Becker, Marc

Burggraf, Siegfried

Czibere, Ludwig

Durner, Jurgen

Fleige, Tobias

Kreig-Schneider, Frank

Madejska, Arleta

WATTS, D.C.

English

A rapid and easily workable alternative (MEP: Munich Extraction Protocol) is outlined to our current RNA purification method for circumstances where there is limited availability of test kits. Details are available upon request

The University of Manchester - Institutional Repository

The University of Manchester ResearchFast and simple high-throughput testing of COVID 19DOI:10.1016/j.dental.2020.04.001Document VersionAccepted author manuscriptLink to publication record in Manchester Research ExplorerCitation for published version (APA):Durner, J., Burggraf, S., Czibere, L., Fleige, T., Madejska, A., WATTS, D. C., Kreig-Schneider, F., & Becker, M.(2020). Fast and simple high-throughput testing of COVID 19. Dental Materials.https://doi.org/10.1016/j.dental.2020.04.001Published in:Dental MaterialsCiting this paperPlease note that where the full-text provided on Manchester Research Explorer is the Author Accepted Manuscriptor Proof version this may differ from the final Published version. If citing, it is advised that you check and use thepublisher's definitive version.General rightsCopyright and moral rights for the publications made accessible in the Research Explorer are retained by theauthors and/or other copyright owners and it is a condition of accessing publications that users recognise andabide by the legal requirements associated with these rights.Takedown policyIf you believe that this document breaches copyright please refer to the University of Manchester’s TakedownProcedures [http://man.ac.uk/04Y6Bo] or contact uml.scholarlycommunications@manchester.ac.uk providingrelevant details, so we can investigate your claim.Download date:09. Jun. 2022 1 Fast and simple high-throughput testing of COVID 19   Jürgen Durner,1,2*# Siegfried Burggraf,1# Ludwig Czibere,1 Tobias Fleige,1 Arleta Madejska,1 David C Watts,4 Frank Krieg-Schneider,3 and Marc Becker1,2    1Laboratory Becker & Colleagues, Führichstr. 70, 81671 Munich, Germany  2Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-Universität München, Goethestr. 70, 80336 Munich, Germany  3R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany  4School of Medical Sciences and Photon Science Institute, University of Manchester, UK.              #Both authors contributed equally to this paper  *Corresponding author:  Prof. Dr. rer. nat. Dr. med. Jürgen Durner Department of Conservative Dentistry and Periodontology, University Hospital Ludwig-Maximilians-University Munich Goethestraße 70 80336 Munich Germany Email: juergen.durner@med.uni-muenchen.de Phone: +49-89-44005-9301  Fax: +49-89-44005-9302       2 Abstract A rapid and easily workable alternative (MEP: Munich Extraction Protocol) is outlined to our current RNA purification method for circumstances where there is limited availability of test kits. Details are available upon request.  Fast and simple high-throughput testing of COVID 19   The outbreak of the new coronavirus SARS-CoV-2 is a significant challenge for the analytical performance of laboratories (1, 2). In addition to the test capacity, this service also includes coping with requirements for increased supply of the necessary reagents.  We hereby present, in outline, a fast and easily workable alternative to our current RNA purification method for circumstances where there is limited availability of test kits. We also report on the increase of test capacity without modifying the equipment pool. This Munich Extraction Protocol (MEP) approach draws upon first principles of molecular biology that are in danger of being lost due to the normal availability of ready-to-use kits.  Nasal and throat swabs that are transported in a special liquid viral medium to the laboratory were used. An aliquot of the sample was transferred into a cavity of a 96-well plate. SARS-CoV-2 RNA extraction was performed either with two commercially available tests (MagNA Pure 96 Roche Diagnostics (Penzberg, Germany) and the Chemagic Viral DNA/RNA 300 Kit H96 (PerkinElmer Chemagen Technologie, Baesweiler, Germany); both tests require about 60 min per 96 samples). In addition we tested a fast method, which is based on a dilution step and heating to 92 °C for 10 minutes. The amplification was done in a Roche LightCycler® 480 II with an RIDA®GENE SARS-CoV-2 RUO assay from R-Biopharm.  Despite significant time saving and the use of readily available reagents, clinically acceptable sensitivity losses were seen with our “Munich Extraction Protocol (MEP)” (Fig 1).   3 With the commercial kits one dilution level better sensitivity could be measured. One reason is that a 10-fold higher amount of template RNA can be used in these kits. This reduction in the amount of RNA used is due to the fact that commercial kits use purified RNA, whereas we use matrix-contaminated RNA. In this case it is common practice to reduce the amount of material used in order to avoid inhibition. We noticed that while the MEP method perfectly performs with standard samples a delay of crossing point (CP) values is observed with samples contaminated with hemoglobin. Due to the much shorter extraction time using MEP, the test capacity can readily be increased by a factor of four.  In a further experiment we transferred aliquots from four 96-well plates to one 384-well plate and adapted the real-time PCR protocol according to (3). The number of positive cases remained the same for both methods. The presented modifications in the analytical process enable laboratories to increase their independence from potential reagent supply bottlenecks and to increase the test capacity without major modifications in the laboratory workflow. The medical quality is maintained. We will be pleased to provide interested colleagues with the detailed methodological protocol.  References 1. Salathe M, Althaus CL, Neher R, Stringhini S, Hodcroft E, Fellay J, et al. Covid-19 epidemic in switzerland: On the importance of testing, contact tracing and isolation. Swiss Med Wkly 2020; 150:w20225. 2. Binnicker MJ. Emergence of a novel coronavirus disease (covid-19) and the importance of diagnostic testing: Why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak. Clin Chem 2020. 3. Czibere L, Burggraf S, Fleige T, Gluck B, Keitel LM, Landt O, et al. High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qpcr. Eur J Hum Genet 2020; 28:23-30.    4 Figure 1  Figure captions  Figure 1. The amplification curves of the different preparation protocols (right side) and the corresponding dilution levels (left side; green: Chemagic Viral Kit, red: MEP; blue: MagNA Pure 96) are shown.   When using the undiluted sample, an increase in fluorescence was observed with the commercial kits from about 15 cycles. When using the MEP, this increase was observed after about 20 cycles. At this number of cycles an increase of the fluorescence signal of the first dilution stage (1:10) could be measured when using the commercial kits. When using the MEP, approximately 1:10 of the template RNA is used compared to the commercial kits.      

Fast and simple high-throughput testing of COVID 19

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Fast and simple high-throughput testing of COVID 19

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