A Portable, 3-D Printed, Microfluidic Device for Multiplexed, Real Time, Molecular Detection of Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus at the Point of Need

Abstract

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) are emerging / reemerging coronaviruses (CoVs) of neonatal pigs that cause great economic losses to pig farms and pork processors. Specific, rapid, and simple multiplex detection of these viruses is critical to enable prompt implementation of appropriate control measures. Conventional methods for molecular diagnosis require skilled personnel, relatively sophisticated equipment, restricting their use to centralized laboratories. We developed a low-cost, rapid, semi-quantitative, field deployable, 3D-printed microfluidic device for auto-distribution of samples; self-sealing; real-time, and reverse transcription-loop-mediated isothermal amplification (RT-LAMP), enabling co-detection of PEDV, TGEV and PDCoV within 30 minutes. Our assay’s analytical performance is comparable with benchtop, real-time RT-LAMP assay as well as the gold standard quantitative reverse transcription- polymerase chain reaction (qRT-PCR) assay with limits of detections of 10 genomic copies / reaction for PEDV and PDCoV, and 100 genomic copies / reaction for TGEV. Evaluation of clinical specimens from diseased pigs with our microfluidic device revealed excellent concordance with both benchtop RT- LAMP and qRT-PCR. Our portable RT-LAMP microfluidic chip will potentially facilitate simple, specific, rapid multiplexed detection of harmful infections in minimally equipped veterinary diagnostic laboratories and on-site in pigs’ farms.

Introduction

The Coronaviridae family of the Nidovirales order comprises four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are members of the Alphacoronavirus genus while porcine deltacoronavirus (PDCoV) is a Deltacoronavirus .¹

Coronaviruses are positive-sense, single-stranded, enveloped RNA viruses and have an RNA genome of 25–30 kb (kilobases); the largest ever reported. The virion RNA contains 5’ and 3’ untranslated regions (UTR), capped at the 5’ end and polyadenylated at the 3’ end. The 5’ terminal two-thirds of the genome contain open reading frames (ORF) 1a and 1ab that encode two replicase polyproteins (pp1a and pp1ab). The 3’-terminal one-third of the genome encodes structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.^{2, 3} PEDV, TGEV and PDCoV are emerging / reemerging coronaviruses (CoVs) of neonatal pigs and cause great economic losses to the pig industry.⁴ All three viruses affect the animals’ enteric system, are antigenically distinct, lack cross immunity protection, and cause acute gastroenteritis in neonatal piglets with similar clinical manifestations. The infected intestinal epithelial cell necrosis causes atrophy of the intestinal villi, diarrhea, growth retardation, dehydration, and death of piglets.^{1, 5} Currently, among these three viral infections, the PEDV infection is the most significant and consequential in the USA. This is likely because PEDV has been only recently introduced to the USA and pigs in USA lack immunity against it.⁴

Effective prevention and control of these diseases can be achieved by stringent biosecurity measures, disease containment within and among farms, and vaccination. Vaccines are available against TGEV and PEDV.^{1, 3, 6} Rapid co-detection of these viruses would be followed by rapid initiation of appropriate control measures, and reduction of the expected economic losses.

Currently, confirmative diagnosis of these diseases is performed in centralized laboratories by enzyme-linked immunosorbant assay (ELISA), virus isolation, virus neutralization (VN) and immunofluorescence techniques, quantitative real-time- reverse transcription- PCR (qRT-PCR), and conventional RT-PCR. However, all the above-mentioned detection methods have challenges for implementing specific, rapid, simple and specific diagnosis in laboratories with poor resources. These challenges stem from excessive labor and time in testing, lack of skilled personnel, insufficient specificity and sensitivity, and costly equipment requirements.^7–17

Recently, RT-loop-mediated isothermal amplification (RT-LAMP) has been demonstrated to have sensitivity equal to RT-PCR for the detection of swine enteric coronaviruses.^18–21 LAMP utilizes four to six primers and strand-displacing DNA polymerase. LAMP is carried out at a constant temperature, without a need for temperature cycling.²² Furthermore, LAMP is compatible with multiple amplicon detection methods, including visual inspection with the naked eye based on color change or increased turbidity; gel electrophoresis; fluorescence; bioluminescence,^23–27 and lateral flow strip immunochromatograph, detecting labeled amplicons using conjugated primer sets. Fluorescence and bioluminescence-based detections enable real-time amplicon monitoring and quasi-quantification.²⁸

To implement diagnostic assays at the point of need, it is desirable to minimize manual operations. This is best accomplished with microfluidic technology that enables integration of various unit operations. Here, we describe a 3-D printed, microfluidic device for real time–reverse transcription-LAMP that enables rapid, on-site molecular multiplexed detection and quasi-quantification of PEDV, TGEV and PDCoV (Fig. 1)

Fig. 1.

Workflow for molecular diagnosis of PEDV, TGEV and PDCoV in pigs. A sample is collected with a rectal swab from a piglet and lysed to release and solubilize pathogenic viral nucleic acids. The extracted nucleic acids are enzymatically amplified with RT-LAMP using the microfluidic chip that is incubated with a portable processor that provides constant temperature. The amplification products are monitored in real-time with intercalating fluorescent dye and a low-cost CCD camera. Alternatively, end-point detection of amplicons using colorimetric dyes is also feasible.

Materials and Methods

2.1. 3D-Printed Chip Design and Fabrication

Our 28 mm (L) × 28 mm (W) × 4 mm (H) microfluidic cartridge ‘chip’ (Fig. 2) features four reaction chambers of (20 ± 1) μL volume each, connecting conduits, and passive capillary valves to assist in flow control and sample distribution among the reaction chambers. The chip is designed with SolidWorks 2018 software (DS SolidWorks^™) producing CAD files in STL format compatible with most 3D printers., The chip is fabricated with a Low Force Stereolithography (LFS) resin-based 3D printer (Formlabs^™, Form 3). The chip material is a clear photopolymer resin (Formlabs^™, FLGPCL04) with a specific mass of 1.1 kg·m⁻³ and thermal conductivity of 0.110 W·m⁻¹K⁻¹. Portions of the 3D-printed chip are left uncapped to enable introduction of reagents. After printing, printing supports are removed, and the chips are thoroughly washed with Form Wash (Formlabs^™) for 15 min, followed by a 15-min post-cure at 60 °C with Form Cure (Formlabs^™). To reduce the absorbance of enzymes and sample to reaction chambers’ surfaces. and thereby avoid adversely affecting nucleic acid amplification efficiency, we coated the inner surfaces of the reactors with a 2% aqueous solution of polyethylene glycol (PEG) 3350.²⁹

Fig. 2.

A schematic depiction of design principles and fluid control with capillary circuits and passive capillary valves. (A) 3D printed chip design. (B1) Driven by capillary force, LAMP solution (including templates) flows into reactors automatically. In the future, the LAM P mix will be pre-stored in the reaction chambers in dry form and only the sample will be introduced into the chip. (B2) By placing an absorption pad at the sample inlet, capillary force helps dry any excess aqueous solution in the common conduit and therefore cut off the connections between different reactors. (B3) The chip is then mated with the processor and heated to its operating temperature (63℃). During the heating process, the PEG 930 (with a melting temperature around 37 ℃) melts and flows into the common conduits driven by capillary force to seal the chip.

Next, primers specific to individual targets were dried in three of the (test) reaction chambers, while a fourth chamber was left primer-free to serve as a negative control. To this end, we pipetted an aqueous solution of the designated primer set specific to each target through the uncapped common conduit into each test reactor (Fig. 3) and then dried the chip at room temperature for 2 hr. Next, we placed the chip on a cooled plate for molten PEG 930 loading. The PEG 930 has a melting temperature at 36 ± 1 °C and solidified in the PEG chamber (opening II, (Fig. 2 and 3) soon after filling. Finally, the chip was capped with a PCR tape (Bio-Rad^™, MSB1001), leaving only two open ports, one for sample introduction and the other to allow air to escape during sample loading (Fig. 3).

Fig. 3.

Chip Preparation: (A) After 3D printing, the chip is washed, post-cured, and coated with PEG 3350. (B) Aqueous solutions of primer sets for PEDV, TGEV and PDCoV are, respectively, pipetted into chambers a, b, and c. Chamber d is left primer-free. (C) Melt of PEG 930 is inserted into the PEG chamber and (D) allowed to solidify. (E) The chip is capped with a PCR tape to seal the open conduits, leaving only the sample inlet, the air vent, and the PEG chambers open. (F) The chip is then ready for sample introduction and LAMP incubation.

2.2. Auto-distribution and Self-sealing:

The microfluidic chip houses capillary circuits, passive capillary valves, and phase change materials to control the flow in the chip, minimizing the need for actuators and manual operations. Since the 3D-printed resin is hydrophilic with a static water contact angle of 47 degrees (Formlabs^™ Inc.), any abrupt enlargement in a conduit’s cross-section provides a passive capillary valve that pins a liquid column’s advancing meniscus,³⁰ and halts flow. Our microfluidic chip has four different capillary valves of various dimensions that require different pressures to overcome, enabling us to achieve our desired flow control.

Our sample includes the LAMP reaction mix and targets (RNA extracted from the samples), but not primers. In the future, the LAMP reaction mix will be dry stored in the reaction chambers and the sample will include only the extracted nucleic acids. The sample is introduced at the sample inlet (Fig. 2A). The liquid is driven into the chip by capillary forces. As the sample imbibes into the chip, it fills the common conduit and the LAMP reaction chambers and is halted by the capillary valves I, II, and III (Fig. 2A). Then, the inlet port is brought in touch with an absorption pad that drains liquid from the common conduit (Fig. 2B). The capillary valves are designed with valve III having the smallest diameter of 0.8 mm (greatest pinning force), followed by valve II with the intermediate hydrodynamic diameter of 1.5 mm (smallest pinning force), and valve I with the hydrodynamic diameter of 1.3 mm. Therefore, during the draining process, valves III pin the menisci and retain the LAMP reaction chambers filled with sample while liquid is removed from the connecting conduit to prevent crosstalk, and cleared from the vicinity of the inlet port to facilitate subsequent sealing with PEG. The chip is now ready for incubation.

Next, we place the chip on a heater and heat it to the LAMP reaction temperature of 63°C. The PEG melts at 36 ± 1 °C and flows by capillary force, effectively isolating the LAMP reaction chambers from the ambient and minimizing evaporation. When the chip is cooled back to room temperature after incubation, the PEG solidifies sealing the LAMP chambers and preventing release of amplicons to the ambient.

2.3. Viruses and Clinical Samples

PEDV (PC22A strain) propagated in Vero cells,¹⁰ TGEV (virulent Miller-M6 strain) propagated in ST cells,⁹ and PDCoV strain OH-FD22 propagated in LLC-PK cells¹² were provided by the Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, United States. RNA was extracted with AM1836 5X MagMax 96 Viral Extraction kit (Life Technologies Corp.) following manufacturer’s instructions.

The number of copies of genomic RNAs of PEDV, TGEV and PDCoV were quantified,³¹ and diluted to 10⁵ genomic copies per μl of each virus. Eleven rectal swabs were collected from piglets that previously tested positive for PEDV, TGEV, or PDCoV using the gold standard virus-specific RT-PCR assays,^{7, 12, 13} and one negative control rectal swab was collected from non-infected gnotobiotic pig. Samples collection from pigs and extracted nucleic acids transportation were carried out in compliance with the ethical guidelines of both Ohio State University and University of Pennsylvania, USA.

2.4. LAMP Primers Design:

Sequences of various PEDV, TGEV and PDCoV strains complete genomes got from the GeneBank and aligned to determine conserved sequences via MEGA X software (http://www.megasoftware.net/). A 277-nt and 233-nt sequence in PEDV and PDCoV nucleocapsid gene, respectively, and 258-nt sequence in the ORF1ab gene of TGEV were targeted as templates due to their homology among the analyzed strains and their divergence from sequences of other organisms. LAMP primers (Fig. 4, 5 and 6) were designed using the PrimerExplorer software (V5, Eiken Chemical Co. Ltd.). The designed primers were checked for cross-hybridization with sequences of other viruses in the NCBI database BLAST (http://www.ncbi.nlm.nih.gov) that infect the digestive tract of pigs, including swine acute diarrhea syndrome-coronavirus (SADS-CoV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), porcine calicivirus, porcine parvovirus and porcine rotaviruses. Cross reaction was not determined with any of these viruses. The LAMP primers were manufactured (IDT Company, Coralville, IA), provided lyophilized, then suspended in nuclease-free water (Invitrogen, Carlsbad, CA, USA) to a concentration of 100 μM.

Fig. 4.

Template regions and sequences of LAMP primers for PEDV: (A) PEDV amplicon gene sequence with the six primers locations: B3, F3, FIP, BIP, LF and LB showed, arrows indicate the direction of extension. (B) Sequence of primers for PEDV LAMP reaction.

Fig. 5.

Target regions and sequences of LAMP primers for TGEV: (A) TGEV polymerase gene sequence with the six primers locations: B3, F3, FIP, BIP, LF and LB showed, arrows indicate the extension di the direction of extension rection. (B) Sequence of parimers for TGEV LAMP reaction.

Fig. 6.

Target regions and sequences of LAMP primers for PDCoV: (A) PDCoV nucleoprotein gene sequence with the six primers locations: B3, F3, FIP, BIP, LF and LB showed, arrows indicate the direction of extension. (B) Sequence of primers for PDCoV LAMP reaction.

2.5. Benchtop RT-LAMP reaction conditions

The RT-LAMP reaction mix with volume of 10 μL contained F3 and B3 primers (0.2 μM of each); LoopF and LoopB primers (0.8 μM of each); FIP and BIP primers (1.6 μM of each); 6 μL of Isothermal MasterMix (ISO-001, OptiGene, USA); 0.4μL 1×EvaGreen® dye (Biotium Inc., Hayward, CA, USA); 0.2 μL of AMV reverse transcriptase (200 U/ μL) (Promega Corp., Madison, WI, USA), 1 μL of extracted RNA template, and nuclease-free water was added to bring the total volume 10 μL. Fluorescence emission of DNA amplicons were monitored with the 7500-Fast Real Time PCR system (Applied Biosystems, Carlsbad, CA, USA) at 65°C for 30 minutes, then analysis of the melting curve from 50°C to 95°C with 1.0°C increment per second was performed. Positive and negative controls were included in each run. Positive controls were PEDV (PC22A strain), TGEV (virulent Miller-M6 strain) and PDCoV (OH-FD22 strain) while non-template rectal swabs from non-infected gnotobiotic pigs were used as negative controls.

2.6. Benchtop qRT-PCR reaction conditions

Each RT-PCR reaction volume comprised of SsoFast EvaGreen^® Supermix (Bio-Rad, USA) (5 μL); primers F3 and B3 (10 μM), 0.2 μL of AMV reverse transcriptase (200 U/ μL), 1 μL of viral RNA, and nuclease-free water to 10 μL final volume. The cycling conditions were as follows: incubation at 50 °C for 30 min (for reverse transcription), followed by incubation at 95 °C for 10 min (melting), and then 45 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by analysis of melting curve in the range of temperature from 50°C to 95°C with 1.0°C increment per second. Monitoring of fluorescence emission was carried out by the 7500-Fast Real Time PCR system (Applied Biosystems, Carlsbad, CA, USA). Positive and negative controls were involved in each run.

2.7. On-chip Detection and Data Analysis

During the test, LAMP master mix is loaded into the 3D-printed microfluidic chip (Fig. 2) via the inlet port, and each LAMP reactor is filled with (20 ± 1) μL of the master mix, which includes 12-μL Isothermal MasterMix (ISO-001, OptiGene, USA), 0.8 μL 1×EvaGreen^® dye, 0.8 μL AMV reverse transcriptase (200 U/μL), along with 1.4 μL RNA template of one of these viruses and nuclease free water to 20 μL.

The chip was then incubated in an inexpensive, homemade, portable heating system. The portable heating system is controlled by a Raspberry Pi (Raspberry Pi Foundation^®, Pi 4 mode B) and equipped with a polyimide Thermofoil^™ heater (Minco^®, HK6911), and a Type-K thermocouple (Omega^®, TT-K-30-SLE). The microcontroller Raspberry Pi, the thin-film heater, and the thermocouple are used to create a closed-loop proportional–integral–derivative (PID) control system. During incubation, the chip is first heated up to 45 °C for 6 min to ensure auto-sealing prior to LAMP amplification. Then, the temperature of the heater is increased to and maintained at 65 °C for 30 minutes to incubate the LAMP reaction.

A portable USB fluorescence microscope (Dino-Lite^®, AM4115T-GFBW) was used to permit ultraviolet light to excite the EvaGreen® dye incorporated in the amplified DNA and read real-time fluorescence signal (Fig. 1). The fluorescence microscope has seven built-in blue LEDs (Light-emitting diodes) for fluorescent excitation and a 510-nm emission filter which is suitable to observe the EvaGreen^® dye intercalated in the amplified DNA. The microscope is mounted on top of the heating system and monitors all LAMP reactors simultaneously (Supplementary Fig. 1).

Images of the chip during incubation were captured with the fluorescence microscope once every minute and regions selected by the user were analyzed with MATLAB (MathWorks^™, R2019a). The camera records the red green blue (RGB) intensity values for each pixel. DinoCapture^™ 2.0 software (AnMo Electronics Corp., Taiwan) bundled with the portable fluorescent microscope then extracts the G (green) value from the RGB matrix of each pixel and calculates the average G value among all pixels included in the region of interest. The average G values of each LAMP reactor represent the relative fluorescence intensities. The averaged and normalized G values are plotted as functions of time. The threshold time is defined as the time that it takes the G value to reach half its peak intensity.

2.8. Limit of detection (LOD)

To determine the smallest copies number of PEDV, TGEV and PDCoV genomes that could be detected with our microfluidic device, benchtop real time RT-LAMP, and qRT-PCR assays, we prepared serial dilutions of the three viruses RNAs suspended in nuclease-free water.

2.9. Clinical performance

We extracted nucleic acids from eleven samples of diseased piglets and one negative control rectal swab from non-infected gnotobiotic pig. These samples were previously tested for PEDV, TGEV and PDCoV with RT-PCR assays.^{7, 12, 13} Among tested samples, 5 samples showed positive results for PEDV, three samples tested positive for TGEV and three samples were PDCoV positive. The negative control sample was negative for all three viruses. We retested all samples concurrently with our microfluidic RT-LAMP, benchtop RT-LAMP and qRT-PCR. Negative and positive controls were included in these tests. Furthermore, we carried out with our microfluidic device co-detection of samples containing nucleic acids from two viruses (e.g., PEDV+TGEV, PEDV+PDCoV, TGEV + PDCoV) and from all three viruses (PEDV +TGEV+PDCoV).

Results:

3.1. Analytical performance of benchtop RT-LAMP and RT-PCR

Fig. 7 (RT-LAMP) and Fig. 8 (RT-PCR) depict the fluorescence emission intensities (arbitrary units) as functions of time for different target concentrations of gRNAs of PEDV, TGEV, and PDCoV, each and the signals’ threshold times / cycles as functions of each target concentration. The threshold time Tt is defined as the time until the amplification curve achieves half its saturation value. The RT-LAMP and RT-PCR threshold times are nearly linear functions of the log of the target concentration. RT-LAMP and RT-PCR have similar limits of detection. The lowest detectable concentration (limit of detection) is 10 genome copies per reaction for PEDV and PDCoV and 10² for TGEV. Analysis of the melting curve revealed a single peak for both RT-LAMP and RT-PCR products (Supplementary Fig. 2), revealing absence of non-specific products such as primers dimmers. Negative controls and non-template controls did not show any signal of positive amplification.

Fig. 7.

Real time RT-LAMP amplification curves of PEDV (A), TGEV (B) and PDCoV (C). The template concentrations range from 0 to 10⁵ genome copies/reaction. The threshold times (minutes) are depicted as functions of PEDV (D), TGEV (E), and PDCoV (F) concentration (genome copies per reaction). N=3.

Fig. 8.

Real time PCR amplification curves off PEDV (A), TGEV (B) and PDCoV (E). The number of templates ranged from 0 to 10⁵ genomic copies/reaction. The threshold times (minutes) are depicted as functions of PEDV (D), TGEV (E), and PDCoV (F) concentration (genome copies per reaction). N=3.

3.2. Microfluidic Chip Analytical Performances

Our microfluidic chip (Fig. 2) comprises four independent isothermal amplification reactors, each with a dry-stored primer set specific for a designated target. The identity of the amplicon is determined by the reaction chamber’s location. All four reactors are within the field of view of the camera, enabling us to concurrently monitor fluorescent emission from all LAMP reactors in real time. When only one of the targets is present in the sample, the corresponding LAMP reactor lights up, while the other reactors and the non-primer control (NPC) remain dark (Fig. 9 A and B). The analytic performance of the chip was performed using template concentrations range from 0 to 10² genome copies/reaction and showed similar results to the benchtop assays.

Fig. 9.

(A) Image of fluorescence emission at the end of the amplification process when the sample contains only PEDV (chamber no. 3). (B) Reaction chambers’ average fluorescence intensities as functions of time when the sample contains only PEDV (C) Detection of co-morbidities, Image of fluorescence emission when the sample contains only both TGEV and PEDV (chamber no. 2 and 3). (D) Amplification curves when the sample contains both PEDV and TGEV. (E) Detection of co-morbidities, Image of fluorescence emission when the sample contains PEDV, TGEV and PDCoV (chambers no. 1, 2 and 3). (F) Amplification curves when the sample contains PEDV, TGEV and PDCoV.

One of the advantages of our chip is its ability to concurrently test for the presence of multiple targets in a sample, and thus indicating co-morbidities. To demonstrate this, we contrived samples spiked with one, two, and all three different viruses. In the presence of co-morbidities, either two test reactors or all three test reactors light up (Fig. 9 C, D, E and F). Our assay co detected successfully PEDV+TGEV, PEDV+PDCoV, TGEV + PDCoV, and (PEDV +TGEV+PDCoV).

3.3. Performance of the benchtop assays with clinical samples

Eleven samples from diseased piglets were tested for PEDV, TGEV and PDCoV with our benchtop RT-LAMP assays, our chips running RT-LAMP assays, and qRT-PCR (Fig. 10 and supplementary Table 1). Based on earlier qPCR, 5 pigs were positive to PEDV, 3 pigs were positive to TGEV, 3 pigs were positive to PDCoV, and one sample was negative to all these pathogens. All samples were retested with qRT-PCR in our lab in proximity with our RT-LAMP tests. All benchtop RT-LAMP and chip-based RT-LAMP assays had 100% selectivity and 100% sensitivity in comparison to our qRT-PCR and with data provided by the laboratory from which the clinical samples originated.

Fig. 10.

Testing of clinical samples from diseased piglets with the microfluidic based-LAMP, benchtop RT-LAMP and benchtop RT-PCR assays. (A) Microfluidic based LAMP-threshold time as a function of qRT-PCR threshold cycle. (B) Benchtop RT-LAMP threshold time as a function of benchtop qRT-PCR threshold time. (C) Chip RT-LAMP threshold time as a function of benchtop RT-LAMP threshold time (● for PEDV samples; ▄ for TGEV samples; ▲ for PDCoV).

The benchtop RT-LAMP and the microfluidic RT-LAMP threshold times were shorter than that of the qRT-PCR and trended similarly (Supplementary Table 1). The threshold times of benchtop RT- LAMP-assay and microfluidic-based RT-LAMP correlated linearly (R² = 0.7). The aggregated data of all threshold times in our chip (regardless of target type) exhibited a weaker correlation (R² = 0.4, Fig. 10A) with the qRT-PCR threshold cycle. A stronger correlation was, however, obtained (R² = 0.8–0.9) when each pathogen was analyzed separately (Supplementary Fig. 3).

Discussion:

Swine enteric viruses such as PEDV, TGEV and PDCoV are a major threat to the pig industry, causing substantial economic losses.^{1, 4} For example, losses due to PED are estimated at $300,000 per year for a single 700-sow farrow-to-finishing herd.³² To rapidly implement suitable control measures to contain the infection, rapid molecular testing of suspected animals is needed. Since PEDV, TGEV and PDCoV are co-endemic and cause similar symptoms in animals, it is highly desirable to co-test for all three pathogens. Currently, diagnosis for PEDV, TGEV and PDCoV in suspected pigs is carried out with laboratory-based RT-PCR assays. A previous report³³ describes a multiplex molecular detection of swine enteric coronaviruses with RT-PCR combined with post-amplification detection with gel electrophoresis. In addition to the complex time-consuming operation, the necessity to open amplicon rich tubes endangers contamination of the workspace. Another report³⁴ describes a multiplexed assay that uses TaqMan-probes for specific detection of enteric coronaviruses targets with real-time qRT-PCR. Such an assay requires an expensive thermal cycler with a multicolor reader; is limited in the level of multiplexing; and has a 10-fold lower sensitivity than the single-plexed assays, presumably because probes emit less light than intercalating dye and/or due to the competition for enzymes.

The various PCR-based assays require well-equipped facilities, possibly expensive instruments, and trained personnel that typically are not available in farms and often are located a large distance away, which complicates testing and delays test results. To facilitate testing in resource poor settings such as rural areas and developing countries, we have developed new benchtop single-plex RT-LAMP assays to detect PEDV, TGEV and PDCoV. RT- LAMP is more suitable for use in resource poor settings than RT-PCR. Since LAMP operates at fixed temperature, ranging from 60 °C to 65 °C, does not require precise incubation temperature, and does not require temperature cycling, it can be incubated with simple instruments such as ESEQuant TubeScanner (GmbH, Stockach, Germany), Genie II (Optigene, Horsham, UK), and a water bath or even instrumentation and electricity-free with heating provided by an exothermic chemical reaction of the type used in military rations and in meals ready-to-eat and temperature regulation provided with a phase change material (PCM)^{35, 36} LAMP’s constant temperature operations also consumes less power than the thermal cycling needed for PCR. LAMP products can be detected with intercalating dye and molecular beacons like PCR products. But, since LAMP produces about an order of magnitude greater number of amplicons than PCR, LAMP products and byproducts can also be detected with colorimetric dyes that are visible to the naked eye, eliminating the need for a reader. Furthermore, fluorescent emission from LAMP can be detected with the ubiquitous smartphone^{35, 36}. Finally, LAMP assays are more tolerant of contaminants than PCR assays,³⁷ simplifying sample preparations. In summary, LAMP assays are highly sensitive, specific, robust, can be carried out over a temperature range (60–65 °C) in less than one hour, and are more suitable for use outside centralized laboratories than PCR.^{38, 39}

Our benchtop PEDV, TGEV and PDCoV RT-LAMP assays perform on par with the gold standard qRT-PCR assays with shorter processing times. Like the benchtop qRT-PCR, our RT-LAMP assays are processed in closed tubes without a need to open the tube and transfer amplification products to a lateral flow strip – a process that risks contaminating the workspace and rendering future negative tests false positives. Each of our RT-LAMP assays for PEDV and PDCoV have a limit of detection of 10 gRNA molecules per reaction. Our RT-LAMP assay for TGEV has a limit of detection of 100 gRNA molecules per reaction. These limits of detection are appropriate for early-stage infection detection.^{15, 40, 41} Our assays’ threshold times correlate linearly with the log of template concentration, enabling estimation of the number of viruses in the sample.

To confirm our assays clinical performance for virus detection, we tested elven clinical specimens from diseased pigs with our benchtop RT-LAMP assays. Our LAMP test results are in excellent agreement with the gold standard qRT-PCR. We believe that our assays are ready for field tests.

To enable convenient co-detection of co-endemic targets, we developed a microfluidic system, comprising of a 3D-printed microfluidic chip and a simple processor. The 3D – printing technology allows us rapid, low-cost prototyping. In departure from earlier works on LAMP on a chip that typically process a single target at a time,^{42, 43} our chip hosts four independent reaction chambers, enabling co-testing for multiple, co-endemic pathogens in a single sample at the point of need. Three of our reaction (test) chambers are customized for testing for each of the pathogens PEDV, TGEV and PDCoV by pre-storing the corresponding primer sets in the reaction chambers. The fourth reaction chamber serves as a negative control. Our chip architecture can readily accommodate a larger number of reaction chambers to enable the co-detection of a larger number of targets, if needed.

The loading and drying operations in our chip are driven by capillary forces and do not require external instruments for flow control, in contrast to many other microfluidic devices that require syringe pumps and centrifugal forces.^{44, 45} We rely on capillary imbibition and capillary valves to aliquot the sample among the various reaction chambers and, after sample filling, to severe the connections among the reaction chambers to eliminate any crosstalk and false positives. Our chips self-seal with temperature-actuated phase change material (PCM). During incubation, the melted PCM prevents both evaporation from the reaction chambers and amplicons escaping and contaminating the workspace. After amplification, the PCM solidifies tombing the reaction mix. Given the limited scope of this study, we were not able to dry store reagents inside our chip. In the future, we will store all reagents inside the chip in dry form for long-term (over one year) refrigeration-free shelf-life, and the sample will include only the extracted nucleic acids.

We demonstrated that our RT-LAMP chip has similar performance to that of our benchtop RT-LAMP assays and that we can detect any of the targeted analytes in a single process as well as comorbidities. The chip required a bit longer processing times than our benchtop RT-LAMP, which we attribute to the lower performance of our heating system compared to the benchtop equipment. We anticipate that with further improvements our microfluidic system would enable affordable molecular testing at the point of need by minimally trained personnel, providing timely and actional information to farmers. In conclusion, our field deployable real time-RT-LAMP assays and microfluidic chips have the potential to facilitate rapid, inexpensive, multiplexed molecular detection and quasi-quantification of PEDV, TGEV and PDCoV in basic veterinary laboratories and in pigs’ farms.