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

Abstract

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.

Feasibility of Rapid Diagnostic Technology for SARS-CoV-2 Virus Using a Trace Amount of Saliva

“…From this, it is considered that the qPCR method is highly useful in the future. Recently, the development of a LAMP test using saliva as a sample [10] and an attempt to place a satellite laboratory in the inpatient ward of a COVID-19 patient to perform a rapid test [11] have been reported. Various other test methods have been developed and studied [12][13][14][15][16], but none have achieved the same sensitivity and specificity as the conventional qPCR test.…”

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

“…These dyes allow visualization of the result with the naked eye. In the assay presented here, we build on the primers previously designed by us [ 16 ] and couple them with a pH-sensitive colorimetric reagent: phenol red [ 28 ]. The primers were screened through the LOD study.…”

“…As seen in previous studies, the Warmstart ® LAMP 2 × Master Mix, which contains phenol red, is characterized by its transition from pink to yellow as the LAMP reaction occurs and the pH decreases [ 28 , 34 ]. Positive and negative results using our selected primers for the detection of P. multocida , M. haemolytica , H. somni , and the pink and yellow distinction can be observed in Figure 2 .…”

“…Since the 2000s [12], LAMP has been a widely used method for the detection of pathogens, including bacterial [18][19][20][21][22] and viral targets [23][24][25][26][27][28]. While a majority of these efforts have been made using fluorescent dyes, there have been some advances in the detection of pathogens by coupling LAMP reagents with dyes that are sensitive to magnesium [29,30] or pH [28,[31][32][33]. These dyes allow visualization of the result with the naked eye.…”

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

“…Herein, we present the fabrication of a paper-based device that uses loop-mediated isothermal amplification (LAMP) to detect nucleic acids of pathogens of interest in complex samples by providing a colorimetric response observable to the naked eye. We have previously demonstrated that this device can be used to detect SARS-CoV-2 in human saliva with a limit of detection of 200 copies/µL saliva [2] . Unlike most nucleic-acid-based biosensors for SARS-CoV-2 detection [ 1 , 3 , 7 ], this device does not require prior RNA extraction or concentration.…”