Abstract
The recent Zika virus (ZIKV) outbreak has prompted the need for field-ready diagnostics that are rapid, easy-to-use, handheld, and disposable while providing extreme sensitivity and specificity. To meet this demand, we developed a wax-printed paper microfluidic chip utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). The developed simple and sensitive ZIKV assay was demonstrated using undiluted tap water, human urine, and diluted (10%) human blood plasma. Paper type, pore size, and channel dimension of various paper microfluidic chips were investigated and optimized to ensure proper filtration of direct-use biological samples (tap water, urine, and plasma) during capillary action-driven flow. Once ZIKV RNA has flowed and reached to a detection area of the paper microfluidic chip, it was excised for the addition of an RT-LAMP mixture with a pH indicator, then placed on a hot plate at 68 °C. Visible color changes from successful amplification were observed in 15 minutes and quantified by smartphone imaging. The limit of detection was as low as 1 copy/μL. The developed platform can also be used for identifying other flaviviruses, such as Chikungunya virus (CHIKV) and Dengue virus (DENV), and potentially other quickly transmitted virus pathogens, towards field-based diagnostics.
Highlights
To facilitate and improve field-ready Zika virus (ZIKV) diagnostics, we aim to demonstrate reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) on a simple and disposable platform, coupled with a pH indicator-based colorimetric assay, a simple hot plate, and subsequent smartphone detection, forgoing an excitation light source, optical filters, and a closed container
65 °C, 68 °C and 70 °C, were investigated using a conventional thermocycler
At 68 °C, the target template was successfully amplified from the target-contained sample, with no amplification from the no target control (NTC)
Summary
Rather than splitting the extended products at high denaturation temperature in preparation for the cycle, loop structures are formed at the end of the amplified products, allowing the stage of primer annealing and subsequent extension, all occurring at the same temperature It is highly specific (4–6 vs 2 primers) and requires only a single temperature (typically 60–70 °C), enabling the process to be simple and rapid[9]. A simpler type of dye is needed that is affected by neither ambient lighting perturbations nor the presence of other amplification-inhibiting molecules in the sample For this purpose, pH indicator-based colorimetric assay kits could be used (e.g., WarmStart colorimetric LAMP master mix by New England Biolabs), which includes a pH indicator (phenol red) in the master mix reagent, requiring no additional step, no excitation light source, and no closed container (identifiable under ambient light)[14]. RT-LAMP reactions were still conducted in a conventional, laboratory-based manner in these studies, making them difficult to be used for field applications
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