Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories

Abstract

SummaryHighly contagious transboundary animal diseases such as foot‐and‐mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen‐side. Here, we report on a study to evaluate a fully automated cartridge‐based real‐time RT‐PCR diagnostic system (Enigma MiniLab®) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real‐time RT‐PCR (rRT‐PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter‐laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge‐based automated real‐time RT‐PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

Advances in the Diagnosis of Foot-and-Mouth Disease

“…Although highly reliable, RT-qPCR often requires laboratory setting with a qPCR thermocycler, a cost factor that limits its usage in the field. To overcome such limitation, on-site devices capable of performing FMDV diagnosis in the field have been developed, such as the fully automated cartridge-based RT-qPCR diagnostic system, Enigma MiniLab® ( 18 ). Another handheld RT-qPCR device, Biomeme two3™ (two3) has also been developed and evaluated as a field-deployable platform for FMDV diagnosis, in which the sensitivity was shown to be almost comparable to RT-qPCR using the ABI7500 platform ( 19 ).…”

“…Generally, the FMDV RNA for diagnostic purposes could be obtained from specimens, which include (i) swabs of oral, nasal, and lesion; (ii) epithelial tissue suspensions; and (iii) oral or vesicular fluid. In a recent study, Goller et al ( 18 ) explored milk as a non-invasive sample for FMDV surveillance using RT-qPCR. They demonstrated that the RT-qPCR on milk sample was capable of detecting FMDV RNA 18 days after contact, which is later than the viral RNA detected in serum samples, suggesting milk as a feasible sample for FMD surveillance ( 18 , 21 ).…”

“…In a recent study, Goller et al ( 18 ) explored milk as a non-invasive sample for FMDV surveillance using RT-qPCR. They demonstrated that the RT-qPCR on milk sample was capable of detecting FMDV RNA 18 days after contact, which is later than the viral RNA detected in serum samples, suggesting milk as a feasible sample for FMD surveillance ( 18 , 21 ). In addition, EDTA-treated blood samples have also been explored as a source of the viral RNA for the diagnosis of FMDV using RT-qPCR, owing to the samples' stability during transportation, as well as the ease of sample processing at the diagnostic laboratory ( 41 ).…”

Fully integrated microfluidic devices for qualitative, quantitative and digital nucleic acids testing at point of care

“…An ideal fully integrated microfluidic device for POCT should be ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and delivery to end users), enabling NAT implemented by non-professional personnel away from central laboratories in a short time. Recently, with the emerging miniaturized and equipment-free NAT technologies ( Li et al, 2020 ), various microfluidic devices consisting of NA extraction, NA amplification and signal readout ( Goller et al, 2018 ; Jovanovich et al, 2015 ) have been proposed to realize fully integrated NATs, which can be mainly classified by paper-based microfluidics ( Choi et al, 2016b ; Lafleur et al, 2016 ; Seok et al, 2017 ; Tang et al, 2017 ) and chip-based microfluidics ( Ganguli et al, 2017 ; Hajian et al, 2019 ; Oh et al, 2016 ; Yeh et al, 2017 ). For instance, the development of membrane-based NA extraction techniques, such as FTA card and FTA Elute card (Whatman), enables the integration of NA extraction process on chips ( Chen et al, 2019 ; Trinh et al, 2020 ; Trinh et al, 2019 ).…”

Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays

“…The assay is reliable, easy and cheap to perform, and readily transferable to standard FMD laboratories. Other methods have been developed for detection and characterisation of FMDV, including field-based detection of capsids and genomes [ 16 – 19 ]. Nevertheless, it is likely that antibody based ELISA will remain important for both FMD diagnosis and serotype differentiation for the foreseeable future.…”