Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment

Abstract

Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 13 (50 genomes/reaction) or 23 (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.

Real-time monitoring of isothermal nucleic acid amplification on a smartphone by using a portable electrochemical device for home-testing of SARS-CoV-2

“…In contrast, the device could judge the detection results and present them to the users on smartphones or its indicator panel by the preset threshold in MCU, rather than judging the results by the users themselves, which would avoid the human errors. Although some automated colorimetric detection devices could also achieve the real-time monitoring of INAATs and judging the detection results, even some of which have been proved to have higher sensitivity than electrochemical detection devices, the high expense of the required sophisticated optics might restrict their extension application [ [36] , [37] , [38] ]. Besides SARS-CoV-2 pseudovirus, another pathogen, Escherichia coli O157:H7 was also employed as a target to evaluate the performance of E-INAATs device on the detection of DNA targets.…”

SMART-LAMP: A Smartphone-Operated Handheld Device for Real-Time Colorimetric Point-of-Care Diagnosis of Infectious Diseases via Loop-Mediated Isothermal Amplification

“…In all, we present a novel handheld device for the diagnosis of infectious diseases via colorimetric real-time LAMP assay. Only a few examples of similar platforms have been described, which rely either on pH indicators [ 55 , 56 ] or Hydroxynaphtol blue dye [ 56 ], but, to the best of our knowledge, none of them are based on MG. Additionally, those devices do not make use of ready-to-use mixes, thus making them potentially less applicable to POC settings. Moreover, we have demonstrated that the SMART-LAMP is applicable, without a specific optimization, for the detection of a number of different pathogens using a single positivity threshold for all the assays, hence providing straightforward applications for new diagnostics.…”

“…Regarding SARS-CoV-2 RNA detection in clinical samples, the platform presented by Papadakis et al [ 56 ] showed a 97% sensitivity and 100% specificity when analyzing a total of 89 samples. On the other hand, Diaz et al [ 55 ] used the commercial BioRanger diagnostic platform modified for colorimetric LAMP for COVID-19 testing in a limited number of simulated samples spiked with either synthetic RNA or inactivated SARS-CoV-2 virus ( n = 20; 10 positives and 10 negatives), correctly detecting the 10 positive samples and 9/10 of the negatives. In both studies, the results obtained were comparable with those yielded by our SMART-LAMP device.…”

Clinical Validation of a Colorimetric Loop-Mediated Isothermal Amplification Using a Portable Device for the Rapid Detection of SARS-CoV-2

“…However, a limitation of the Hilab Molecular is the sample number that can be analyzed at a time; only one sample plus the positive, negative, and intern control. Another device, with similar technology and space for eight microtubes, has been described elsewhere [ 26 ].…”