Radvanszka, Monika, Paul, Evan D., Hajdu, Roman, Borsova, Kristina, Kovacova, Viera ORCID: 0000-0003-4581-4254, Putaj, Piotr, Birova, Stanislava ORCID: 0000-0002-7448-8868, Cirkova, Ivana, Carnecky, Martin, Buranovska, Katarina, Szobi, Adrian, Vojtassakova, Nina, Drobna, Diana, Cabanova, Viktoria, Slavikova, Monika, Lickova, Martina, Vanova, Veronika, Havlikova, Sabina Fumacova, Lukacikova, L'ubomira, Kajanova, Ivana ORCID: 0000-0003-0182-7037, Koci, Juraj ORCID: 0000-0002-5560-806X, Rusnakova, Diana, Sedlackova, Tatiana, Max, Klaas E. A., Tuschl, Thomas, Szemes, Tomas ORCID: 0000-0002-0900-2534, Klempa, Boris ORCID: 0000-0002-6931-1224 and Cekan, Pavol (2022). Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B. Microb. Biotechnol., 15 (7). S. 1995 - 2022. HOBOKEN: WILEY. ISSN 1751-7915

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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 Charite 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.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Radvanszka, MonikaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Paul, Evan D.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hajdu, RomanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Borsova, KristinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kovacova, VieraUNSPECIFIEDorcid.org/0000-0003-4581-4254UNSPECIFIED
Putaj, PiotrUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Birova, StanislavaUNSPECIFIEDorcid.org/0000-0002-7448-8868UNSPECIFIED
Cirkova, IvanaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Carnecky, MartinUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Buranovska, KatarinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Szobi, AdrianUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Vojtassakova, NinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Drobna, DianaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Cabanova, ViktoriaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Slavikova, MonikaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lickova, MartinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Vanova, VeronikaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Havlikova, Sabina FumacovaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lukacikova, L'ubomiraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kajanova, IvanaUNSPECIFIEDorcid.org/0000-0003-0182-7037UNSPECIFIED
Koci, JurajUNSPECIFIEDorcid.org/0000-0002-5560-806XUNSPECIFIED
Rusnakova, DianaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Sedlackova, TatianaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Max, Klaas E. A.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Tuschl, ThomasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Szemes, TomasUNSPECIFIEDorcid.org/0000-0002-0900-2534UNSPECIFIED
Klempa, BorisUNSPECIFIEDorcid.org/0000-0002-6931-1224UNSPECIFIED
Cekan, PavolUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-687472
DOI: 10.1111/1751-7915.14031
Journal or Publication Title: Microb. Biotechnol.
Volume: 15
Number: 7
Page Range: S. 1995 - 2022
Date: 2022
Publisher: WILEY
Place of Publication: HOBOKEN
ISSN: 1751-7915
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
SENSITIVITY; VIRUSMultiple languages
Biotechnology & Applied Microbiology; MicrobiologyMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/68747

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