Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips

TLDR

The recent Zika virus outbreak has created a need for rapid, easy‑to‑use, handheld, disposable diagnostics that deliver extreme sensitivity and specificity. To meet this demand, we developed a wax‑printed paper microfluidic chip that performs reverse transcription loop‑mediated isothermal amplification (RT‑LAMP). The chip’s paper type, pore size, and channel dimensions were optimized for filtration of direct‑use samples, and after RNA flowed to the detection zone it was excised, mixed with RT‑LAMP reagents and a pH indicator, then heated at 68 °C. The assay detected ZIKV in tap water, urine, and 10 % plasma within 15 minutes,.

Abstract

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.