Abstract
SummaryAccurate, timely diagnosis is essential for the control, monitoring and eradication of foot‐and‐mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple‐to‐use technologies, including molecular‐based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)‐specific reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) and real‐time RT‐PCR (rRT‐PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory‐based rRT‐PCR. However, the lack of robust ‘ready‐to‐use kits’ that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT‐PCR and RT‐LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real‐time, and for the RT‐LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.
Highlights
Foot-and-mouth disease (FMD) is a highly infectious vesicular disease affecting both domesticated and wild cloven-hooved animals
This study describes the lyophilization, laboratory validation and field testing in endemic settings (Tanzania and Kenya) of a previously published FMD virus (FMDV)-specific real-time RT-PCR (rRT-PCR) (Callahan et al, 2002; Madi et al, 2012) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay (Dukes et al, 2006; Waters et al, 2014)
To determine the effect of lyophilizing rRT-PCR reagents, a comparison between wet and lyophilized reagents was performed on a decimal dilution series of FMDV RNA in 0.1 lg/ll carrier RNA
Summary
Foot-and-mouth disease (FMD) is a highly infectious vesicular disease affecting both domesticated and wild cloven-hooved animals. Caused by FMD virus (FMDV), FMD affects over 100 countries worldwide, with disease distribution roughly reflecting economic development (Jamal and Belsham, 2013). The case-fatality rate of FMD is generally below 5%, the disease can be economically devastating: the annual global impact of FMD. Rapid Molecular Field Detection of FMDV in terms of production losses and vaccination in endemic regions alone is estimated between US$ 6.5 and 21 billion (Knight-Jones and Rushton, 2013). Endemic infection represents a constant threat for FMD-free countries, with outbreaks incurring severe economic losses: for example the UK 2001 outbreak is estimated to have cost the national economy US$ 9.2 billion (FAO, 2002). Identification of FMDV in susceptible host populations is essential to minimize the impacts of FMD. The development of technologies to provide rapid, sensitive and in situ FMD diagnosis is an ongoing research priority
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