Abstract
The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.
Highlights
The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level
The polymerase used in Loop-mediated isothermal amplification (LAMP) (Bst) is more robust than that used in traditional PCR and can function in the presence of PCR inhibitors frequently found in bodily fluids such as saliva and viral transport media (VTM), without the need to first purify the RNA
Our results indicate that reverse transcription loop mediated isothermal amplification (RT-LAMP) has the 75% (9/12) positive percentage agreement (PPA) below limit of detection (LoD), 100% (8/8) PPA within LoD, and 100% (10/10) negative percentage agreement (NPA) (Table 2)
Summary
The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient onestep testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings. We sought to develop and share a highly-sensitive, one-step assay for SARS-CoV-2 that could be performed directly on clinical samples as a pointof-care test utilizing readily available reagents and equipment
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