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Updated: Oct 27, 2025

Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples
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SARS-CoV-2 detection in wastewater using multiplex quantitative PCR.

Anna Navarro1, Livia Gómez1, Isabella Sanseverino1

  • 1European Commission Joint Research Centre, Via E. Fermi 2749, 21027 Ispra, VA, Italy.

The Science of the Total Environment
|July 23, 2021
PubMed
Summary
This summary is machine-generated.

A new multiplex reverse transcription quantitative PCR (RT-qPCR) method simultaneously detects multiple SARS-CoV-2 genes. This robust and cost-effective assay accurately identifies the virus in environmental samples, reducing testing time and reagent use.

Keywords:
Environmental surveillanceMultiplex assayRT-qPCRSARS-CoV-2Wastewater

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Area of Science:

  • Environmental microbiology
  • Molecular diagnostics
  • Virology

Background:

  • Accurate detection of SARS-CoV-2 is crucial for public health surveillance.
  • Existing methods for SARS-CoV-2 detection can be time-consuming and reagent-intensive.
  • Multiplex assays offer potential for increased efficiency in pathogen detection.

Purpose of the Study:

  • To develop and validate a multiplex RT-qPCR assay for simultaneous detection of SARS-CoV-2 genes.
  • To assess the performance of the multiplex assay compared to singleplex assays.
  • To evaluate the applicability of the multiplex assay for detecting SARS-CoV-2 in environmental samples.

Main Methods:

  • Designed a multiplex RT-qPCR assay targeting three SARS-CoV-2 genes: nucleocapsid (N1, N3) and spike (S).
  • Assessed assay performance using standard calibration curves for absolute quantification.
  • Tested the multiplex assay on four wastewater samples collected from a wastewater treatment plant.

Main Results:

  • The multiplex assay demonstrated comparable cycle threshold (Ct) values to singleplex PCR.
  • SARS-CoV-2 was detected in all four wastewater samples, with an increasing trend observed in February.
  • The multiplex approach confirmed the presence and relative abundance of SARS-CoV-2 in the samples, highlighting its robustness.

Conclusions:

  • The developed multiplex PCR assay is a robust and reliable method for SARS-CoV-2 detection.
  • This multiplex assay is faster and more cost-effective than single qPCR, reducing reactions threefold.
  • The method is suitable for both laboratory and field applications, including environmental monitoring.