Multiplexing dPCR for Indoor Bioaerosol Surveillance and Detection

Multiplexing dPCR for Indoor Bioaerosol Surveillance and Detection
Watanabe, Patricia Hitomi1; Agarwal, Nehul1; Haines, Sarah1
1Department of Civil and Mineral Engineering, University of Toronto, Toronto, ON, Canada
Keywords: Microbes, dust, filters, fungi, bacteria.

Topic(s): Bioaerosols and Disease, Instrumentation

Indoor bioaerosols, in the form of airborne particles or liquid droplets, constitute a primary exposure pathway of microbes (viable and non-viable) to building occupants, that contribute to acute and chronic respiratory health effects.

Bacteria and fungi are common indoor microbes that host a range of health implications for occupant respiratory health. For example, exposure to fungi in indoor environments is associated with asthma while bacterial cell components are coupled with increased inflammatory respiratory outcomes (Pongracic et al., 2010; Thorn & Rylander, 1998). Furthermore, the health impacts of fungal-bacterial interactions remain poorly understood. Rapid, accurate, and simultaneous quantification and characterization of these microorganisms is therefore essential for developing effective mitigation strategies to prevent harmful bioaerosol exposures within the built environment.

Quantitative polymerase chain reaction (qPCR) has long served as the standard for detecting and quantifying bioaerosol DNA/RNA in indoor environments. However, current qPCR-based quantification of indoor bioaerosol samples relies on external standards, introducing variability in-between studies and often compromising precision. Furthermore, it lacks sufficient sensitivity for short-term bioaerosol sample analyses usually of low biomass. This study evaluates nanoplate-based dPCR systems for quantifying fungal and bacterial bioaerosols from diverse indoor sampling methods, including vacuum-collected dust, indoor air cleaner filters, and surface swabs. Following the establishment of a universal single-target protocol, this research further explores the multiplexing capabilities of dPCR to simultaneously profile fungal and bacterial targets (and potentially viral targets) in indoor bioaerosol samples.

Synthetic gBlock DNA carrying universal fungal and bacterial target sequences were used to standardize comparisons across qPCR and plate-based dPCR platforms. Preliminary single-assay evaluations were conducted on HVAC filter samples from a commercial building air-handling unit. So far, dPCR has not guaranteed improved sensitivity for low biomass samples for both bacterial and fungal quantifications. Continued refinement of single-target protocol will support multiplex assays for increased throughput of indoor sampling.

References
Pongracic, J. A., O’Connor, G. T., Muilenberg, M. L., Vaughn, B., Gold, D. R., Kattan, M., Morgan, W. J., Gruchalla, R. S., Smartt, E., & Mitchell, H. E. (2010). Differential effects of outdoor versus indoor fungal spores on asthma morbidity in inner-city children. Journal of Allergy and Clinical Immunology, 125(3), 593–599. https://doi.org/10.1016/j.jaci.2009.10.036
Thorn, J., & Rylander, R. (1998). Inflammatory response after inhalation of bacterial endotoxin assessed by the induced sputum technique. Thorax, 53(12), 1047–1052. https://doi.org/10.1136/thx.53.12.1047