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

Abstract

The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 °C for 30 minutes, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.

Comparison of RT-PCR, RT-LAMP, and Antigen Quantification Assays for the Detection of SARS-CoV-2

“…The specificity of RT-LAMP was 100% for crude RNA samples (10), which is consistent with our results and indicates that there are few false positives when using the RT-LAMP test. On the other hand, false positives have been reported to occur with low frequency when using a crude sample with saliva (12) and NPS (13).…”

“…Using crude RNA (purification-free method), it was difficult to detect less than 250 RNA copies. A previous report showed no positives with a Ct greater than 30 using the purification-free RT-LAMP method (10). Since 250 copies of RNA gave a Ct value of approximately 31 under our experimental conditions, the detection limit of the RT-LAMP method with crude RNA was about Ct 31.…”

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

“…After these modifications, RT-LAMP achieved 77.2% overall clinical sensitivity and 97% specificity, with 93.2% sensitivity in saliva containing at least 10 2 viral copies/µL (Kappa = 0.895), which is on par or more sensitive compared to most of the direct RT-LAMP approaches reported so far [8,15,[25][26][27][28][29][30][31][32][33]. Moreover, the high agreement (>97.6%) observed between color interpretation and specific amplification plots in rtRT-LAMP demonstrates reliability on end-point color readout if real-time analysis is not possible.…”

A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset

“…After these modifications, RT-LAMP achieved 77.2% overall clinical sensitivity and 97% specificity, with 93.2% sensitivity in saliva containing at least 10 viral copies/µL (Kappa = 0.895), which is on par or more sensitive compared to most of the direct RT-LAMP approaches reported so far (7,12,(22)(23)(24)(25)(26)(27)(28)(29)(30). Moreover, the high agreement (>97.6%) observed between color interpretation and specific amplification plots in rtRT-LAMP demonstrates reliability on end-point color readout if real-time analysis is not possible.…”