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Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B |
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| Authors: | Monika Radvánszka Evan D Paul Roman Hajdu Kristína Bor?ová Viera Ková?ová Piotr Putaj Stanislava Bírová Ivana ?irková Martin ?arnecký Katarína Buranovská Adrián Szobi Nina Vojta??áková Diana Drobná Viktória ?abanová Monika Sláviková Martina Li?ková Veronika Vaňová Sabína Fuma?ová Havlíková ?ubomíra Luká?iková Ivana Kajanová Juraj Ko?i Diana Rusňáková Tatiana Sedlá?ková Klaas E A Max Thomas Tuschl Tomá? Szemes Boris Klempa Pavol ?ekan |
| Institution: | 1. MultiplexDX, s.r.o., Comenius University Science Park, Ilkovi?ova 8, Bratislava, 841 04 Slovakia;2. MultiplexDX, s.r.o., Comenius University Science Park, Ilkovi?ova 8, Bratislava, 841 04 Slovakia
MultiplexDX, Inc, One Research Court, Suite 450, Rockville, MD, 20850 USA College of Medical, Veterinary and Life Sciences, School of Life Sciences, University of Glasgow, University Avenue, Glasgow, G12 8QQ UK These authors contributed equally to this work.;3. Biomedical Research Center, Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, 845 05 Slovakia;4. MultiplexDX, s.r.o., Comenius University Science Park, Ilkovi?ova 8, Bratislava, 841 04 Slovakia MultiplexDX, Inc, One Research Court, Suite 450, Rockville, MD, 20850 USA;5. Geneton s.r.o., Comenius University Science Park, Ilkovi?ova 8, Bratislava, 841 04 Slovakia;6. Geneton s.r.o., Comenius University Science Park, Ilkovi?ova 8, Bratislava, 841 04 Slovakia Comenius University Science Park, Ilkovi?ova 8, Bratislava, 841 04 Slovakia;7. Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065 USA |
| Abstract: | Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic. |
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