Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

• Published in: Microbial biotechnology Vol. 15; no. 7; pp. 1995 - 2021
• Main Authors: Radvánszka, Monika, Paul, Evan D., Hajdu, Roman, Boršová, Kristína, Kováčová, Viera, Putaj, Piotr, Bírová, Stanislava, Čirková, Ivana, Čarnecký, Martin, Buranovská, Katarína, Szobi, Adrián, Vojtaššáková, Nina, Drobná, Diana, Čabanová, Viktória, Sláviková, Monika, Ličková, Martina, Vaňová, Veronika, Fumačová Havlíková, Sabína, Lukáčiková, Ľubomíra, Kajanová, Ivana, Koči, Juraj, Rusňáková, Diana, Sedláčková, Tatiana, Max, Klaas E. A., Tuschl, Thomas, Szemes, Tomáš, Klempa, Boris, Čekan, Pavol
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01-07-2022; John Wiley and Sons Inc; Wiley
• Subjects: Cold storage; Coronaviruses; COVID-19; Design; DNA probes; DNA-directed RNA polymerase; Genomes; Influenza; Influenza A; Laboratories; Nucleic acids; Nucleotides; Pandemics; Protocol; Public health; Ribonuclease P; Ribonucleic acid; RNA; RNA polymerase; Room temperature; Sensitivity analysis; Sensitivity enhancement; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Technological change; Technology; Vaccination; Virology; Workflow
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.14031

Summary
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.
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, added an RNase P internal control, utilized locked nucleic acid nucleotides and dual probe technology to enhance sensitivity and specificity, optimized an RNA‐extraction free workflow, and multiplexed the assays to detect influenza. 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.