Table 1.
Molecular diagnostic assays for detection of FMDV infection.
| Methods | Description | Detection limit | Tested clinical samples | References |
|---|---|---|---|---|
| RT-PCR | Amplification of genome fragments from IRES to the end of the ORF, followed by the Sanger sequencing for serotyping | • 5 log10 dilution of viral RNA of FMDV O/MOG/7/2010 | Bovine saliva, porcine podal vesicle, ovine saliva, caprine serum samples | (10) |
| An RT-PCR based on a novel primer (FM8/9) targeting the 3D region of the FMDV | • 100.2 to 10−2.8 TCID50/mL of FMDV depending on the strains | Serum, and saliva samples from pigs and cows | (11) | |
| RT-iiPCR | Utilizes a commercially available compact, portable POCKIT™ Nuclei Acid Analyser (GeneReach, USA) for rapid (<2 h) detection of all seven serotypes of FMDV. Coupling to the taco™ mini extraction kit (GeneReach, USA), the detection assay detected 63 different FMDV strains representing all seven serotypes. Detection of the FMDV RNA from vesicular fluid samples is possible without nuclei acid extraction | • ≥9 copies of in vitro–transcribed FMDV O1 Manisa/69 3D RNA | Nasal and oral swabs from calves, sheep, and piglets, oral fluid, epithelial tissues, and vesicular fluids from piglets | (12) |
| RT-ddPCR | Targets FMDV 3D region based on the water-oil emulsion droplet technology for partition of nanoliter droplets containing viral cDNA. This assay enables absolute quantification of the FMDV RNA without the need of reference standards. Detects serotypes O, A, and C | • 101.4 TCID50/mL and 26.5 copies of viral RNA determined using FMDV A24 Cruzeiro and a plasmid containing the 3D-FMDV sequences, respectively | Epithelium and esophageal–pharyngeal fluids of bovine origin | (13) |
| RT-RPA | An assay based on isothermal DNA amplification and the use of combinatory enzymes and proteins. The assay was reported to detect 3D RNA of FMDV within 4–10 min | • 1,436 copies of in vitro–transcribed FMDV 3D RNA •Diagnostic sensitivity: 98% |
Heart, blood, serum, milk, saliva, and vesicular samples from cattle, buffaloes, and sheep | (14) |
| RT-qPCR | A multiplex assay targeting the VP1 region of FMDV for specific and simultaneous detection of FMDV of O, A, and Asia 1 circulating in the Middle East | • 1.78 to 2.74 copies of in vitro–transcribed FMDV RNA depending on serotypes | Vesicular epithelium, saliva, and heart tissue homogenates from animals | (15) |
| Addition of 5′-tails to the primers targeting 3D and 5′ UTR region of FMDV was demonstrated to enhance detection of FMDV. The RT-qPCRs using the tailed forward and reverse primers targeting 3D and 5′ UTR were performed in parallel in a triplex one-step protocol. Both assays detected all seven serotypes of FMDV with enhanced overall performance | • The detection limit of RT-qPCR (with tailed primers) targeting 3D and 5′ UTR of FMDV are −0.72 and −0.35 log10 TCID50/mL of FMDV O1 Manisa, respectively | Serum samples from cattle, pigs, and sheep | (16) | |
| An RT-qPCR assay based on the SYBR green I dye for detection of FMDV of all seven serotypes. Primers used in this assay were carefully selected using multiple in silico approaches to enhance amplification efficiency | • 1–10 copies/μL of in vitro–transcribed FMDV RNA depending on the target regions | Epithelium and vesicular fluid samples from cattle | (17) | |
| A pen-side, fully automated diagnostic tool (Enigma MiniLab®) which integrate both nucleic acid extraction and downstream RT-qPCR for rapid detection (<1.5 h) of FMDV. The assay was shown to produced comparable results to the standard RT-qPCR assay recommended by the OIE | • 10−5 to 10−6 dilution of the FMDV O/UAE 2/2003 stock depending on the nuclei acid extraction kits. | Saliva and epithelium of bovine, porcine, and ovine origin. Milk (from bovine) spiked with FMDV | (18) | |
| A field-deployable RT-qPCR-based diagnostic system (Biomeme two3™) with Biomeme proprietary nucleic acid extraction kit (M1) for rapid detection of FMDV, within 30–60 min. This system was reported to detect isolates representing six serotypes (O, A, Asia 1, SAT 1, SAT 2, and SAT 3) of FMDV. Detection of FMDV RNA in various samples was possible without nucleic acid extraction steps, but at lower sensitivity | • 10−4, 10−3, 10−2, 10−5, 10−3, and 10−3 dilutions of FMDV O, A, Asia 1, SAT 1, SAT 2, and SAT 3 stocks, respectively | Serum, vesicular fluid, tissue suspension, oral fluid, oral, and nasal swab samples from sheep, pigs, and cattle | (19) | |
| This study compared the performance of two commercially available one step RT-qPCR systems, TaqMan® Fast Virus 1-Step Master Mix (Applied Biosystems®) and Superscript III Platinum® One-Step qRT-PCR Kit (Invitrogen™) in detection of FMDV RNA from milk samples, a non-invasive alternative for detection and typing of FMDV | • The detection limit of Superscript III Platinum® One-Step qRT-PCR Kit and TaqMan® Fast Virus 1-Step Master Mix are 10−6 and 10−5 dilutions of FMDV A/KEN/6/2012, respectively | Serum, milk, vesicular epithelium or fluid samples of bovine origin | (20, 21) | |
| RT-PCR- Microarray | This assay was capable of detecting and serotyping FMDV and VSV in addition to detecting VESV and SVDV. Multiplex RT-PCR was able to detect viruses representing all seven serotypes of FMDV. Typing of the FMDV was achieved by slide microarray containing serotype-specific probe | • The detection limit of multiplex RT-PCR and microarray are 46 and 4.6 TCID50/mL of FMDV O1 Manisa, respectively •The diagnostic sensitivity and specificity: 92.6 and 100%, respectively |
Oral swabs from calves | (9) |
| This assay detects and differentiates FMDV, SVDV, VESV, ASFV, CSFV, PRRSV, and PCV2. Samples are amplified with multiplex RT-PCR and then applied to automated electronic microarray assay. This approach is less laborious and utilizes a single instrument that integrates and automates capture probe printing, hybridization, washing, and reporting on a disposable electronic microarray cartridge | • The detection limits of multiplex (seven-plex) RT-PCR and microarray for ASFV, PCV2 and PRRSV were reported to be at a range between one copy in PCR and 10 copies in microarray, respectively, followed by SVDV, CSFV and VESV at approximately 10 copies in PCR and 100 copies in microarray and >100 copies for FMDV in PCR and >10,000 copies in microarray | Biological material spiked with viruses, serum, and nasal and oral swabs from pigs | (22) | |
| RT-LAMP | An assay for rapid detection (within 45 min) and typing of FMDV of serotype Asia 1. This assay targets the conserved region of VP1 sequence of FMDV serotype Asia 1 | • NA | Infected pig samples (not specified) | (23) |
| An assay for rapid detection and typing of FMDV of serotype C. This assay targets the conserved region of VP1 sequence of FMDV serotype C and can be completed in an hour | • 0.325 ng/mL RNA template harvested from cell culture infected with FMDV of serotype C | Infected cells and blood samples from animals | (24) | |
| A multiplex RT-LAMP assay that utilizes combined primer sets from different individual RT-LAMP assays to compensate high sequence variability of FMDV. The assay was demonstrated to be superior or at least as good as individual RT-LAMP assay | • Detection limit: 10−3 dilution of RNA template harvested from FMDV O1 Manisa TUR/8/69 infected epithelial suspensions •Diagnostic sensitivity and specificity: 98.0 and 98.1%, respectively |
Epithelial suspension and esophageal–pharyngeal fluid samples from animals | (25) | |
| RT-LAMP assay utilizes swarm primers in addition to the standard six primers for improved sensitivity. This assay was demonstrated to specifically detect VP3 gene of FMDV (O serotype) | • 102 TCID50/mL or 103 copies/μL of in vitro–transcribed O/Andong/KOR/2010 3D RNA | Serum, saliva, and epithelial tissue samples from animals | (26) | |
| A real-time RT-LAMP assay targeting the 3D region for rapid detection of FMDV serotypes A, O, and Asia 1. It uses hydroxyl naphthol blue (HNB) dye for visual detection of positive sample in addition to the turbidity change | • 4.2 × 10−4, 2 × 10−6 and 1.1 × 10−4 TCID50/mL for FMDV serotypes O, A and Asia1, respectively | Tongue epithelium and semen samples from infected bulls | (27) | |
| The study evaluated RT-LAMP assay that utilized lyophilized reagents for detection of FMDV. Lyophilized reagents were shown to have no negative impact on amplification | • 10 copies/μL of in vitro–transcribed O/UKG/35/2001 3D RNA | Epithelial tissue, serum, and esophageal–pharyngeal fluid samples of bovine origin | (28) | |
| An RT-LAMP assay targeting the 3D region for rapid detection of FMDV. This assay targets the VP1 region of FMDV for specific detection of FMDV serotypes A, O, and Asia 1 | • 10 copies of DNA | NA | (29) | |
| Ag-RT-LAMP | An assay that utilizes FMDV genotype-specific IgG immobilized on a tube to capture the virus prior to RT-LAMP amplification. The assay can be completed within 3 h but was negatively affected by high viral load in the samples | • 0.58 x 102 copies of FMDV O/Akesu/58 | Vesicle fluid samples from cattle | (30) |
| qRT-LAMP | A real time RT-LAMP assay targeting the 3D region for rapid detection of FMDV. This assay also targets the VP1 region of FMDV for specific detection of FMDV serotypes A, O, and Asia 1 | • 10−5 dilution of FMDV RNA template harvested from infected epithelial suspensions •The detection limits of FMDV serotypes Asia 1 and O is 10−3 TCID50/mL, and 10−5 TCID50/mL for serotype A |
Epithelial suspension, tongue, and foot epithelium from animals | (29, 31) |
RT-PCR, reverse transcription-polymerase chain reaction; RT-iiPCR, reverse transcription-insulated isothermal polymerase chain reaction; RT-ddPCR, reverse transcription-droplet digital polymerase chain reaction; RT-RPA, reverse transcription-recombinase polymerase amplification; RT-qPCR, real-time reverse transcription-polymerase chain reaction; RT-LAMP, reverse transcription-loop-mediated isothermal amplification; Ag-RT-LAMP, antigen-capture reverse transcription-loop-mediated isothermal amplification; qRT-LAMP; real-time reverse transcription-loop-mediated isothermal amplification; IRES, internal ribosome entry site; ORF, open reading frame; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; FMDV, foot-and-mouth disease virus; SVDV, swine vesicular disease virus; VESV, vesicular exanthema of swine virus; ASFV, African swine fever virus; CSFV, classical swine fever virus; PRRSV, porcine respiratory and reproductive syndrome virus; PCV2, porcine circovirus type 2; VSV, vesicular stomatitis virus; TCID50, 50% tissue culture infectious dose; UTR, untranslated region; IgG, immunoglobulin G; NA, data not available.