Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies/μL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex® backing via an ARclean® double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.

Paper-based biosensors are microfluidic analytical devices used for the detection of biochemical substances. The unique properties of paper-based biosensors, including low cost, portability, disposability, and ease of use, make them an excellent tool for point-of-care testing. Among all analyte detection methods, nucleic acid-based pathogen detection offers versatility due to the ease of nucleic acid synthesis. In a point-of-care testing context, the combination of nucleic acid detection and a paper-based platform allows for accurate detection. This review offers an overview of contemporary paper-based biosensors for detecting nucleic acids from pathogens. The methods and limitations of implementing an integrated portable paper-based platform are discussed. The review concludes with potential directions for future research in the development of paper-based biosensors.

Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations are towards clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm²) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.

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.

Foodborne outbreaks caused by fecal contamination of fresh produce represent a serious concern to public health and the economy. As the consumption of fresh produce increases, public health officials and organizations have pushed for improvements in food safety procedures and environmental assessments to reduce the risk of contamination. Visual inspections and the establishment of "buffer zones" between animal feeding operations and producing fields are the current best practices for environmental assessments. However, a generalized distance guideline and visual inspections may not be enough to account for all environmental risk variables. Here, we report a baseline measurement surveying the background Bacteroidales concentration, as a quantitative fecal contamination indicator, in California's Salinas Valley. We collected a total of 1632 samples from two romaine lettuce commercial fields at the time of harvesting through two seasons in a year. The quantification of Bacteroidales concentration was performed using qPCR, revealing a notably low concentration (0-2.00 copies/cm²) in the commercial fields. To further enhance the applicability of our findings, we developed a user-friendly method for real-world fecal contamination risk assessment that seamlessly integrates with industry practices. Through the generation of heatmaps that visually illustrate varying risk levels across fields, this approach can identify site-specific risks and offer fresh produce stakeholders a more comprehensive understanding of their land. We anticipate this work can encourage the use of Bacteroidales in the fresh produce industry to monitor fecal contamination and prevent future foodborne outbreaks.