key: cord-1031806-7z6a7jwa
authors: Israeli, Ofir; Beth-Din, Adi; Paran, Nir; Stein, Dana; Lazar, Shirley; Weiss, Shay; Milrot, Elad; Atiya-Nasagi, Yafit; Yitzhaki, Shmuel; Laskar, Orly; Schuster, Ofir
title: Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction
date: 2020-08-10
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.08.015
sha: 798fe10b45aa1f13170eaf647b4bab000af7e60c
doc_id: 1031806
cord_uid: 7z6a7jwa

Abstract SARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies support the elimination of the extraction step. Herein, we assessed the RNA extraction necessity, by comparing RT-qPCR efficacy in several direct approaches vs. the gold standard RNA extraction, in detection of SARS-CoV-2 from laboratory samples as well as clinical Oro-nasopharyngeal SARS-CoV-2 swabs. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified more than 60% of positive clinical specimens.

The COVID-19 pandemic caused by SARS-CoV-2 produced significant morbidity and mortality worldwide.

At the time of writing, more than six million cases and over 370,000 deaths were reported [1] . The pandemic has created an acute need for rapid, cost effective and reliable diagnostic screening. COVID-19 genetic diagnostics process include RNA extraction from Oro-nasopharyngeal swabs followed by reverse transcriptase quantitative PCR (RT-qPCR) targeting viral genes [2] . However, the global demand for reagents has placed extensive strain on supply chains for RT-qPCR kits and to an even greater extent, on RNA isolation reagents. Potentially, eliminating RNA extraction would greatly simplify the diagnostic procedure, reducing both cost and time to answer, while allowing testing to continue in case of reagent shortages. Previous studies demonstrated that several lysis buffers might allow the elimination of RNA extraction [3] [4] [5] . Very recently, two studies [ 7 -6 ] used a direct no-buffer RT-qPCR approach which identified <90% of the tested clinical samples.

In this study, we tested the diagnostic efficiency following thermal inactivation (65°C for 30min and 95°C for 10min) without addition of lysis buffers ("no buffer") or following lysis by three buffers (Virotype, QuickExtract and 2% Triton-X-100) and compared it to diagnosis after standard RNA extraction. Samples after inactivation at 95°C for 10min or 65°C for 30min.

RT-qPCR assays were performed using the SensiFAST Probe Lo-ROX One-Step kit (Bioline). Primers and probe for SARS-CoV-2 E gene were taken from the Berlin protocol [2] . (Table 1 ). The alternative buffers exhibited much lower detection levels:

Triton (both inactivation protocols) detected a single positive sample (5% detection); OuickExtract and Virotype had 35-40% detection rates (both inactivation protocols). Surprisingly, direct no-buffer approach was superior with 50% and 70% for the 65°C and 95°C inactivation protocols, respectively. Detection was reversely-correlated to samples' Ct, with efficiency dropping from 100% for Ct < 32 to 25% for samples with higher Ct. The 95°C no buffer approach was further evaluated in a larger set of known positive samples producing similar results (n total =43, 63% detection).

Our results demonstrate that RNA extraction significantly improves comprehensive and sensitive clinical diagnosis of SARS-CoV-2. We suggest that clinical samples (which include a multitude of nucleic acids and proteins) might significantly hamper detection. Although being previously reported to facilitate viral detection [3] [4] [5] , the tested buffers severely compromised the limit of detection (to a maximum of 40%). This is surprising, considering that direct analysis without adding buffers achieved a 63% detection level.

This no-buffer direct approach could potentially be used with some success in times of need to achieve screening for high-titer samples. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. J o u r n a l P r e -p r o o f ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

No funding source is applicable to this study.

Pre-existing samples were used and de-identified. This work was therefore determined to be "not human subjects' research".

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

A 5-min RNA preparation method for COVID-19 detection with RT-qPCR

Comparison of SARS-CoV-2 Indirect and Direct Detection Methods

SARS-CoV-2 detection by direct rRT-PCR without RNA extraction. Clin Virol

An alternative workflow for molecular detection of 357 SARS-CoV-2 -escape from the NA extraction kit-shortage

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

Acknowledgments SARS-CoV-2 was kindly provided by Bundeswehr Institute of Microbiology, Munich, Germany. The authors would like to thank Itai Glinert for his fruitful reviewing of this manuscript.