• Nadine Lübke, Tina Senff, Sara Scherger, Sandra Hauka, Marcel Andrée, Ortwin Adams, Jörg Timm, and Andreas Walker.
• Institute of Virology, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany. Electronic address: nadine.luebke@med.uni-duesseldorf.de.
• J. Clin. Virol. 2020 Sep 1; 130: 104579.

BackgroundFast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed.ObjectivesTo establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials.Material And MethodsDifferent SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene.ResultsThe protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 μl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing.ConclusionDirect RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.Copyright © 2020 Elsevier B.V. All rights reserved.

30 keywords...

hide…