Abstract
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.
Highlights
A novel coronavirus disease 2019 (Covid-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China in December 2019 [1]
The lowest Cq was observed at 17.37 ± 0.04, which corresponded to 1.2 × 108 RNA copies per reaction (Figure 1A)
When RNA was spiked in nasal exudate, we found the limit of detection (LoD) of 12 RNA copies per reaction with a Cq of 38.72 (Table 1)
Summary
A novel coronavirus disease 2019 (Covid-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China in December 2019 [1]. Organization (WHO) has declared Covid-19 a pandemic, triggering various travel restrictions, border control, quarantine and social distancing measures in many countries [2] Despite these interventions, the pandemic continues to spread globally, with its epicenter moving across continents [3], including in countries with well-developed public health systems and aggressive implementation of measures [4]. The first SARS-CoV-2 genome was published and deposited in NCBI database as Wuhan-Hu-1, GenBank accession number MN908947 on 14 January 2020 [8]. This allowed several laboratories around the world, including our laboratory, to develop nucleic acid amplification tests (NAAT) to detect SARS-CoV-2 genetic materials [9].
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