The effects of RT-qPCR standards on reproducibility and comparability in monitoring SARS-CoV-2 levels in wastewater

Abstract

Reverse transcription-quantitative PCR (RT-qPCR) is widely used for monitoring viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in wastewater. Various materials, including plasmid DNA, synthetic nucleic acids, PCR amplicons, genomic DNA, and cDNA, are currently used for SARS-CoV-2 quantification by generating standard curves. We assessed three common standards on quantifying SARS-CoV-2 RNA across nine wastewater treatment plants in Finland, as part of the national wastewater surveillance effort. We pairwise compared RT-qPCR results from 148 wastewater samples, using both IDT (#10006625, IDT, USA) and CODEX standards (#SC2-RNAC-1100, CODEX DNA), and 179 samples using both IDT and EURM019 standards (#EURM-019, European Commission, Joint Research Centre) in our assessment. Amongst the tested standards, the CODEX standard consistently yielded more stable results than either the IDT or EURM019 standards. We found that SARS-CoV-2 levels were higher with the IDT standard (4.36 Log₁₀ GC/100 mL) compared to the CODEX standard (4.05 Log₁₀ GC/100 mL). Similarly, quantification using the IDT standard was higher (5.27 Log₁₀ GC/100 mL) than values obtained with the EURM019 (4.81 Log₁₀ GC/100 mL). SARS-CoV-2 RNA quantified with IDT and CODEX standards exhibited stronger concordance (Spearman's correlation rho median of 0.79) compared to those quantified with IDT and EURM019 standards (rho median of 0.59). This study highlights the significant impact of standard material selection on SARS-CoV-2 RNA quantification, emphasizing the need for harmonization in standard material.

Keywords: Real-time quantitative PCR; SARS-CoV-2; Standard control; Wastewater surveillance; Wastewater-based epidemiology.

Fig. 1

SARS-CoV-2 RNA copy numbers detected using the IDT plasmid, and CODEX and EURM019 RNA standards from wastewater (a) and using IDT plasmid standard and EURM019 RNA (b). Wastewater samples collected from nine wastewater treatment plants in Finland. The number of samples per WWTP ranged from 12 to 20 (see Table 3). The IDT standard consistently yielded significantly higher copy numbers than the CODEX standard in all samples together (*p < 0.001, Wilcoxon signed-rank test). Similarly, the IDT standard consistently yielded significantly higher virus copy numbers than EURM019 standard (p < 0.001, Wilcoxon signed-rank test). Since small sample number of individual WWTP, we did not carryover statistical test for them. IDT, CODEX and EURM019 indicated in light green and light blue and orange boxes, respectively.