Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.

Electricity-free chemical heater for isothermal nucleic acid amplification with applications in COVID-19 home testing.

Molecular detection of pathogenic nucleic acids from patient samples requires incubating biochemical reactions at specific temperatures to amplify DNA. This incubation is typically carried out with an electrical heater and a temperature controller. To reduce test cost, to eliminate the need for manufacturing incubators, which may require significant time, and to enable electricity-free operation, we use energetic compounds such as an Mg(Fe) alloy mixed with a phase-change material (PCM) that undergoes phase transformation at the desired incubation temperature. We dubbed this composite Energetic Phase Change Material (EPCM). When the EPCM is brought into contact with water, the magnesium alloy interacts with the water to produce heat. The EPCM heats up to its phase transition temperature. Any excess heat is absorbed as latent heat and the system is maintained at its desired incubation temperature, independent of ambient temperatures, long enough to facilitate enzymatic amplification. The EPCM together with colorimetric amplicon detection facilitates an inexpensive, disposable, point-of-need diagnostic test that does not require any electric power. We demonstrate the feasibility of our approach by detecting SARS-Cov-2 in saliva samples either without any instrumentation or with a palm-size CCD camera that enables us to follow the amplification process in real time.

A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry (preprint)

The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training.