Amy Ellis, PhD, from the University of Wisconsin–Madison, speaks about using Standard BioTools™ technology to test air samples in schools and ultimately bolster public health knowledge
In our connected world, monitoring and controlling infectious diseases has become an essential part of public health stewardship. Threats like disease outbreaks and antimicrobial resistance mean that accurate, fast pathogen detection is essential in transforming research and positioning a laboratory as a center of excellence.
Pathogen surveillance using singleplex qPCR
Since 2021, Shelby O’Connor’s lab at the University of Wisconsin–Madison has been performing air surveillance to determine pathogens, primarily SARS-CoV-2, present in congregate settings. Today, the O’Connor Lab is focused on testing air in K–12 schools and has expanded its scope to include other pathogens, such as seasonal coronaviruses, RSV and influenzas.
Amy Ellis, PhD, is a senior scientist in the O’Connor Lab. Her team’s goal is to better educate the public on community spread of viruses. Each week, researchers collect air using air samplers, which have now been placed in 32 schools in the Dane County, WI, area. After processing, the lab shares its data on a weekly basis to public health dashboards.
O’Connor Lab sample processing pipeline:
- Run air sampler
- Remove cartridges
- Elute substrates in PBS
- Extract nucleic acids
- Multi-pathogen PCR assay detection/metagenomics/qPCR or dPCR assays
Multi-pathogen PCR assays are a crucial tool in Ellis’s air-sample research. Other more sensitive and more quantitative methods like qRT-PCR or dPCR don’t support surveilling a large number of targets, and the cost is inhibitive. Air sample volume is also “extremely limited,” somewhere between 50–100 µL per sample. “There’s also not a lot of genetic material collected on the air sampler,” Ellis says. “So we typically have to figure out what to do with it in a realistic fashion.”
The O’Connor Lab was using the CARMEN assay, a CRISPR/Cas13-based PCR assay developed out of the Broad Institute during the COVID-19 pandemic, originally for use with nasal swabs. Ellis and team adapted the assay for air samples in the 2023/2024 school year, identifying 15 targets, including SARS-CoV-2, metapneumovirus, human parainfluenza virus, RSV, influenzas and adenovirus.
CARMEN assay pipeline:
- Perform reverse transcription PCR on nucleic acids with multiplexed primers
- Target/template DNA for next step
- Run 192.24 integrated fluidic circuit (IFC) on the Biomark™ X9 System for High-Throughput Genomics
- Template DNA from PCR (with T7 promoter)
- Target RNA
- CRISPR RNA of interest recognizes target RNA
- Cas13 enzyme binds to and cleaves target RNA
The lab compared CARMEN data to qPCR data for the 2023/2024 school year. Concordance was observed between CARMEN and qPCR for SARS-CoV-2. However, the lab saw no concordance for influenza A, even after specific assay expansion, showing that “there are some limitations” to the assay, Ellis says. The CARMEN assay has other shortcomings for air surveillance: It requires an inefficient preamplification step due to long primers; the Cas13 enzyme is very sensitive to PCR inhibitors, which are prevalent in air samples; and it is difficult to design CRISPR RNAs for viruses that have high sequence diversity, such as rhinovirus or enterovirus.
Ellis and her team wanted to explore other multi-pathogen detection panels, which led them to design a custom respiratory pathogen panel (RPP) with help from Standard BioTools. The RPP assay has 17 targets, with room for expansion up to 48.
RPP assay workflow:
- Perform preamplification RT-PCR step on nucleic acids
- Load samples onto 48.48 IFC on Biomark X9 System
How does CARMEN compare to RPP?
The team took 40 air samples from the 2023/2024 school year and performed qPCR, RPP and CARMEN assays, using SARS-CoV-2 (N) as a common target. Regardless of the stringency of analysis, the researchers concluded that RPP and qPCR data agree more frequently overall.
Wanting to look at a variety of other targets, Ellis and team moved on to samples from the 2024/2025 school year. Comparing CARMEN and RPP assay air sample data from August 2024 to March 2025, the researchers observed that the RPP assay in general was able to identify a higher frequency of percent positive samples for individual sampling time periods compared with the CARMEN assay. For example, the RPP assay picked up an increased frequency of air sampler cartridges that were positive for human coronavirus HCoV OC43, influenza A, influenza B and RSV where the CARMEN assay did not.
The team concluded that the CARMEN and RPP assays can detect similar pathogens from air samples in similar time periods in the 2024/2025 school-year samples; in some cases, such as flu B, the RPP assay seems to have improved detection. Additionally, the RPP assay’s expansion potential will allow for detection of a higher number of pathogens.
“As far as our future directions, we do plan in the 2025–26 school year to move to this assay exclusively for air sampling,” Ellis says. The team also routinely runs the RPP assay on emergency department and clinic air samples, and used the assay for a household study in a local school district that examined household transmission in known cases of respiratory illnesses.
Learn more
Timely detection and accurate identification of infectious pathogens are essential for outbreak surveillance, environmental monitoring and public health decision-making. The Biomark X9 System for High-Throughput Genomics and Dynamic Array™ IFCs provide a cost-effective and scalable microfluidics-based approach to pathogen detection, enabling simultaneous multi-pathogen testing while providing the flexibility to include tracking of new variants, antimicrobial resistance genes and host gene responses – all in one run.
Learn more about pathogen detection using microfluidics
Read about using our tools and technologies to tackle infectious diseases