Abstract
Abstract Objectives Fast and precise detection of SARS-CoV-2 RNA in infected patients is essential for treatment decisions. Methods A diagnostic strategy by analyzing nasopharyngeal swabs to detect SARS-CoV-2 RNA in individuals was established. The negative impacts of the individual buffer components on RT-qPCR analysis was reviewed and overcome by RNA purification. To investigate the functionality of the improved protocol we compared the novel diagnostic strategy to a Bead-based RNA extraction method using previously positive tested samples. Results A method to extract purify RNA molecules from SARS-CoV-2 was established. We examined the significance of nucleic acid purification and the need for an RNase inhibitor. Evaluation of 3,664 samples from March 23rd until May 18th in 2020 showed the incidence of COVID-19 infections in Thuringia, Germany. Additionally, benefits and limits of three RT-qPCR kits were assessed. Conclusions Our study suggests that virus capsid lysis combined with nucleic acid purification enables a viable alternative for the molecular diagnostics of SARS-CoV-2 infections. Due to the current delivery delays from different companies, this method offers the possibility to continue diagnosis and to handle the large number of samples.
Highlights
In December 2019, the novel coronavirus disease 19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was affecting several million individuals in countries around the world [1]
Within eight weeks 3,627 collagen swabs and 37 bronchoalveolar lavages (BAL) were analyzed via room temperature (RT)-qPCR resulting in 121 individuals infected with SARS-CoV-2
We reviewed possible negative effects of the individual buffer components on the RT-qPCR analysis
Summary
In December 2019, the novel coronavirus disease 19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was affecting several million individuals in countries around the world [1]. COVID-19 infection results in different clinical features include fever, cough, sore throat, headache, fatigue, myalgia and breathlessness. The clinical features are varied, ranging from asymptomatic state to acute respiratory distress syndrome and multi-organ dysfunction [9]. There is no current evidence to recommend any specific anti-SARS-CoV-2 treatment for people with a COVID-19 infection. Molecular test for rapid detection of this virus are urgently needed for early identification of infected people. For SARS-CoV-2, the most commonly employed targets include the structural nucleocapsid (N ) and spike (S) genes, as well as the nonstructural RNA-dependent RNA polymerase (RdRp) genes [3, 12]
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