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Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic

Paul R. GrantiD, Melanie Turner, Gee Yen ShiniD, Eleni NastouliiD, Lisa J. Levett

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Abstract

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19), a respiratory tract infection. The standard molecular diagnostic test is a multistep process involving viral RNA extraction and real-time quantitative reverse transcriptase PCR (qRT-PCR). Laboratories across the globe face constraints on equipment and reagents during the COVID-19 pandemic. We have developed a simplified qRT-PCR assay that removes the need for an RNA extraction process and can be run on a real-time thermal cycler. The assay uses custom primers and probes, and maintains diagnostic sensitivity within 98.0% compared to the assay run on a high-throughput, random-access automated platform, the Panther Fusion (Hologic). This assay can be used to increase capacity for COVID-19 testing for national programmes worldwide.

In-depth summary

Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (9 Apr 2020) · CC BY 4.0

Adding 2 μL of unextracted swab transport medium directly to a SARS-CoV-2 qRT-PCR gave 98.0% sensitivity and 100% specificity against an extraction-based Panther Fusion assay.

Standard SARS-CoV-2 testing relies on RNA extraction on a small number of automated platforms, and their reagents, consumables and cartridges ran short in the UK and Europe during the pandemic. This work drops the extraction step so the same in-house N gene primers and probes can run on a real-time thermal cycler, which many laboratories already have.

Key results

  1. ShownPipetting 2 μL of unheated viral transport medium (VTM) straight into the RT-PCR gave the lowest cycle threshold (Ct) values of all the methods tested. It was chosen as the protocol.

    Median Ct was 25.74 for 2 μL of direct, unheated VTM vs 29.51 for RNA extracted on the EZ1 (Qiagen). Figure 1 shows the medians and interquartile ranges. · Results; Figure legends

  2. ShownCompared with the laboratory's standard Panther Fusion Open Access assay, the extraction-free PCR kept high diagnostic agreement.

    Diagnostic sensitivity was 98.0%, specificity 100% and overall accuracy 98.8% against the Panther Fusion assay (Table 2). The text does not give the number of samples tested. · Results; Conclusion; Tables

  3. ShownSample volume and heating mattered. Adding 10 μL of VTM blocked amplification, and heating the sample before adding it raised the Ct.

    No amplification occurred at 10 μL, with or without heating at 95 °C for 10 min. At smaller volumes, direct addition gave lower median Ct than heat-treated samples and was reported as equivalent to EZ1 extraction (Figure 1). · Direct sample transfer; Sample heating prior to direct transfer; Results; Figure legends

  4. ClaimedThe assay was reported not to cross-react with seasonal coronaviruses or a range of other respiratory viruses.

    Negative results are reported for the four seasonal coronaviruses (OC43, NL63, 229E, HKU1), for influenza A and B, and for RSV, enterovirus, rhinovirus, adenovirus, metapneumovirus and parainfluenza viruses. No sample numbers or Ct data are shown for this test. · Results

  5. ClaimedThroughput is set by swab handling rather than by the PCR. The authors argue that this lets laboratories without extraction equipment add testing capacity.

    Samples run in batches of 96, and each thermal-cycler run takes 56 minutes. Swab processing is the rate-limiting step. Capacity depends on how many safety cabinets and real-time cyclers a laboratory has. · Discussion

Methods, data and reproducibility

Swab samples in VTM were added directly to a one-step TaqMan qRT-PCR on a QuantStudio 5 at 10, 5 or 2 μL, either unheated or after 95 °C for 10 min. The results were compared with RNA extracted on Qiagen platforms and with an in-house N gene assay run on the Panther Fusion Open Access channel.

  • Panel of SARS-CoV-2 positive and negative clinical swab samples in viral transport medium · human (clinical swab samples); target virus SARS-CoV-2
  • Positive clinical sample serially diluted to end point for analytical sensitivity (Table 3) · human (clinical swab sample); target virus SARS-CoV-2 · n = 1 sample
  • Respiratory viruses tested for cross-reactivity: seasonal coronaviruses (OC43, NL63, 229E, HKU1), influenza A and B, RSV, enterovirus, rhinovirus, adenovirus, metapneumovirus and parainfluenza · virus (multiple respiratory viruses)
Methods & tools
TaqMan Fast Virus 1-step Master Mix (Applied Biosystems), QuantStudio 5 real-time PCR system (ThermoFisher Scientific), Panther Fusion with Open Access RNA/DNA enzyme cartridge (Hologic), Hologic internal RNA IC control primers and probe, QIAsymphony SP with QIAsymphony DSP Virus/pathogen Mini kit (Complex 200 protocol), EZ1 Advanced XL with EZ1 DSP virus kit (Qiagen), Qiagen Lysis buffer (guanidinium-based)
Reproducibility gaps
There is no data availability statement. The primer and probe sequences (Table 1), the diagnostic accuracy counts (Table 2) and the analytical sensitivity results (Table 3) are referred to, but their contents are not in the text provided.
Limitations (4)
  • Assay sensitivity depends on PCR efficiency, so other PCR reagents or protocols would need careful evaluation before being used with the extraction-free approach. · stated by the authors
  • Capacity is limited by swab processing in safety cabinets and by the number of real-time thermal cyclers available. · stated by the authors
  • The text gives neither the panel size nor the number of positive samples, and the accuracy and analytical sensitivity tables are not reproduced. The precision of the 98.0% sensitivity figure therefore cannot be judged. · noted in this summary
  • This is a single-laboratory comparison. The reference is an in-house Panther Fusion assay that uses the same N gene primers and probes, so it cannot reveal failures caused by the primers themselves. · noted in this summary

Read this if you run SARS-CoV-2 qRT-PCR diagnostics and want to skip RNA extraction when extraction kits, cartridges or platforms are in short supply.

Citations
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