and evaluation of lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19

“…Despite the important results observed, these papers showed no lyophilization protocol screening other than trehalose concentration, such as other cryopreservative molecules and freezing-time. A recent report showed a point-of-care and roomtemperature PCR mix in a micro uidic display with a more complex formulation using trehalose, mannitol and PEG 20,000, stable for 1-2 years in room-temperature 22 . The ideal lyophilization time proved to be an important variable since it was identi ed as an important part of the process in the protocol.…”

Electrochemical Diffusion Study in Poly(Ethylene Glycol) Dimethacrylate-Based Hydrogels

“…To make microfluidics operational for various applications in molecular diagnostics, the integration of reagents is a fundamental challenge. Various concepts have been pursued for this purpose, such as blister bags, freeze-dried pellets, functionalized beads, sponges, paper strips, biopolymers, and hydrogels [ 14 , 15 , 16 , 17 , 18 ]. For the large-scale production of microfluidics, automated pick-and-place tools, lamination systems, and spotting and printing technologies have been developed for the assembly process [ 14 ].…”