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.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7–100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60–100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

This work develops a loop-mediated isothermal amplification (LAMP) assay that detects the presence of bacterial pathogens (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) for bovine respiratory disease complex (BRD) in crude nasal samples. Diagnosing BRD involves a physical examination of cattle expressing physical symptoms linked to the disease. Unfortunately, these symptoms are not unique to BRD alone and do not identify specific pathogens to treat. Nucleic acid-based diagnostics, like polymerase chain reaction (PCR), identify BRD pathogens by amplifying species-specific genes present in samples. However, PCR-based approaches require (i) expensive equipment for successful operation and (ii) DNA extractions to remove PCR inhibitors in samples. LAMP offers an accurate, inhibitor-resistant approach to detecting BRD pathogens in a point-of-care format. This developed LAMP assay is 97% accurate in pure DNA samples, 99% sensitive, and 89% specific in DNA-spiked bovine nasal samples (with 10 4 DNA copies/reaction).

No abstract