Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

Abstract

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT—PCR (Reverse transcription—Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT—LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross‐reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called “COVIDISC” to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

“…Dilution reduces the buffering capacity of saliva and decreases the concentration of inhibitory components, both of which would delay colorimetric reporting. We found dilution preferable to more commonly used pre-treatment steps found in a variety of studies, such as pre-treatment with proteases ( Janíková et al, 2021 ; Wei et al, 2021b ), Chelex® 100 ( Yaren et al, 2021 ) or RNA extraction steps to inactivate inhibitory components of saliva ( Garneret et al, 2021 ; Yamazaki et al, 2021 ), as it is less complex on the end-user.…”

Integrated Microfluidic-Based Platforms for On-Site Detection and Quantification of Infectious Pathogens: Towards On-Site Medical Translation of SARS-CoV-2 Diagnostic Platforms

“…Both types of microfluidic systems can be leveraged as a complementary component to existing diagnostic tools to attain complete or partial automation, multiplexing and high throughput [ 32 , 33 ]. These medical diagnosis steps generally include sample preparation, sample collection and labeling, signal reading and data analysis.…”

Population (Antibody) Testing for COVID-19—Technical Challenges, Application and Relevance, an English Perspective

“…The mainstay or recognized Gold Standard of COVID-19 confirmation is the detection of SARS-CoV-2 nucleic acid in appropriately taken specimens [ 8 , 9 ]. Typically, polymerase chain reaction (PCR) methods have been used; however, other methodologies such as loop mediated isothermal amplification (LAMP) have given promising results [ 9 , 10 ]. Virus antigen tests using lateral flow technology are also widely available offering the means to screen for infection in non-laboratory settings in real time [ 11 , 12 ].…”