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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The development of RT PCR methodology has facilitated greatly the genetic characterisation of many rabies viruses (RVs), distinct strains of which persist in certain host species reservoirs within geographically defined regions. The relative temporally conserved nature of certain regions of the RV genome, particularly the N gene, permits development of rapid molecular methods for RV typing. Two main strategies have been applied to viral discrimination: (1) restriction fragment length polymorphism (RFLP) of PCR products and (2) strain-specific PCR (SS-PCR), in which sequences of specific viral strains are amplified differentially using strain-specific primers. Both these approaches have yielded methods of value to rabies epidemiological studies and control programs in Ontario. These procedures have facilitated the identification of intra-strain variants of the arctic fox strain, the only terrestrial RV strain persisting in the area, and they allow rapid discrimination of this strain from those circulating in insectivorous bat reservoirs and from the foreign raccoon strain, which continues to spread throughout the northeastern US and threatens to enter Ontario. Such methods can be adapted readily for use in other regions harbouring multiple overlapping RV reservoirs.