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RNA Extraction

RNA Extraction

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
Introduction

The emergence of the novel human coronavirus in late 2019 in the Wuhan region of China rapidly evolved into a global pandemic. The high transmission rate and high proportion of asymptomatic infections led to a massive, worldwide need for rapid, affordable, and efficient diagnostic tests, that can be performed in clinical and non-clinical settings1,2.

Currently, the widely used method of SARS-CoV-2 detection in clinical diagnostics is an RT-PCR assay, detecting the presence of viral RNA in patient samples. Although RT-PCR is widely implemented for the detection of pathogens, including viruses3 in clinical samples, the implementation of the specific assay for the detection of SARS-CoV-2 has only recently been established. The most commonly used protocol4 was developed and optimized for the detection of the novel coronavirus at the Charité University Hospital, in collaboration with institutes in Germany, the Netherlands, China, France, the United Kingdom, and Belgium. A different test protocol was developed by the Center for Disease Control (CDC) in the United States through comparison and validation of various kits for nucleic acid extraction and the use of alternative probe and primer sets for SARS-CoV-2 detection in clinical samples5,6. Routinely, the application of quantitative PCR (qPCR) for the relative quantification of an RNA of interest is preceded by (1) the isolation and purification of total RNA from the sample, (2) elution and possible concentration of the material, and (3) the use of purified RNA in a reverse-transcription (RT) reaction resulting in complementary DNA (cDNA) from the template RNA which is then utilized for the qPCR reaction. However, nucleic acid purification and RT of the resulting RNA into cDNA are not only laborious and time-consuming, but the additional steps requiring manual handling can result in experimental errors. In the case of clinical sampling and diagnostics, the use of a single-reaction kit combining the RT and qPCR reactions is therefore customary. Although single-reaction RT-PCR removes the need for a separate RT reaction, RNA isolation from clinical samples constitutes a major bottleneck in the diagnostic process, as it remains both manually laborious and expensive. Specifically, both the Charité University Hospital and the CDC protocols require the use of RNA purification kits, which not only results in a significant cost increase but led to a major supply shortage of such kits. It is therefore crucial that a new test is not only affordable, quick, and efficient, but also that it keeps the use of industrial kits to the minimum. Recent attempts have been made to circumvent RNA extraction in COVID-19 detection7,8,9.

Here, we establish routines for SARS-CoV-2 RNA-extraction-free single-reaction RT-PCR testing (Fig. 1) on heat-inactivated nasopharyngeal swab samples in transport medium and compared the results with clinically diagnosed patient samples, demonstrating the viability of extraction-free SARS-CoV-2 diagnostics. In addition, we evaluate various buffer formulations as alternative transport media, and we provide data showing that SARS-CoV-2 RT-PCR can be performed in presence of high concentration of detergent, allowing testing directly on sample lysates. Importantly, our method builds on clinically established protocols and could easily be integrated to expand ongoing testing pipelines. It is also modular and can be incorporated into alternative approaches of detection utilising PCR.
Fig. 1: Schematic overview of SARS-CoV-2 RT-PCR testing procedure.
figure1

The currently widely used procedure for COVID-19 testing involves: a Collection of patient material and deposition of potential SARS-CoV-2 viral particles in transport medium. b Inactivation of the virus by detergent/chaotropic reagents or by heating. c RNA extraction. d, e Transfer to PCR-plate (96/384-well) format in which cDNA synthesis by RT and detection by qPCR may take place. Alternatively, detection can be made by sample barcoding and high-throughput DNA sequencing. f, g Unlike the widely used approach, which includes an RNA extraction step (c) using industrial RNA extraction kits, direct sample testing circumvents this process by omitting extraction. Instead, after clinical samples are deposited in transport medium, viral particles are inactivated either through heating or by direct lysis in detergent-containing buffer. The inactivated samples are then used for the downstream RT-PCR diagnostic reaction.

https://www.nature.com/articles/s41467-020-18611-5/figures/1

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