Abstract
A multiplex reverse transcription quantitative PCR (RT-qPCR)-based method was designed for the simultaneous detection of different SARS-CoV-2 genes. In this study, we used three target genes encoding for the nucleocapsid 1 and 3 (N1, N3), and the spike (S) proteins, all commonly used in the detection of SARS-CoV-2 in human and environmental samples. The performance of the multiplex assay, compared to the single assay was assessed for the standard calibration curve, required for absolute quantification, and then, for the real environmental samples to detect SARS-CoV-2. For this latter, four environmental samples were collected at a local wastewater treatment plant (WWTP). The results showed that the cycle threshold (Ct) values of the multiplex were comparable to the values obtained by the singleplex PCR. The amplification of the three target genes indicated the presence of SARS-CoV-2 in the four water samples with an increasing trend in February and these results were confirmed in the multiplex approach, showing the robustness of this method and its applicability for the relative abundance analysis among the samples. Overall, both the laboratory and field work results demonstrated that the multiplex PCR assay developed in this study could provide a method for SARS-CoV-2 detection as robust as the single qPCR, but faster and cost-effective, reducing by three times the number of reactions, and consequently the handling time and reagents.
Highlights
SARS-CoV-2, like other coronaviruses, shows low stability in the environment (La Rosa et al, 2020a), the presence of viral genetic material in the stool of positive subjects could reflect the abundance of SARS-CoV-2 RNA content in untreated wastewaters (Foladori et al, 2020; Wurtzer et al, 2020a) and surface waters to which wastewaters are directly discharged (Guerrero-Latorre et al, 2020)
Polymerase chain reaction (PCR) based methods currently employed for accurate and sensitive detection of SARS-CoV-2 target genes in environmental samples mainly rely on quantitative PCR and reverse transcription qPCR (RT-qPCR)
This study presents the development of a multiplex method for a simultaneous detection of three target genes (N1, N3 and spike (S)), which are commonly used for the detection of SARS-CoV-2 (Ahmed et al, 2020; La Rosa et al, 2020b; Medema et al, 2020b; Sherchan et al, 2020)
Summary
SARS-CoV-2, like other coronaviruses, shows low stability in the environment (La Rosa et al, 2020a), the presence of viral genetic material in the stool of positive subjects could reflect the abundance of SARS-CoV-2 RNA content in untreated wastewaters (Foladori et al, 2020; Wurtzer et al, 2020a) and surface waters to which wastewaters are directly discharged (Guerrero-Latorre et al, 2020). The gene N1 is the most frequently used as an indicator for SARS-CoV-2 detection in wastewater samples followed by N2 gene, and N3 This latter showed to be more sensitive than N2 (Hamouda et al, 2021; Medema et al, 2020b) and for this reason, it was included in the present study. Differences in sensitivity between the target genes and the gene copy number variations over a threemonth period (December 2020–February 2021) have been determined and confirmed by the multiplex approach These results showed that the multiplex method provides a robust, rapid and reliable approach applicable to monitoring SARS-CoV-2 in wastewater samples
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