Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings

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.

Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform

“…However, Ag‐LFDs have only been validated for use with epithelial suspension and vesicular fluid, and limited analytical sensitivity restricts their usefulness to the acute stage of infection (Ferris et al., , ). As a consequence, attempts to transfer highly sensitive rRT‐PCR assays onto portable detection platforms have been investigated (Hearps et al., ; Howson et al., ; Madi et al., ; Monwina et al., ; Paixão et al., ), but are either not commercially available or are only suitable for research purposes (i.e., not diagnostic use). This study evaluates the performance of a commercially available, lyophilized FMDV pan‐serotype‐specific assay, in combination with a commercially available portable thermocycler, in laboratory and East African field settings.…”

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus

“…In contrast, PCR‐based assays such as RRT‐PCR assays are highly sensitive (>95%) and specific and therefore widely used for routine laboratory‐based detection of FMDV. Over the last decade, a number of attempts were made to transfer the laboratory‐based RRT‐PCR assays to portable PCR equipment that can be operated in the field (Callahan et al., ; Hearps et al., ; Risatti et al., ; Tomlinson et al., ; Rasmussen et al., ; Takekawa et al., ; Madi et al., ; Molsa et al., , ; Liu et al., ; Howson et al., ). These portable PCR instruments, however, depend on precision thermocycling, are highly sophisticated and therefore expensive and delicate for field use.…”

“…FMD outbreaks have to be quickly detected and contained, and therefore, onsite detection tools are highly desirable. To date, a number of field‐deployable molecular assays have been developed and evaluated for their potential to use in the field to support local decision‐making (Dukes et al., ; Chen et al., ,b; Madi et al., ; Abd El Wahed et al., ; Yamazaki et al., ; Kasanga et al., ; Ranjan et al., ; Waters et al., ; Howson et al., ), and some of them have been successfully tested in the field (Madi et al., ; Abd El Wahed et al., ; Howson et al., ). To our knowledge, none of those assays are commercially available yet.…”

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus

“…() and Howson et al. (); the lyophilization of the assay reagents, as shown by Howson et al. (); and the development of an alternative visualization strategy as described by Waters et al.…”