The 2019 novel coronavirus (COVID-19) epidemic is a growing public health emergency of international concern. As of February 25, 2020, the virus has spread to over 33 countries, sickened more than 80,000 people worldwide, and killed over 2,600 people. Current methods for qualitative detection of COVID-19 primarily depend on reverse transcription-PCR (RT-PCR). However, RT-PCR tests can produce false negative results due to insufficient detection sensitivity for patients with a low viral load. Moreover, RT-PCR requires well-equipped laboratories and skilled personnel. Due to the rapid spread of the virus and limited capacity of hospitals, a more accurate, rapid, and easy-to-use molecular diagnostic for COVID-19 is urgently needed.
In a recent preprint manuscript posted on ChemRxiv™, El-Tholoth and colleagues at the University of Pennsylvania have developed simple, point-of-care assays for qualitative COVID-19 detection using one-stage LAMP (loop-mediated isothermal amplification) or two-stage Penn-RAMP (recombinase polymerase amplification plus LAMP). Specificity was accomplished by amplifying a conserved sequence of ORF1ab from the COVID-19 genome with high homology to the virus and high divergence from all other coronaviruses. The one-stage LAMP assay contains an isothermal master mix with EvaGreen® dye for reliable and robust amplification. Results show the one-stage LAMP assay performs comparably to RT-PCR methods for COVID-19 detection in tests with purified template and mock patient samples. Results for the two-stage Penn-RAMP assay showed detection sensitivity of 100 times higher than the one-stage LAMP or RT-PCR methods in similar mock samples. Moreover, the Penn-RAMP assay can be performed entirely in a closed tube and is suitable for colorimetric detection by LCV (leuco crystal violet). Given the simplicity, high-sensitivity, and minimal instrumentation required by Penn-RAMP, the authors propose further validation for its use as a point-of-care COVID-19 diagnostic.
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References
Coronavirus Map: Tracking the Spread of the Outbreak – The New York Times. Available at: https://www.nytimes.com/interactive/2020/world/asia/china-wuhan-coronavirus-maps.html. (Accessed: 25th February 2020)
El-Tholoth, Mohamed; Bau, Haim H.; Song, Jinzhao (2020): A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.11860137.v1