Sensitivity analysis of RT-qPCR and RT-ddPCR for SARS-CoV-2 detection with mutations on N1 and E primer-probe region.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that undergoes rapid mutation. Based on viral whole genome sequencing analysis in Hebei Province, China, we identified several essential single nucleotide variants (SNVs) on primer-probe regions accumulating within some Omicron variants' genomes. In this study, we focused on three SNVs, C28290T, T28297C, and C28311T emerging on 2019-nCoV-N1 (CDC-N1) primer-probe regions, recommended by CDC in 2020, and two SNVs, C26270T, A26275G emerging on E (Charité-E) primer-probe regions recommended by Charité, Germany. Our findings revealed that the presence of one or two SNVs in the primer or probe region affected the sensitivity of reverse transcription-quantitative polymerase chain reaction and droplet digital PCR to varying extents. This discovery underscores the importance of continuously monitoring the whole genome sequences of SARS-CoV-2 variants, especially the primer-probe targeting regions, and correspondingly updating commercial test kits or recommended primer-probe sequence sets. IMPORTANCE: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection.

Surveillance of SARS-CoV-2 in wastewater by quantitative PCR and digital PCR: a case study in Shijiazhuang city, Hebei province, China.

In this study, we reported the first long-term monitoring of SARS-CoV-2 in wastewater in Mainland China from November 2021 to October 2023. The city of Shijiazhuang was employed for this case study. We developed a triple reverse transcription droplet digital PCR (RT-ddPCR) method using triple primer-probes for simultaneous detection of the N1 gene, E gene, and Pepper mild mottle virus (PMMoV) to achieve accurate quantification of SARS-CoV-2 RNA in wastewater. Both the RT-ddPCR method and the commercial multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) method were implemented for the detection of SARS-CoV-2 in wastewater in Shijiazhuang City over a 24-month period. Results showed that SARS-CoV-2 was detected for the first time in the wastewater of Shijiazhuang City on 10 November 2022. The peak of COVID-19 cases occurred in the middle of December 2022, when the concentration of SARS-CoV-2 in the wastewater was highest. The trend of virus concentration increases and decreases forming a "long-tailed" shape in the COVID-19  outbreak and recession cycle. The results indicated that both multiplex RT-ddPCR and RT-qPCR are effective in detecting SARS-CoV-2 in wastewater, but RT-ddPCR is capable of detecting low concentrations of SARS-CoV-2 in wastewater which is more efficient. The SARS-CoV-2 abundance in wastewater is correlated to clinical data, outlining the public health utility of this work.HighlightsFirst long-term monitoring of SARS-CoV-2 in wastewater in Mainland ChinaCOVID-19 outbreak was tracked in Shijiazhuang City from outbreak to containmentWastewater was monitored simultaneously using RT-ddPCR and RT-qPCR methodsTriple primer-probe RT-ddPCR detects N1 and E genes of SARS-CoV-2 and PMMoV.