Research Article: Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva

Date Published: February 5, 2018

Publisher: Public Library of Science

Author(s): Maite Sabalza, Rubina Yasmin, Cheryl A. Barber, Talita Castro, Daniel Malamud, Beum Jun Kim, Hui Zhu, Richard A. Montagna, William R. Abrams, Ruslan Kalendar.


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.

Partial Text

Multiple outbreaks of infectious diseases, including yellow fever, severe acute respiratory syndrome (SARS), Ebola, dengue, chikungunya, are occurring with increasing frequency. At least three major factors have influenced the increasing incidence of infectious disease epidemics: population concentration at the epicenter of an infection, the underlying characteristics and vulnerabilities of the population at the epicenter [1] and extensive air travel to spread the disease globally [2–5]. The most recent is Zika virus (ZIKV), which is an emerging arbovirus in the genus Flavivirus and family of Flaviviridae, related to yellow fever, dengue, West Nile, Japanese encephalitis, and tick-borne encephalitis viruses. Zika virus is transmitted among humans primarily by mosquito vectors (Aedes spp., including Ae. aegypti and Ae. Albopictus) [6–8] and humans serve as the primary amplification hosts in areas where there are no non-human primates [9]. ZIKV was first identified in Uganda in 1947, and since then outbreaks have been recorded in Africa, Southeast Asia, Pacific with the last one in Brazil in early 2015, which spread to other countries in Central and South America, the Caribbean, and the Southern United States [10–12]. In all these areas autochthonous and travel associated infection has been reported [6–8, 13].

The recent ZIKV outbreak is an example of the course of emerging infectious diseases. ZIKV infection had been considered to only be a modest public health concern with only local outbreaks [31, 32]. However reports from the recent outbreak in Brazil and the Americas during 2015–2016, indicated that infections during pregnancy are associated with microcephaly and other neurological disorders in newborns and Guillain-Barré Syndrome (GBS) in adults pointing to potentially more serious health impacts [33, 34]. On 1 February 2016, the World Health organization (WHO) declared ZIKV and its link to birth defects a public health emergency of international concern and it lasted during 10 months [35]. ZIKV diagnosis is a challenge because it shares vectors, geographic distribution and symptoms with Dengue virus and Chikungunya infection [36] and the three illnesses are often misdiagnosed. Given the risk for adverse pregnancy outcomes in women infected with ZIKV during pregnancy, it is particularly important to distinguish between the 3 viruses. This recent ZIKV outbreak confirm that we need an effective surveillance and diagnostic program to reduce the impact of future emerging infectious diseases. Towards the goal of increasing the speed of making effective diagnostics available, we have developed a generic protocol that can be adapted for the next emerging or remerging disease.




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