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

Abstract

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems.

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

“…The iiPCR technology and a commercially available field-deployable device (POCKIT™ combo system), which includes a taco™ mini Automatic Nucleic Acid Extraction System and a POCKIT™ Nucleic Acid Analyzer (GeneReach USA, Lexington, MA, USA), allow automatic detection and interpretation of PCR results within 1-1.5 h [36]. It has been shown that iiPCR or RT-iiPCR assays have excellent sensitivity and specificity for the detection of various targets, including swine pathogens, such as classical swine fever virus (CSFV), FMDV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV) [38][39][40][41], and various pathogens in shrimp, dogs, cats, poultry, ruminants, and horses [42][43][44][45][46][47][48][49][50][51][52].…”

Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus

“…1]). A number of iiPCR-based assays have been developed for detection of human and animal pathogens [56,57,[59][60][61][62][63][64][65][66][67][68][69][70] with two of the most recent additions being directed against all serotypes of DENV and MERS-CoV [56,58]. The sensitivity and specificity of all iiPCR-based assays have demonstrated to be comparable with other diagnostic methods currently in use (e.g.…”

“…in human specimens [56][57][58]. The iiPCR is highly sensitive and specific for the detection of both DNA and RNA not only from human, but also various animal pathogens [59][60][61][62][63][64][65][66][67][68][69][70]. The PCR reaction (denaturation, annealing, and extension) is accomplished in a capillary vessel (R-tube™; GeneReach USA, Lexington, MA, USA) heated through the bottom end of the tube where, based on the Rayleigh-Bénard convection principle, the fluids cycle through temperature gradients.…”

A field‐deployable insulated isothermal RT‐PCR assay for identification of influenza A (H7N9) shows good performance in the laboratory

“…This system has recently been developed to rapidly detect pathogenic viruses, including white spot syndrome virus, classical swine fever, foot and mouth disease, equine influenza, bluetongue virus, and MERS-CoV. [5][6][7][8][9][10] Taking advantage of the iiRT-PCR portable system, the aim of this study was to validate the performance of the iiRT-PCR portable system for H7N9 detection.…”