Transbound Emerg Dis. 2019;66:1789–1795. wileyonlinelibrary.com/journal/tbed  |  1789 Received: 5 March 2019  |  Revised: 6 May 2019  |  Accepted: 7 May 2019 DOI: 10.1111/tbed.13227 S H O R T C O M M U N I C A T I O N Foot‐and‐mouth disease virus detection on a handheld real‐time polymerase chain reaction platform Kate Hole1 | Charles Nfon1,2 1National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Manitoba, Canada 2Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada Correspondence Charles Nfon, National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, 1015 Arlington Street, Winnipeg, MB, Canada. Email: charles.nfon@canada.ca Funding information Canadian Food Inspection Agency, Grant/ Award Number: TD WIN-A-1701 Abstract Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that re- quires rapid control. Early detection is critical but transportation of samples to lab- oratory delays testing. Sensitive and specific field-deployable assays are therefore desirable. Real-time reverse transcription polymerase chain reaction (RRT-PCR) and RRT-loop-mediated isothermal amplification assays for FMDV on portable platforms have been described but none of these are handheld. In this report, we have evalu- ated a handheld Biomeme two3™ Real-Time PCR Thermocycler (two3) as a field- deployable platform for FMDV RRT-PCR targeting the 3D gene segment. Two3's performance was compared with the laboratory-based reference assay on the ABI7500 platform. RNA extraction using a rapid Biomeme proprietary sample prep technology (M1) was compared with MagMax RNA extraction. Two3 successfully de- tected FMDV isolates for six serotypes (O, A, Asia 1, SAT 1, 2 and 3). Serotype C was excluded since it has not been detected in the field since 2004. The limits of detec- tion for serial 10-fold dilutions of cell culture isolates were equal or one log different between two3 and ABI7500. Furthermore, two3 detected FMDV RNA in multiple sample types including serum, vesicular fluid, tissue suspensions, oral fluid, oral and nasal swabs. Two3 also detected FMDV RNA directly in vesicular fluid and other samples without prior RNA extraction. Comparison of the time to first detection of a positive result in serial samples in MagMax RNA extraction/ABI7500 (MgMx/ABI) system vs. M1 RNA extraction/Two3 system revealed similar or slightly better ana- lytical sensitivity for the MgMx/ABI system. Overall, RNA extraction by M1 yielded good results and FMDV RNA detection on two3 was not significantly different from the ABI7500. Therefore, two3 could potentially enable sensitive penside detection of FMDV within an hour using M1-extracted RNA or direct testing of vesicular fluid and swabs without RNA extraction thereby ensuring prompt implementation of ap- propriate control measures. K E Y W O R D S Foot-and-mouth disease virus, handheld, polymerase chain reaction Reproduced with the permission of the Minister of Health. © 2019 Her Majesty the Queen in Right of Canada Transboundary and Emerging Diseases © 2019 Blackwell Verlag GmbH www.wileyonlinelibrary.com/journal/tbed mailto: https://orcid.org/0000-0003-0899-3986 mailto:charles.nfon@canada.ca http://crossmark.crossref.org/dialog/?doi=10.1111%2Ftbed.13227&domain=pdf&date_stamp=2019-06-05 1790  |     HOLE and nFOn 1  | INTRODUC TION Foot-and-mouth disease (FMD) is a highly contagious vesicu- lar disease of cloven-hoofed animals caused by the FMD virus (FMDV). Seven distinct serotypes of FMDV exist, namely A, O, C, Asia 1, South African Territories (SAT) 1, SAT 2 and SAT 3. Foot- and-mouth disease outbreaks result in severe economic damages, especially if not detected and rapidly controlled (Alexandersen, Quan, Murphy, Knight, & Zhang, 2003). Effective control relies on rapid diagnosis. Routine assays often require transfer of samples to the lab, thereby increasing the turnaround time from clinical sus- picion to test results. Furthermore, in resource limited countries, the transportation of samples can be challenging and the quality of samples upon arrival at a lab cannot be guaranteed. Highly sen- sitive and specific field-deployable tests are therefore desirable for rapid and accurate detection of FMDV. Antigen detection and molecular assays have been evaluated for this purpose. Lateral flow strip tests (LFSTs) for FMDV antigen detection are available, some of which are being used in the field (Ferris et al., 2010, 2009; Yang, Caterer, Xu, & Goolia, 2015; Yang, Goolia, Xu, Bittner, & Clavijo, 2013; Yang, Mudabuka, Quizon, & Nfon, 2019). LFST was used for FMD detection during the 2007 FMD outbreak in the UK (Ryan et al., 2008). However, LFSTs have low sensitivity, meaning a negative result still requires laboratory confirmation before any decision-making. Real-time reverse transcription polymerase chain reaction (RRT- PCR) assays for FMDV are highly sensitive (Callahan et al., 2002; Howson, Armson, et al., 2017; Moniwa, Clavijo, Li, Collignon, & Kitching, 2007; Rasmussen, Uttenthal, Stricker, Belak, & Storgaard, 2003) but are mostly restricted to specialized laboratories. Nevertheless, significant progress has been made on field-deploy- able molecular assays for FMDV (Ambagala et al., 2017; Dukes, King, & Alexandersen, 2006; Howson et al., 2018; Howson, Armson, et al., 2017; Howson, Kurosaki, et al., 2017; Kasanga et al., 2014; Waters et al., 2014). However, a sensitive handheld platform has not been described for FMDV. Here, we report the evaluation of a handheld Biomeme two3™ Real-Time PCR Thermocycler (Biomeme Inc.) as a field-deployable platform for FMD detection by RRT-PCR targeting the 3D gene segment, comparing the performance of the device to reference laboratory-based ABI7500 (ThermoFisher Scientific Inc.). Furthermore, RNA extraction using a simple, rapid Biomeme pro- prietary sample prep technology (M1) that can be performed in the field was compared with MagMax RNA extraction (ThermoFisher Scientific Inc.). The Biomeme two3™ (two3) is operated using a smart phone App and does both real-time PCR and isothermal molecular detection of nucleic acids in a variety of sample types. Testing from sample-to-re- sults can be achieved in 30–60 min when the M1 sample prep tech- nology is combined with the two3 instrument (https ://shop.biome me.com/produ cts/two3-real-time-pcr-therm ocycler). Two3 was used for the detection and monitoring of Flavobacterium psychrophi‐ lum in water, with significant correlation in the results between two3 and a lab-based platform (Nguyen et al., 2018). Furthermore, two3 and a CFX96 real-time PCR detection system (BioRad) detected Venezuelan equine encephalitis virus in a mosquito pool with Cts 33.92 and 30.63 respectively. The authors concluded that two3 was ‘an effective, ultra-portable platform for initial triaging of mosquito samples in the field’ (Russell et al., 2018). 2  | MATERIAL S AND METHODS Viruses used in this study (Tables 1 and 2) were obtained from the World Reference Laboratory for FMDV, The Pirbright Institute, UK. Clinical samples (Tables 3 and 4) were obtained from sheep, pigs and cattle experimentally infected with FMDV as previously described (Ambagala et al., 2017; Senthilkumaran et al., 2017; Yang, Parida, et al., 2015). Serotype C was excluded because it has not been detected in the field since 2004 (EuFMD, 2019). Samples were collected as previ- ously described (Ambagala et al., 2017; Senthilkumaran et al., 2017). Total RNA was extracted using MagMAX™-96 Viral RNA Isolation Kit (AMB1836-5, Life Technologies) and the MagMAX™ Express-96 Magnetic Particle Processor (Life Technologies) follow- ing manufacturer's protocol. The extraction used 55 µl of each sam- ple and the extracted RNA was eluted into 50 μl of elution buffer. RNA extraction with M1 was performed according to manufactur- er's protocol (Biomeme Inc.) starting with 150 µl of the sample and eluting in 150 µl of buffer. The M1 kit utilizes a filtration method whereby total nucleic acid selectively bound to the silica membrane TA B L E 1  Foot-and-mouth disease virus (FMDV) genome detection in cell culture isolates FMDV isolate ABI7500 Ct Two3 Ct O UKG 11/2001 16.0 17.4 O TAW 10/97 20.9 27.7 O VIT 1/2012 14.6 17.0 O SAU 1/2016 15.9 17.1 A24 Cruzeiro 13.9 14.6 A22 IRQ 24/64 13.4 14.9 A IRN 1/2005 15.8 16.3 A SAU 2/2015 16.2 15.6 Asia 1 Shamir 25.6 24.2 Asia 1 PAK 51/2011 14.9 15.2 SAT1 KEN 4/98 13.4 14.1 SAT1 BOT 11/15 14.1 14.3 SAT2 SAU 2/2000 15.2 15.9 SAT2 ETH 2/07 15.7 16.4 SAT3 ZIM 4/81 17.2 17.1 SAT3 SAR 1/06 14.0 14.4 Note: RNA extracted from cell culture isolates by the MagMAX™-96 Viral RNA Isolation Kit was tested on ABI7500 and a handheld Biomeme two3™ Real-Time PCR Thermocycler. Ct, crossing threshold. Ct < 35.99 or < 40 is considered positive for FMDV for ABI7500 and two3 respectively. 18651682, 2019, 4, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/tbed.13227 by C anadian A griculture L ibrary A griculture & A gri-Food C anada, W iley O nline L ibrary on [25/02/2026]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://shop.biomeme.com/products/two3-real-time-pcr-thermocycler https://shop.biomeme.com/products/two3-real-time-pcr-thermocycler      |  1791HOLE and nFOn inside Biomeme's proprietary M1 Sample Prep Columns is sequen- tially washed with proprietary buffers prior to elution. Previously described primers/probe sets for FMDV and β-actin RRT-PCR were used (Moniwa et al., 2007). For ABI7500, a 4x TaqMan Fast Virus 1-step Master Mix (Applied Biosystems™) was used in a FMDV/ β-actin duplex mixture comprising 11.75 µl RNase-free water, 6.25 µl of 4x TaqMan Fast Virus 1-step Master Mix, 1 µl of 25X FMDV primers/probe mix (0.5 μM each of forward and reverse primers and 0.2 μM FAM-labelled probe), 1 µl of 25X β-actin primers/probe mix (1.0 μM each of forward and reverse primers and 0.2 µM VIC-labelled probe) and 5 µl RNA template in a final volume of 25 µl. For two3, a 2x qscript XLT 1-step toughmix (Quanta BioSciences, Inc) was used in a FMDV/β-actin duplex mixture comprising 5.5 µl RNase-free water, 12.5 µl of 2x qscript XLT 1-step toughmix (no ROX), 1 µl of 25X FMDV primers/probe mix (0.5 μM each of forward and reverse primers and 0.2 µM FAM-labelled probe), 1 µl of 25X β- actin primers/probe mix (1.0 μM each of forward and reverse prim- ers and 0.2 µM Quasar 670-labelled probe) and 5 µl RNA template in a final volume of 25 µl. The cycling parameters for each platform were kit-specific based on manufacturer's recommendations: For the ABI7500 stage 1(5 min at 50°C), stage 2 (20 s at 95°C), stage 3 (15 s at 95°C, 45 s at 60°C repeated 40 times with collection of fluorescence) with a crossing threshold (Ct) < 35.99 was considered positive and Ct 36–40 was considered suspicious for FMDV genome (Moniwa et al., 2007). For two3 stage 1(10 min at 50°C), stage 2 (1 min at 95°C), stage 3 (3 s at 95°C, 60 s at 30°C repeated 40 times with collection of fluores- cence). Nucleic acids from negative samples (50) were tested on the two3 and no amplification detected after 40 cycles (data not shown). Hence, the two3 was programmed to report positive results for samples with a Ct < 40. Samples were tested in singles. Data sets were analysed by two way t-test and correlation/association tests between ABI7500 vs. two3 were performed using the Spearman correlation coefficient test in XLSTAT (version 2017.1). 3  | RESULTS AND DISCUSSION Cell culture isolates were used in preliminary evaluation of equiva- lency between two3 and ABI7500. Two3 accurately detected FMDV in cell culture isolates for six serotypes tested (Table 1) with compa- rable Ct values between two3 and ABI7500, hence significant corre- lation between the two platforms for FMDV detection in cell culture isolates (Figure 1a). Similarly, the last dilution of a 10-fold dilution series of cell culture isolates to give positive result was equivalent or one log different between two3 and ABI7500 (Table 2). Furthermore, some dilutions gave Ct = 36 < 40 on ABI7500. These are considered suspicious for FMDV genome and are usually retested if no other sample from the same epidemiological unit tests positive. Two3 also correctly detected FMDV from clinical samples when RNA was extracted using MagMax (Table 3) giving com- parable Ct values and significant correlation between two3 and ABI7500 (Figure 1b). With M1 RNA extraction, 18/19 samples were positive on both two3 and ABI7500 (Table 3), with signifi- cant correlation between the two platforms (Figure 1c). Syringe filters used for M1 RNA extraction were easily clogged by sera thereby reducing the efficiency of RNA extraction from sera. Sera were therefore excluded for statistical comparison of extraction methods. There was no statistically significant difference between M1 and MagMax extraction methods when comparing Ct values obtained by testing RNA from these extraction methods on two3 (p = 0.265) and ABI7500 (p = 0.061). FMDV genome was also de- tected in vesicular fluids and other samples tested directly by RRT- PCR without prior RNA extraction (Table 3), also with significant correlation (p = 1.5 x 10−6) between the two platforms (Figure 1d). Virus was more readily detected in directly tested vesicular fluid (lower Ct values) compared to sera and other samples. There was TA B L E 2  Analytical sensitivity of ABI7500 and a handheld Biomeme two3™ Real-Time PCR Thermocycler for detection of foot-and-mouth disease virus (FMDV) genome in cell culture isolates Dilution ABI7500 Ct Two3 Ct O UKG 11/2001 10−2 27.6 27.5 10−3 32 32.7 10−4 37.3 36.1 10−5 >40.0 >40.0 A22 IRQ 24/64 10−2 27.5 28.5 10−3 31.7 35.6 10−4 38.2 >40.0 10−5 >40.0 >40.0 ASIA 1 Shamir 10−1 32.2 33.9 10−2 >40 38.5 10−3 >40 >40 SAT1 KEN 4/98 10−2 25.2 26.6 10−3 29 29.9 10−4 34.4 33.2 10−5 >40 37.3 10−6 >40 >40 SAT2 SAU 2/2000 10−2 26.3 27.9 10−3 30.6 30.9 10−4 35.2 >40 10−5 >40 >40 SAT3 ZIM 4/81 10−1 26.6 27.5 10−2 30 31.3 10−3 34.9 35 10−4 >40 >40 Note: Serial 10-fold dilutions of cell culture isolates of FMDV were tested, RNA extracted by the MagMAX™-96 Viral RNA Isolation Kit and tested on the two platforms. Ct = crossing threshold. Ct < 35.99 or < 40 is considered positive for FMDV for ABI7500 and two3 respectively. Ct = 36 < 40 is considered suspicious for ABI7500. Grey cells represent the last positive dilution for each isolate. 18651682, 2019, 4, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/tbed.13227 by C anadian A griculture L ibrary A griculture & A gri-Food C anada, W iley O nline L ibrary on [25/02/2026]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 1792  |     HOLE and nFOn a significant difference in Ct values for directly tested samples (sera not included) and M1 extracted RNA when run on two3 (p = 0.021). Remarkably, there was no significant difference in Ct values for directly tested samples and M1 extracted RNA when run on ABI7500. However, M1 extraction improved RRT-PCR sen- sitivity (lower Ct values) for oral fluids, oral and nasal swabs com- pared to direct testing on both platforms. Conversely, MagMax extraction significantly increased sensitivity (p < 0.05) compared to direct testing of samples on both two3 and ABI7500. Analytical sensitivity (time to first detection of positive) of the reference lab system [MagMax RNA extraction/ABI7500 testing( MgMx/ABI)] and the field-deployable system [M1 RNA extraction/ two3 testing (M1/two3)] were determined by testing DPI 0–3 sam- ples from experimentally infected pigs, cattle and sheep (n = 2 each). Both systems gave positive results for nasal swabs from two pigs and one cattle starting at DPI 1, but the rest of the DPI 1 nasal swabs were positive only on the MgMx/ABI (Table 4). Detection in oral swabs was mostly similar between the two systems (Table 4). RRT-PCR assays for FMDV have high sensitivity and specific- ity and these have been transferred onto portable platforms in recent years. Full validation of these platforms for field use will provide reliable rapid assays for onsite diagnosis of FMD. Here we have demonstrated that two3, a handheld real-time PCR plat- form, is capable of detecting FMDV with diagnostic and analyt- ical sensitivity comparable to the reference lab-based platform. With further field validation, the results from two3 could poten- tially be accepted with the same level of confidence as the ref- erence lab-based platforms, especially for oral and nasal swabs and specifically vesicular flu acquired during the acute phase of infection. Similar results have been reported for FMDV on other field-deployable real-time molecular assays/platforms (Howson et al., 2018; Howson, Armson, et al., 2017; Howson, Kurosaki, TA B L E 3  Foot-and-mouth disease virus (FMDV) genome detection in clinical samples from FMDV-infected animals Serotype Sample type Animal DPI RNA extracted by MagMax RNA extracted by M1 Direct testing (no RNA extraction) ABI 7500 Two3 ABI 7500 Two3 ABI 7500 Two3 A24 VF Pig 3 11.7 12.9 21.6 20.7 17.5 22.6 A IRN 1/2009 VF Pig 4 20.8 19.0 22.9 20.7 25.3 25.9 A IRN 1/2009 VF Pig 4 17.1 16.3 20.0 15.1 23.2 18.1 SAT2 EGY 6/2012 VF Pig 3 12.8 13.6 16.6 15.2 14.8 19.0 ASIA1 PAK 19/14 Serum Cattle 4 25.3 25.7 35.6 36.0 33.8 34.0 ASIA1 PAK 19/14 Serum Cattle 4 28.6 30.0 39.4 >40 36.4 38.0 ASIA1 PAK 19/14 Serum Cattle 4 25.9 26.4 35.4 33.2 34.1 39.1 ASIA1 PAK 19/14 Serum Cattle 4 18.0 18.2 30.4 28.2 27.5 31.6 O UKG 11/2001 OS Pig 4 26.2 27.4 25.4 25.1 27.9 29.5 O UKG 11/2001 OS Pig 5 24.1 24.1 23.2 24.7 27.7 28.5 O UKG 11/2001 OS Pig 4 25.5 25.3 28.5 29.6 30.6 32.6 ASIA1 PAK 19/14 NS Cattle 3 20.1 20.8 23.6 22.3 26.7 25.5 ASIA1 PAK 19/14 NS Cattle 3 22.4 21.5 26.4 25.1 28.9 29.5 ASIA1 PAK 19/14 NS Cattle 4 23.9 26.5 25.3 26.5 28.7 27.7 ASIA1 PAK 19/14 NS Cattle 4 22.3 22.1 25.9 25.1 29.0 28.4 O UKG 11/2001 OF Group A (pigs) 2 25.3 25.4 27.4 26.7 29.9 31.7 O UKG 11/2001 OF Group B (pigs) 3 26.0 26.2 28.2 27.1 32.0 36.8 O UKG 11/2001 OF Group C (pigs) 3 24.2 25.4 27.0 26.7 31.1 39.7 O UKG 11/2001 OF Group D (pigs) 3 23.8 24.19 27.0 27.9 30.8 32.5 Note: RNA extracted by MagMAX™-96 Viral RNA Isolation Kit (MagMax), Biomeme proprietary sample prep technology (M1) or no RNA extraction was tested on ABI7500 and Biomeme two3™ Real-Time PCR Thermocycler. Ct = crossing threshold. Ct < 35.99 or < 40 is considered positive for FMDV for ABI7500 and two3 respectively. Ct = 36 < 40 is considered suspicious for ABI7500. DPI, days post-infection, VF, vesicular fluid, OS, oral swab, NS, nasal swab, OF, oral fluids (saliva and oral mucosa exudates collected by squeezing cotton ropes that pigs have chewed on; Group A–D described in Senthilkumaran et al, 2016). Grey shading represents Ct above cut off for each platform. 18651682, 2019, 4, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/tbed.13227 by C anadian A griculture L ibrary A griculture & A gri-Food C anada, W iley O nline L ibrary on [25/02/2026]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense      |  1793HOLE and nFOn et al., 2017; Moniwa et al., 2007). The fact that two3 detected FMDV in the absence of RNA extraction further potentiates its utility for onsite testing. Furthermore, serum cannot be tested on LFST whereas two3 can directly detect FMDV in serum. Our results for direct detection of FMDV in a variety of samples are in agreement with other studies (Ambagala et al., 2017; Howson et al., 2018). Diagnostic sensitivity was reduced with direct test- ing of samples on two3, implying that RNA extraction may be required, particularly if a pre-clinical case is suspected due to epidemiological links. RNA extraction has previously been the main limiting factor to full exploitation of field-deployable mo- lecular assays. M1 showed great promise as a rapid, user-friendly RNA extraction technology that can be combined with two3. RNA was successfully extracted from a variety of samples in ap- proximately 5 min. However, M1 technology may not be appro- priate for serum samples because they clog the syringe filters used in M1 technology, possibly due to the high protein content in serum. RNA extraction with other sensitive field methods previously described (Ambagala et al., 2017; Howson et al., 2018) can also be evaluated for pairing with two3. Some of the field-deployable molecular assays for FMD have been proven field ready (Howson et al., 2018; Howson, Armson, et al., 2017; Howson, Kurosaki, et al., 2017). However, two3 being a handheld, smart phone-operated device, it is very easy to transport and does both real-time PCR and isothermal molecular detection of nucleic acids with run programming that is customizable by the end-user, providing results within 30–60 min. Also, data can be uploaded to a cloud data- base with GPS coordinates thus allowing the reference lab to access the data obtained on the field at the end of the run. Nevertheless, its high portability comes at the expense of throughput, as it can only run three samples at a time. Therefore, in field testing, samples have to be prioritized, starting with those that can potentially give the most valu- able results. Hence, when present, vesicular fluid should get top prior- ity followed by swabs of freshly ruptured vesicles, nares or oral cavity. In conclusion, two3 is a truly portable real-time PCR platform that has great potential for FMDV detection in the field. The TA B L E 4  Foot-and-mouth disease virus (FMDV) genome detection in clinical samples from FMDV-infected animals [Colour table can be viewed at wileyonlinelibrary.com] System DPI 0 DPI 1 DPI 2 DPI 3 Nasal swabs P13 M1/Two3 40.00 33.53 24.23 25.53 MgMx/ABI7500 40.00 32.95 19.86 23.58 P14 M1/Two3 40.00 26.82 26.08 29.75 MgMx/ABI7500 40.00 28.45 21.55 22.81 C42 M1/Two3 40.00 40.00 40.00 40.00 MgMx/ABI7500 40.00 40.00 30.66 25.39 C47 M1/Two3 40.00 31.76 35.65 22.23 MgMx/ABI7500 40.00 31.35 23.79 28.84 S24 M1/Two3 40.00 40.00 40.00 37.53 MgMx/ABI7500 40.00 32.80 27.23 23.22 S55 M1/Two3 40.00 40.00 40.00 40.00 MgMx/ABI7500 40.00 27.51 29.16 34.05 Oral swabs P13 M1/Two3 40.00 34.51 26.19 27.73 MgMx/ABI7500 40.00 39.03 24.79 25.39 P14 M1/Two3 40.00 40.00 27.7 26.99 MgMx/ABI7500 40.00 40.00 26.69 25.23 C42 M1/Two3 40.00 40.00 38.86 32.38 MgMx/ABI7500 40.00 40.00 40.00 33.31 C47 M1/Two3 40.00 40.00 36.13 34.68 MgMx/ABI7500 40.00 40.00 32.02 21.37 S24 M1/Two3 40.00 40.00 37.49 21.9 MgMx/ABI7500 40.00 40.00 30.95 21.97 S55 M1/Two3 40.00 40.00 32.41 33.56 MgMx/ABI7500 40.00 40.00 31.13 40.00 Note: RNA extracted by MagMAX™-96 Viral RNA Isolation Kit and tested on the ABI7500 (MgMx/ABI), Biomeme proprietary sample prep technol- ogy (M1) and tested on the Biomeme two3™ Real-Time PCR Thermocycler (M1/Two3). Ct = crossing threshold. Ct < 35.99 or < 40 is considered positive for FMDV for ABI7500 and two3 respectively (green cells). Abbreviations: C, cattle; DPI, days post-infection; P, pig; S, sheep. 18651682, 2019, 4, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/tbed.13227 by C anadian A griculture L ibrary A griculture & A gri-Food C anada, W iley O nline L ibrary on [25/02/2026]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense www.wileyonlinelibrary.com 1794  |     HOLE and nFOn complementary M1 technology provides rapid RNA extraction capability for specific sample types but FMDV can also be directly detected in most samples during the acute phase of infection with no need for RNA extraction. The RRT-PCR reagents in this assay can be lyophilized to eliminate the need for cold chain and further enhance the field utility of this platform. The next step will be to further validate this system in the field using outbreak samples and apply this technology for FMD diagnosis in endemic countries. Note: After submission of this manuscript the two3 was up- graded to the three9 which is still handheld, with same smartphone- operated technology and can test up to nine samples at a time. The two3 is still being supported by the company. In addition, a compar- ison of two3 and three9 gave similar or slightly improved results for three9 (Table S1 and S2). ACKNOWLEDG EMENTS This project was funded in part by the Canadian Food Inspection Agency Technology Development fund (TD WIN-A-1701). We thank the Animal Care staff for their help with animal experiments. We also thank Romaine Edirmanasinghe, Diana Lusansky, Thanuja Ambagala for technical support and Drs. Alfonso Clavijo and John Copps for reviewing the manuscript. ORCID Charles Nfon https://orcid.org/0000-0003-0899-3986 R E FE R E N C E S Alexandersen, S., Quan, M., Murphy, C., Knight, J., & Zhang, Z. (2003). Studies of quantitative parameters of virus excretion and transmis- sion in pigs and cattle experimentally infected with foot-and-mouth disease virus. Journal of Comparative Pathology, 129, 268–282. https :// doi.org/10.1016/S0021-9975(03)00045-8 Ambagala, A., Fisher, M., Goolia, M., Nfon, C., Furukawa-Stoffer, T., Ortega Polo, R., & Lung, O. (2017). Field-Deployable Reverse Transcription- Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus. Transboundary and Emerging Diseases, 64, 1610–1623. Callahan, J. D., Brown, F., Osorio, F. A., Sur, J. H., Kramer, E., Long, G. W., … Nelson, W. M. (2002). 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Insert in each figure represents Spearman correlation matrix [Colour figure can be viewed at wileyonlinelibrary.com] 18651682, 2019, 4, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/tbed.13227 by C anadian A griculture L ibrary A griculture & A gri-Food C anada, W iley O nline L ibrary on [25/02/2026]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://orcid.org/0000-0003-0899-3986 https://orcid.org/0000-0003-0899-3986 https://doi.org/10.1016/S0021-9975(03)00045-8 https://doi.org/10.1016/S0021-9975(03)00045-8 https://doi.org/10.2460/javma.2002.220.1636 https://doi.org/10.2460/javma.2002.220.1636 https://doi.org/10.1007/s00705-005-0708-5 https://doi.org/10.1016/j.jviromet.2009.09.022 https://doi.org/10.1016/j.jviromet.2009.09.022 https://doi.org/10.1016/j.jviromet.2008.09.009 https://doi.org/10.1111/tbed.12684 https://doi.org/10.1111/tbed.12684 https://doi.org/10.1111/tbed.12451 https://doi.org/10.1016/j.jviromet.2017.08.013 https://doi.org/10.4102/ojvr.v81i2.727 https://doi.org/10.4102/ojvr.v81i2.727 www.wileyonlinelibrary.com      |  1795HOLE and nFOn between three real-time instruments. 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Clinical and Vaccine Immunology : CVI, 22, 389–397. https ://doi.org/10.1128/CVI.00594-14 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article.  How to cite this article: Hole K, Nfon C. Foot-and-mouth disease virus detection on a handheld real-time polymerase chain reaction platform. Transbound Emerg Dis. 2019;66:1789–1795. https ://doi.org/10.1111/tbed.13227 18651682, 2019, 4, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/tbed.13227 by C anadian A griculture L ibrary A griculture & A gri-Food C anada, W iley O nline L ibrary on [25/02/2026]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://doi.org/10.1007/s00705-003-0145-2 https://doi.org/10.1007/s00705-003-0145-2 https://doi.org/10.1038/s41598-018-23641-7 https://doi.org/10.1038/s41598-018-23641-7 https://doi.org/10.1136/vr.163.5.139 https://doi.org/10.1371/journal.pone.0105630 https://doi.org/10.1371/journal.pone.0105630 https://doi.org/10.1016/j.jviromet.2015.05.001 https://doi.org/10.1186/1743-422X-10-125 https://doi.org/10.1186/1743-422X-10-125 https://doi.org/10.1128/CVI.00594-14 https://doi.org/10.1111/tbed.13227