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    Assessment of Nucleic Acid Extraction Kits for SARS-CoV-2 Surveillance in Wastewater Samples
    (2025) Sait, Ahmet; Korkmaz, Serol; Parmaksız, Ayşe; Bayraktar, Bülent; Aslan, İsmail
    Objective: The aim of this study is to evaluate the effectiveness of three commercial nucleic acid extraction kits (kit A, B and C) in isolating SARS-CoV-2 viral RNA from wastewater samples. Method: In this study, water samples were collected in March 2021 from three wastewater treatment plants located in different parts of Istanbul, and it was confirmed that they were negative for SARS-CoV-2. Different concentrations of the SARS-CoV-2 virus, previously inactivated at the BSL-3 laboratory of the Pendik Veterinary Control Institute, were added to the wastewater samples. RNA extraction and quantification were performed using commercial nucleic acid extraction kits and and RT-qPCR kit specific to SARS-CoV-2. Results: At the end of the study, it was determined that kit C yielded the highest total RNA and produced more consistent results, significantly outperforming the other two kits in terms of RNA yield and purity. Statistical analysis revealed significant differences in RNA concentrations (p < 0.05) and gene copy numbers (p < 0.01) between the kits, and kit C demonstrated superior linearity and reproducibility. Conclusion: According to the findings, although all three evaluated kits are suitable for detecting SARS-CoV-2 RNA in wastewater samples, kit C provides the most efficient and reliable performance, especially for high-throughput studies. Additionally, this study highlights the importance of selecting appropriate nucleic acid extraction methods for wastewater surveillance, which serves as an early warning system for outbreaks that threaten public health.
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    Investigation of the recovery efficiency of CeUF method through RT-qPCR quantification of inactivated SARS-CoV-2 in untreated wastewater
    (Elsevier Science Inc, 2022) Sait, Ahmet; Korkmaz, Serol; Parmaksiz, Ayse; Bayraktar, Bulent
    Wastewater-based disease monitoring is an early warning system and a surveillance tool for infectious disease outbreaks regarding pathogens with pandemic potential. This study aimed at investigating the recovery efficiency of centrifugal ultrafiltration (CeUF), which is one of the most-used virus concentration methods, for inactive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) added to wastewater. Inactivated SARS-CoV-2 was inoculated into untreated wastewater at different concentrations (4 x 10(3), 8 x 10(3), 16 x 10(3), 24 x 10(3) and 32 x 10(3) gene copy/mu L) and concentrated through ultrafiltration with a disposable centrifugal filter device. Total nucleic acids in concentrated filtrates were extracted and isolated by an automated system. In isolates, total RNA was measured by a UV/VIS spectrophotometer, and the recovered virus was quantified by RT-qPCR with two gene regions (N1 and N2). The recovery rates were between 11% and 17.8% (mean 15.1%, CV below 15%). While there were positive correlations among the inoculated virus, total RNA and recovered virus, there was no correlation and linearity between the recovery rates. Despite limited recovery rates, CeUF integrated with RT-qPCR quantification can be a valid assay for monitoring SARS-CoV-2 in wastewater, and an early warning system.