Fabrication of a paper-based colorimetric molecular test for SARS-CoV-2.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused unprecedented damage to the global economy. Diagnostic testing is a key factor in limiting virus transmission and safeguarding public health. We present the fabrication process of a paper-based device that uses reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 in complex matrices by providing a colorimetric response apparent to the naked eye. Because of LAMP's functionality, this device just requires a simple heat source (e.g., water bath, incubator), it can be deployed in resource-constrained areas and can be used as a supplement to current point-of-care (POC) and community testing procedures. Since the test is based on nucleic acids, the testing platform itself lends to further applications including food safety monitoring, animal diagnostics, etc. simply by changing the specific primers.•We developed a platform capable of on-paper detection of SARS-CoV-2 using colorimetric reporters that produce responses visible to the naked eye.•The platform is easily reconfigurable to target different pathogens by changing the primer sets, and multiplexing is possible by adding additional reaction sites to the device.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

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.

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva.

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.