High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Goodman, Laura B.; Anderson, Renee R.; Slater, Marcia; Ortenberg, Elen; Renshaw, Randall W.; Chilson, Brittany D.; Laverack, Melissa A.; Beeby, John S.; Dubovi, Edward J.; Glaser, Amy L.

The results described above were representative of all the targets on the plate with the exception of Mycoplasma cynos, which showed considerably more variation in analytic performance. This was likely due to the sub-optimal melting temperature (T m ) of the primers, which should ideally be 58-60 °C (the ideal probe Tm is 68-70 °C). A limitation of this platform is that it takes longer to design and manufacture the plates than for ordering individual probes, which limits the ability to quickly modify sequences. Another limitation of real-time PCR in general is the inability to detect novel or unexpected pathogens. This can be overcome to some extent by designing assays that match sequences in multiple species, but unbiased whole genome sequencing based methodologies are more suited for discovery of novel agents. 11, 12 Nanoscale real-time PCR allows a new paradigm for syndrome-based rather than species-based testing, which is useful for reducing reagent and labor costs in high-throughput molecular diagnostics. Large-scale panel testing by this approach can facilitate OneHealth surveillance efforts such as those described by Dunne and Gurfield 13 and Moutailler et al. 14 . Collection of swab samples early, generally within 3 days of clinical onset, provides the best chance to identify the presence of respiratory pathogens. Infectious disease emergence is often unpredictable, and tests that can be performed on different species without the need for modification of reagents are ideal for preparedness. Future applications of this technology are likely to be in pathotyping, antimicrobial resistance profiling, and further syndrome-based clinical diagnostic panels. Nanoscale real-time PCR is most useful for rapid, high-throughput screening of multiple sample and pathogen types, and would be complemented by unbiased or partially biased sequencing-based approaches for identifying new and emergent pathogens.