Jiangshan Wang

African swine fever virus (ASFV) poses a major transboundary threat to global swine production, underscoring the need for rapid and field-deployable diagnostic tools. Although quantitative polymerase chain reaction (qPCR)-based assays are the standard molecular assay for ASFV detection, their reliance on centralized laboratory infrastructure, multi-step sample preparation, and trained personnel limit their utility for timely decision-making at the point of need (PON). Here, we report a portable molecular diagnostic platform that enables colorimetric quantitative loop-mediated isothermal amplification (qLAMP) directly from diluted whole blood on microfluidic paper-based analytical devices (μPADs). The assay targets the conserved ASFV viral protein 72 (VP72) and topoisomerase II (TOPII) genes and incorporates objective image-based colorimetric signal analysis to reduce user-dependent interpretation. Using plasmid DNA spiked into whole blood diluted to 5% (v/v) in 5% D-mannitol, the μPAD-LAMP assay achieved a limit of detection (LOD) of 25 copies per reaction (67 copies/μL of whole blood sample) for VP72 targets with no observed cross-reactivity against nine common swine pathogens, demonstrating 100% analytical sensitivity and specificity during in-house testing and 90% and 92% analytical sensitivity and specificity respectively in an external laboratory evaluation. The complete assay was performed within 60 minutes using a portable heating and imaging platform. Together, these results demonstrate a simple, DNA extraction-free molecular diagnostic approach that enables rapid and reliable ASFV detection from whole blood applicable to field-relevant conditions.

Fresh produce encounters pathogens at various stages of production and supply, with the harvesting process serving as one of these stages. To evaluate contamination associated with harvesting, we systematically swabbed zone 1 harvester surfaces and quantified Bacteroidales as a fecal biomarker using quantitative polymerase chain reaction (qPCR). Baseline contamination was dominated by non-detects, with occasional low-level detections (2 ) near the assay limit of detection (LoD). Detection occurred more frequently post-harvest (overall ∼4% pre-harvest and 10% post-harvest), while microbial loads remained low, indicating that harvesting primarily affected the likelihood of low-level contamination rather than increasing contamination abundance. Additionally, we developed and field-deployed a portable loop- mediated isothermal amplification (LAMP) assay for rapid harvester hygiene assessment and benchmarked its field performance against qPCR. Together, these results support a practical molecular tool for monitoring fecal contamination and informing cleaning and sanitization decisions.

Microfluidic paper-based analytical devices (µPADs) are an attractive format for colorimetric nucleic acid amplification tests (NAATs) because they enable low-cost, portable diagnostics in resource-limited settings. However, researchers often optimize colorimetric assays in liquid reactions in tubes before translating them to µPADs. Since both formats require separate instruments for incubation and real-time sensing, direct comparison of reactions between the two formats is difficult. To address these cross-platform limitations, we developed ThermiQuant™ AquaStream, a portable benchtop device (15 × 20 × 16 cm, ~ 5 kg; cost: USD 327) that supports seamless colorimetric loop-mediated isothermal amplification (LAMP) reactions in both µPADs and tubes under a common workflow. The system enables real-time reaction tracking (every 30 seconds) through onboard image processing, precise isothermal control (±0.5 °C) using a repurposed consumer-grade sous-vide heater, and medium-throughput (24 tubes or 42 µPADs). Testing with synthetic SARS-CoV-2 orf7ab DNA fragments demonstrated a limit of detection corresponding to a 95% probability of detection (LOD95) of 110 copies per reaction in tube (22 copies/µL) and 39 copies per reaction in µPADs (5 copies/µL), estimated using probit regression. In both formats the limit of quantification (LOQ), defined as the lowest concentration yielding a coefficient of variation (CV) of quantification time (Tq) ≤ 10%, was 250 copies/reaction resulting in a strong linear (R ² = 0.98 & R ² = 0.96 respectively for tube and µPADs) standard calibration curves. ThermiQuant™ AquaStream provides an affordable and versatile benchtop platform capable of supporting both tube- and µPAD-based colorimetric LAMP assays, serving as a proof-of-concept research tool for assay development and molecular diagnostics in One Health settings such as clinics, farms, and field environments.

Microfluidic paper-based analytical devices (µPADs) are an attractive format for colorimetric nucleic acid amplification tests (NAATs) because they enable low-cost, portable diagnostics in resource-limited settings. However, researchers often optimize colorimetric assays in liquid reactions in tubes before translating them to µPADs. Since both formats require separate instruments for incubation and real-time sensing, direct comparison of reactions between the two formats is difficult. To address these cross-platform limitations, we developed ThermiQuant™ AquaStream, a portable benchtop device (15 × 20 × 16 cm, ∼5 kg; cost: USD 327) that supports seamless colorimetric loop-mediated isothermal amplification (LAMP) reactions in both µPADs and tubes under a common workflow. The system enables real-time reaction tracking (every 30 seconds) through onboard image processing, precise isothermal control (±0.5 °C) using a repurposed consumer-grade sous-vide heater, and medium-throughput (24 tubes or 42 µPADs). Testing with synthetic SARS-CoV-2 orf7ab DNA fragments demonstrated a limit of detection corresponding to a 95% probability of detection (LOD95) of 110 copies per reaction in tube (22 copies/µL) and 39 copies per reaction in µPADs (5 copies/µL), estimated using probit regression. In both formats the limit of quantification (LOQ), defined as the lowest concentration yielding a coefficient of variation (CV) of Tq ≤ 10%, was 250 copies/reaction resulting in a strong linear (R ² = 0.98 & R ² = 0.96 respectively for tube and µPADs) standard calibration curves. ThermiQuant™ AquaStream provides an affordable and versatile benchtop platform capable of supporting both tube- and µPAD-based colorimetric LAMP assays, serving as a proof-of-concept research tool for assay development and molecular diagnostics in One Health settings such as clinics, farms, and field environments.

Propidium monoazide (PMA) is a dye that distinguishes between live and dead cells in molecular assays like the Polymerase Chain Reaction (PCR). It works by cross-linking to the DNA of cells that have compromised membranes or extracellular DNA upon photoactivation, making the DNA inaccessible for amplification. Currently, PMA is used to detect viable pathogens and alleviate systemic bias in the microbiome analysis of samples using 16S rRNA gene sequencing. In these applications, treated samples consist of different amounts of dead bacteria and a range of bacterial strains, variables that can affect the performance of PMA and lead to inconsistent findings across various research studies. To evaluate the effectiveness of PMA, we used a sensitive qPCR assay and post-treatment sample concentration to determine PMA cross-linkage and activity accurately under varying sample conditions. We report that PMA is unreliable for viability assays when the concentration and composition of the bacterial mixtures are unknown. PMA is suitable only for qualitatively assessing viability in samples containing a known number of dead microbes or extracellular DNA.

Nucleic-acid biosensors have emerged as useful tools for on-farm detection of foodborne pathogens on fresh produce. Such tools are specifically designed to be user-friendly so that a producer can operate them with minimal training and in a few simple steps. However, one challenge in the deployment of these biosensors is delivering precise sample volumes to the biosensor’s reaction sites. To address this challenge, we developed an innovative drop dispenser using advanced 3D printing technology, combined with a hydrophilic surface chemistry treatment. This dispenser enables the generation of precise sample drops, containing DNA or bacterial samples, in volumes as small as a few micro-liters (∼20 to ∼33 μL). The drop generator was tested over an extended period to assess its durability and usability over time. The results indicated that the drop dispensers have a shelf life of approximately one month. In addition, the device was rigorously validated for nucleic acid testing, specifically by using loop-mediated isothermal amplification (LAMP) for the detection of Escherichia coli O157, a prevalent foodborne pathogen. To simulate real-world conditions, we tested the drop dispensers by integrating them into an on-farm sample collection system, ensuring they deliver samples accurately and consistently for nucleic acid testing in the field. Our results demonstrated similar performance to commercial pipettors in LAMP assays, with a limit of detection of 7.8×10⁶ cells/mL for whole-cell detection. This combination of precision, ease of use, and durability make our drop dispenser a promising tool for enhancing the effectiveness of nucleic acid biosensors in the field.