Journal:Point-of-care RNA-based diagnostic device for COVID-19

Full article title	Point-of-care RNA-based diagnostic device for COVID-19
Journal	Diagnostics
Author(s)	Yang, Ting; Wang, Yung-Chih; Shen, Ching-Fen; Cheng, Chao-Min
Author affiliation(s)	National Tsing Hua University, National Defense Medical Center, National Cheng Kung University
Primary contact	Email: chaomin at mx dot nthu dot edu dot tw
Year published	2020
Volume and issue	10(3)
Article #	165
DOI	10.3390/diagnostics10030165
ISSN	2075-4418
Distribution license	Creative Commons Attribution 4.0 International
Website	https://www.mdpi.com/2075-4418/10/3/165/htm
Download	https://www.mdpi.com/2075-4418/10/3/165/pdf (PDF)

Point-of-care RNA-based diagnostic device for COVID-19

At the end of 2019, the novel coronavirus disease 2019 (COVID-19), a fast-spreading respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was reported in Wuhan, China and has now affected over 123 countries globally. As of March 14, 2020, the death toll has exceeded 5,400, and there have been 145,000 confirmed cases, causing not only a huge medical health burden but also tremendous economic losses worldwide. Unlike previous coronaviruses that caused large-scale epidemics, such as the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), the transmission rate for COVID-19 is much higher, with an average of two to three people becoming infected for every already infected person. Currently, there are two primary methods for diagnosing COVID-19: (1) a lateral flow immunoassay, which is a common point-of-care (POC) diagnostic approach that detects antibodies against specific viruses (e.g., SARS-CoV-2) in patient samples; and (2) a molecular-based assay. The current standard approach for screening COVID-19 requires a reverse real-time polymerase chain reaction (rRT-PCR) assay, which can be carried out using a variety of clinical specimens, including bronchoalveolar lavage fluid, fibrobronchoscope brush biopsies, sputum, nasal swabs, nasopharyngeal swabs, feces, or blood. This approach relies on expensive facilities, well-trained staff, and is often time-consuming, leaving a rapidly rising number of potential cases untested and opening a gaping hole in disease prevention efforts. Moreover, traveling to a clinical setting for testing increases the risk of spreading the disease and adds strain to a resource-limited healthcare system. For these reasons, an alternative, rapid, inexpensive, easy-to-use, and sensitive COVID-19 diagnostic tool must be developed for use by nonclinical individuals in their homes.

A recent study conducted by El-Tholoth et al. at the University of Pennsylvania in mid-February of 2020 describes a novel closed-tube COVID-19 assay that includes pathogen nuclei acid amplification and detection. Because viral samples are small and standard practices require RT-PCR, false-negative test results are possible and patients may consequently become more seriously ill. [5] To improve COVID-19 diagnostic test sensitivity, El-Tholoth’s group developed a closed-tube Penn-RAMP, a two-stage isothermal dsDNA amplification method that utilized both recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) techniques in a single tube. To perform the LAMP test specifically for COVID-19, El-Tholoth et al. selected conserved COVID-19 sequences using Clustal X and further designed LAMP primers. In order to make the detection process simpler, they used leucocrystal violet (LCV) dye as a chromogenic reagent, providing an obvious, deep violet color change that could be observed with the naked eye. The entire diagnostic process was relatively simple and required only a single tube for reaction. In this process, the RPA mixture was loaded onto the inside of the tube lid and the LAMP mixture (ratio of 1:9) was placed within the tube itself. The tube was subsequently sealed and incubated at 38 degrees Celsius for 15 to 20 minutes in a thermal cycler to facilitate the RPA reaction. The tube was then inverted several times and incubated at 63 degrees Celsius for 40 minutes. Because they did not have access to actual COVID-19 samples, El-Tholoth et al. used samples of healthy nasal mucosa spiked with inactivated HIV particles and other pathogens as their test materials. The Penn-RAMP process provided greater sensitivity than RT-PCR or LAMP alone. When using limited viral load, Penn-RAMP provided 100 times better sensitivity than a single LAMP test. Compared to a LAMP assay, which requires sophisticated equipment and must be run at a fixed temperature, the Penn-RAMP process requires less energy cost, is easier to execute, and can be completed in clinical or home settings.

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added.