Journal Article

Rapid isothermal amplification and portable detection system for SARS-CoV-2

Ganguli, Anurup, Sun, Fu, Valera, Enrique, Mostafa, Ariana, Berger, Jacob, Bashir, Rashid, de Ramirez, Sarah A. Stewart, Cunningham, Brian T., King, William P., Aydin, Mehmet Y.

2020

Overview

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

• Publisher: National Academy of Sciences
• Subject: Assaying / Betacoronavirus - genetics / Betacoronavirus - pathogenicity / Biological Sciences / Cartridges / Coronavirus Infections - diagnosis / Coronaviruses / COVID-19 / COVID-19 infection / detection limit / Diagnostic systems / diagnostic techniques / Engineering / Humans / infrastructure / Laboratories / Limit of Detection / Medical Sciences / Molecular Diagnostic Techniques - instrumentation / Molecular Diagnostic Techniques - methods / Molecular Diagnostic Techniques - standards / Nasal Mucosa - virology / nose / Pandemics / Physical Sciences / Pneumonia, Viral - diagnosis / point-of-care systems / Point-of-Care Testing - standards / Polymerase chain reaction / Portable equipment / Reproducibility of Results / Reverse Transcriptase Polymerase Chain Reaction - instrumentation / Reverse Transcriptase Polymerase Chain Reaction - methods / Reverse Transcriptase Polymerase Chain Reaction - standards / Reverse transcription / reverse transcription loop-mediated isothermal amplification / Ribonucleic acid / RNA / SARS-CoV-2 / Severe acute respiratory syndrome coronavirus 2 / Smartphone / Transport media / Viral diseases / Viruses