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Open AccessArticle

Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore
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Genes 2020, 11(6), 664; https://doi.org/10.3390/genes11060664
Received: 9 May 2020 / Revised: 2 June 2020 / Accepted: 16 June 2020 / Published: 18 June 2020
There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting. View Full-Text
Keywords: Point-of-care; Diagnostics; DIRECT-PCR; portable PCR; SARS-CoV-2; Covid-19 Point-of-care; Diagnostics; DIRECT-PCR; portable PCR; SARS-CoV-2; Covid-19
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Wee, S.K.; Sivalingam, S.P.; Yap, E.P.H. Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler. Genes 2020, 11, 664.

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