key: cord-0954774-xurlwnn0
authors: Eichhoff, O. M.; Bellini, E.; Lienhard, R.; Stark, W. J.; Bechtold, P.; Grass, R. N.; Bosshard, P. P.; Levesque, M. P.
title: Comparison of RNA extraction methods for the detection of SARS-CoV-2 by RT-PCR
date: 2020-08-14
journal: nan
DOI: 10.1101/2020.08.13.20172494
sha: 2bfa6b2dcc110689635d9831f6459ef35e3179a1
doc_id: 954774
cord_uid: xurlwnn0

Objectives: The SARS-CoV-2 pandemic outbreak has stressed health care systems as well as medical supply chains, but diagnostic testing is an essential public health measure to control viral spread. Here we test the suitability of different RNA extraction methods for integration into a diagnostic workflow for coronavirus testing. Methods: We applied six RNA extraction methods on the same 24 SARS-CoV-2 positive patient samples and quantified their results by subsequent reverse-transcriptase PCR (RT-PCR) of three viral genes. These methods included a) column-based extraction, b) phenol-chloroform extraction, as well as c) extraction using magnetic beads (i.e., one commercial kit as well as three different magnetic beads in combination with home-brewed buffers and solutions). Results: We achieved diagnostic-quality RT-PCR results with all methods, and there was no significant difference between the tested methods, except for one magnetic bead protocol with home-brewed buffers, in which the number of positive tested genes was significantly lower. Conclusions: Five of the six RNA extraction methods are interchangeable in a diagnostic workflow. Since some methods are more scalable than others, and have comparable results on RT-PCR quantitation, they may be more amenable to high-throughput sample processing pipelines.

With over 4 million confirmed active cases worldwide, the coronavirus pandemic is a historical outbreak with enormous consequences for national health care systems and economies. On December 31st 2019, the World Health Organization (WHO) was informed about the detection of a cluster of cases of pneumonia with unknown origin appearing in Wuhan, Hubei Province of China [1] . Investigation of patients suffering from this new respiratory disease revealed that the cause was a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which caused the coronavirus disease 2019 (COVID-19) [2, 3] . In a short period, SARS-CoV-2 spread to a dozen countries and within a few months developed into a pandemic outbreak [4] . Until an efficacious and safe vaccine is available, the only way to prevent further spread of the virus is to dramatically reduce infection rates. The WHO recommends several public health measures: (i) rapid diagnosis and immediate isolation of cases, (ii) rigorous tracking, and (iii) precautionary self-isolation [5] . These strategies also mean that testing must be widely available and the barriers to testing have to be as low as possible. Thus, hundreds of thousands of tests need to be available daily worldwide, which challenges global supply chains and the production of reagents necessary for diagnostic testing. In order to reduce supply chain vulnerabilities and limit dependencies on single suppliers, we compared different RNA extraction protocols to establish our coronavirus diagnostics workflow at the Department of Dermatology, University Hospital of Zurich, which could be used for subsequent detection of viral RNA by reverse-transcriptase quantitative PCR (RT-PCR).

We received 24 nasal or mucosal swabs from confirmed SARS-CoV-2 positive patients from the ADMED laboratory, Switzerland.

Nasal or mucosal swabs were provided in Amies medium and were diluted 1:1 with 

We compared the feasibility of different commercially available and home-brewed RNA isolation techniques for a SARS-CoV-2 diagnostics workflow with the aim to test whether these techniques can be inter-changeable in crises when supply-chains are unreliable. We chose to evaluate the RNA extraction efficiency of each method via the multiplex RT-PCR TaqPath COVID-19 CE-IVD RT-PCR Kit, which was approved as a diagnostic tool by the FDA and as a CE mark throughout Europe. We found that all methods tested here, except using magnetic-beads produced by BeaverBio, could be used to make diagnostic-quality RNA extractions, as there was no statistically significant difference between the results as tested with Fisher's exact tests. Using BeaverBeads, however, required two samples to be repeated due to inconclusive results (e.g., 2 out of 3 genes negative, Figure 1+2 ) and the overall number of positive tested genes was significantly lower as compared to the TRIzol method (64/72 versus 72/72; p = 0.0064), which detected all three genes in each sample. All other methods did not significantly differ from TRIzol (p > 0.5), but in all other methods single genes were not recognized, increasing the risk of false negative results. Importantly, methods that require intensive manual pipetting (TRIzol) or allow only low-scale throughput (Qiacube, 12 samples/run) are not optimal for a daily routine process with 200+ samples and therefore magnetic-bead based extraction using a KingFisher instrument is advantageous, since it processes 96 samples/run. In conclusion, two of the three magnetic beads with home-brewed buffers and solutions can be equally used in comparison to the MagMax manufacturer's kit. Thus, the dependency on suppliers in times of crisis can be decreased by implementing comparable techniques for the isolation of viral RNA in a laboratory diagnostic workflow. 

Emerging coronaviruses: Genome structure, replication, and pathogenesis

A Novel Coronavirus from Patients with Pneumonia in China

WHO Declares COVID-19 a Pandemic

Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections