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

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.

Keywords: Covid-19; DIRECT-PCR; Diagnostics; Point-of-care; SARS-CoV-2; portable PCR.

Figure 1

Amplification plot of SARS-CoV-2 N gene ssRNA control in (A) water, (B) spiked in sputum, and (C) spiked in nasal exudate conducted in the same thermocycler run. Numbers indicated the log₁₀ copy number of the template present. No amplification was observed in the NTC. (D) Comparison of DIRECT-PCR assay amplification of SARS-CoV-2 N gene ssRNA control (blue line), N gene spiked in sputum (red line) and N gene spiked in nasal exudate (black line). Templates were ten-fold diluted in 8 orders of magnitude. Two microliters of template was used in 20 µL of PCR mastermix on the benchtop thermocycler. The amplification efficiency (E) was determined by plotting of mean C_q values against log₁₀ copy number calculated using theoretical molarity of templates.

Figure 2

Fast DIRECT-PCR assay amplification efficiency of SARS-CoV-2 N gene plasmid spiked in sputum (red line) and N gene plasmid spiked in nasal exudate (black line) using the benchtop thermocycler (solid line) and the portable thermocycler (dotted line). Templates were ten-fold diluted in 6 orders of magnitude. One microliter of template was used in 10 µL of PCR mastermix.

Figure 3

Workflow of fast DIRECT-PCR protocol for the direct detection of SARS-CoV-2. Covid-19 infection can be confirmed in less than an hour after sample collection and addition to mastermix for amplification and detection.