Abstract
In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.
Highlights
Multiple outbreaks of infectious diseases, including yellow fever, severe acute respiratory syndrome (SARS), Ebola, dengue, chikungunya, are occurring with increasing frequency
We demonstrated that the RT-loop-mediated isothermal amplification (LAMP) assay using primers targeting the Zika virus (ZIKV) capsid gene could detect multiple ZIKV Asian-lineages strains
We determined the limit of detection of the reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay using ten-fold serial dilutions of purified genomic RNA from 4 different ZIKV strains of the Asian-lineage
Summary
Multiple outbreaks of infectious diseases, including yellow fever, severe acute respiratory syndrome (SARS), Ebola, dengue, chikungunya, are occurring with increasing frequency. Kim and Hui Zhu) are employees of Rheonix, Inc. Rheonix, Inc. provided support in the form of salaries for authors RM, RY, BJK and HZ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the "author contributions" section
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