PubMed · 8049358
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L Raeymaekers. Comments on quantitative PCR.. https://pubmed.ncbi.nlm.nih.gov/8049358/
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Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information-such as identifying circulating variants-is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000 bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method-consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs-yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.
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A method to synthesize competitor RNAs as internal standards for competitive RT-PCR is improved by using the long accurate PCR (LA-PCR) technique. Competitor templates synthesized by the new method are almost the same in length, and possibly in secondary structure, as target mRNAs to be quantified except that they include the short deletion within the segments to be amplified. This allows the reverse transcription to be achieved with almost the same efficiency from both target mRNAs and competitor RNAs. Therefore, more accurate quantification can be accomplished by using such competitor RNAs.