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

Abstract

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.

Keywords: COVID-19; Isothermal amplification; RAMP; Two stage,; closed-tube; nested amplification.

Figure 1:. Comparison of LAMP, RT-PCR, and Closed-Tube Penn-RAMP for COVID-19 Detection.

(A) LAMP, (B) PCR, (C) closed-tube Penn-RAMP detection of 70000, 7000, 700, 70, 7, and 0 (no template control) copies per reaction. The limits of detection of LAMP, PCR, and closed-tube Penn-RAMP are, respectively 70, 70, and 7 copies per reaction. The threshold time of (D) LAMP, threshold cycle of (E) PCR, and threshold time of (F) one-tube Penn-RAMP as functions of the log of nCoV-2019 target concentration (n = 3).

Figure 2:. LAMP primers for the COVID-19 are specific.

Only samples with COVID-19 nucleic acid show positive signal, while negative coronaviruses controls (PEDV, TGE, PDCoV, and IBV) and non-template control (NTC) do not show any signal during incubation.

Figure 3:. Home Test for COVID-19.

(A) Visual detection of COVID-19 with our one-tube Penn-RAMP with LCV dye. Negative: 0 copies of COVID-19 synthesized DNA; Positive: 100 copies of synthesized DNA. (B) Sequence of operations of the home test. The reactions can be incubated either in a block heater or in a domestic oven with temperature control.