Wastewater SARS-CoV-2 Surveillance via qRT-PCR

Wastewater specimens were collected as 24-h flow-dependent composite samples and processed as previously described (14 (link)). Debris of 100–200 mL of sewage samples was pelleted and the supernatant was concentrated by using 100 kDa Centricon ultrafilters (Millipore Sigma, https://www.emdmillipore.com); in vitro–transcribed dengue virus type-2 RNA was added as an internal extraction control. RNA was extracted by using the Nuclisens kit (bioMérieux, https://www.biomerieux.com) and KingFisher purification system (Thermo Fisher Scientific, https://www.thermofisher.com) (14 (link)). RNA was screened by quantitative reverse transcription PCR (qRT-PCR) with 5 primer–probe sets targeting the SARS-CoV-2 nucleocapsid (N) gene (N1–N3) (24 ), envelope (E) gene for all sarbecoviruses (25 (link)), and the internal control.

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Izquierdo-Lara R., Elsinga G., Heijnen L., Munnink B.B., Schapendonk C.M., Nieuwenhuijse D., Kon M., Lu L., Aarestrup F.M., Lycett S., Medema G., Koopmans M.P, & de Graaf M. (2021). Monitoring SARS-CoV-2 Circulation and Diversity through Community Wastewater Sequencing, the Netherlands and Belgium. Emerging Infectious Diseases, 27(5), 1405-1415.

Publication 2021

Centricon ultrafilters Nuclisens kit KingFisher purification system

Dengue Dengue virus Gene Nucleocapsid Primer Reverse transcription Sarbecoviruses Sars cov 2 Sewage Virus rna

Corresponding Organization :

Other organizations : KWR Water Research Institute, Erasmus MC, University of Edinburgh, Technical University of Denmark

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Variable analysis

independent variables

Wastewater specimen collection method (24-h flow-dependent composite samples)

dependent variables

Presence and concentration of SARS-CoV-2 RNA in wastewater samples (measured by qRT-PCR)

control variables

Addition of in vitro-transcribed dengue virus type-2 RNA as an internal extraction control

controls

Positive control: in vitro-transcribed dengue virus type-2 RNA added as an internal extraction control
Negative control: Not explicitly mentioned

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