A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19

Abstract

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

“…In this study, we confirm that one of the fastest, simplest, cheapest and most scalable protocols available for detecting SARS-CoV-2 nucleic acid is so far reliable for strong positives only, but highly specific compared to RT-PCR detection. Other studies have shown similar reduced sensitivity but high specificity [ 23 , [25] , [26] , [27] , [28] ]. One early study found that RT-LAMP was more sensitive than RT-PCR at detecting virus in an asymptomatic carrier monitored for several days [ 29 ].…”

Field-deployable, rapid diagnostic testing of saliva for SARS-CoV-2

“…The equipment costs for performing the HP-LAMP assay are very low; performing the assay requires only a pipette, a mini centrifuge, a vortexer, and two heat blocks that retail for ~ 250 USD each. In contrast, the equipment costs for RT-PCR based methods is > 45,000 USD while the automated Roche cobas 6800 unit costs ~ 350,000 USD 27 , 28 . If purchasing the consumable reagents individually using off-the-shelf components, the costs for the HP-LAMP assay is ~ 80 USD per assay, and ~ 16 USD per assay when pooling five samples.…”

“…The cost for pre-made HP-LAMP cocktails is 20–25 USD per assay (Sorrento Therapeutics). The consumable cost for RT-PCR based methods is 20–60 USD per assay 27 .…”

Current status, advances, challenges and perspectives on biosensors for COVID-19 diagnosis in resource-limited settings

“…Easy readable results [36] N/A PCR fluorometer Pseudovirus samples ORF1b gene, N gene RT-PCR ~ 30 min 10 copies of pseudovirus Magnetic NP-based RNA extraction [55] N/A N/A Nasopharyngeal swab N gene RT-LAMP < 35 min 1.0 × 10 1 copies/μL Simple turbidity results [57] N/A N/A N/A Synthesized ORF1ab gene RT-LAMP > 20 min 10 copies/µL Isothermal amplification, simple colorimetric readout [60] N/A N/A N/A Synthetic ORF1ab gene Penn-LAMP < 1 h 7 copies/reaction Isothermal reaction, high sensitivity, [65] N/A Portable test strip N/A ORF1ab and S gene SHERLOCK < 1 h 10 copies/µL. POC paper stick, easy colorimetric readout [81] N/A Mobile unit Throat swab, sputum, and nasopharyngeal samples ORF3a and E gene RT-LAMP < 2 h N/A Van-sized unit; suitable for quarantine camp [82] N/A Customized instrument Nasopharyngeal swabs RdRp gene RT-LAMP ~ 30 min 10 2 copies/reaction Eye-observable and artificial intelligence-assisted color readout; gel electrophoresis double-check [83] N/A PCR fluorometer Clinical sample RdRp gene RT-PCR < 30 min 11.2 copies/reaction High sensitivity and specificity …”