Detection of nucleic acids as the biomarkers of hazardous agents in bioaerosols

The transmission of hazardous agents via bioaerosols has been long-acknowledged as an important risk, yet it has been poorly studied and understood, due to paltry data, methodological heterogeneity and limitation. Therefore, this thesis mainly chose 2 different kind of hazardous agents: antibiotic resistance genes (ARGs) and human respiratory viruses as examples to attempt to narrow several gaps in bioaerosol research. Antibiotic resistance genes (ARG) have been considered as a global emerging threat to public health systems. As special locations where both antibiotics and ARGs are directly used, biology laboratories are poorly studied but potential important emission sources where not only the environmental stress is strong but also obtaining resistance is much easier comparing to other well studied hot spots including farms, hospitals, wastewater treatment plants and landfills where antibiotics but not ARGs are used or discharged. Therefore, Chapter 2 focused on emission source identification .11 Swiss biology laboratories working on different fields and located in the city center, suburb and rural area were studied to reveal the abundance and diversity of airborne ARGs in them and their surrounding areas with Colony-forming units (CFU) cultivation and quantitative Polymerase Chain Reaction (qPCR). Most biology laboratories did not discharge significant amounts or varieties of ARGs and cultivable bacteria via air. No correlation was found between the number of CFUs and the abundance of 16S rRNA, but two clusters of correlated airborne ARGs, the animal husbandry related cluster, and city and hospital related cluster were identified in this study. Although most biology laboratories may not be the emission sources of a wide variety of airborne ARGs, the ARGs in the animal husbandry related cluster which are abundant in the animal laboratories and aadA1 which is abundant in the laboratories working on other eukaryocytes need to be furtherly studied to make sure if they are potential health risks for the researchers. Another gap in bioaerosol research is methodology standardization. While the pandemic of coronavirus disease 2019 (COVID-19) continues to threaten public health, various primers of reverse transcription quantitative polymerase chain reaction (RT-qPCR) have been swiftly developed. Comparing their performances is very necessary for the optimization and standardization the detection and quantification method to assist the disease control. Furthermore, for developing countries, cheaper alternative molecular methods for SARS-CoV-2 identification can be crucial to prevent the next wave of infections. Therefore, in Chapter 2, we evaluated the 12 primer sets recommended by the World Health Organization (WHO) on testing both clinical patient and environmental samples with the gold standard diagnosis method: TaqMan-based RT-qPCR and a cheaper alternative method: SYBR Green-based RT-qPCR. We found that using suitable primer sets, such as ORF1ab, 2019_nCoV_N1 and 2019_nCoV_N3, the performance of the SYBR Green approach was comparable or even outperformed the TaqMan approach, even when considering the newly dominating or emerging variants, including Delta, Eta, Kappa, Lambda and Mu. ORF1ab and 2019_nCoV_N3 were found to be the best combination for sensitive and reliable SARS-CoV-2 molecular diagnostics due to their high sensitivity, specificity and broad accessibility. Based on the results of Chapter 2, we furtherly collected outdoor airborne particulate matter (PM) samples from November 2019 to April 2020 in Bern, Lugano, and Zurich to study the correlations between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses, between viruses and environmental factors, and between viruses and human behavior changes due to the public health measures against COVID-19. Because similar studies are scarce on air surveillance of the virus in outdoor non-healthcare environments which is also a current gap of the understanding of viral bioaerosols, although virus-laden particles have been commonly detected and studied in the aerosol samples from indoor healthcare settings. Among 14 detected viruses, influenza A, HCoV-NL63, HCoV-HKU1, and HCoV-229E were abundant in air. SARS-CoV-2 and enterovirus were moderately common, while the remaining viruses occurred only in low concentrations. SARS-CoV-2 was detected in PM10 (PM below 10 µm) samples of Bern and Zurich, and PM2.5 (PM below 2.5 µm) samples of Bern which exhibited a concentration positively correlated with the local COVID-19 case number. The concentration was also correlated with the concentration of enterovirus which raised the concern of coinfection. The estimated COVID-19 infection risks of an hour exposure at these two sites were generally low but still cannot be neglected. This study demonstrated the potential functionality of outdoor air surveillance of airborne respiratory viruses, especially at transportation hubs and traffic arteries. However, there is no available detection and quantification method capable of fast, sensitive, specific and convenient onsite measuring for air surveillance and risk alarming. CRISPR diagnostics systems are potential candidates because they are fast and specific. Yet, current CRISPR diagnostics are either moderate sensitive enough or supersensitive, and rely heavily on pre-amplification which hinders their potential for quantification. To overcome this limitation, in Chapter 4, a new ultrasensitive CRISPR-Cas based nucleic acid detection system, named Paired-CRISPR-Cas-Only Identification Relay Origination Technique (POIROT) was developed to enable CRISPR-Cas12 cascade the signal to CRISPR-Cas13 for nucleic acid detection. The system was tested with two different DNA targets: the viral DNA of human monkey pox virus and floR, an antibiotic resistance gene against florfenicol. This system has an impressive low limit of detection (LoD) of 1 copy/μl and a short responding time of 15 minutes. The system has potential for use in both diagnosis and environmental surveillance, and may provide new insights into the forms of DNAs in the environment. While the system has some limitations that need to be addressed, it offers promising potential for the development of high-performance artificial nucleic acid detection methods with CRISPR-Cas, particularly in environmental surveillance. The findings in this thesis have improved the understanding of bioaerosols from several angles, and offered potential choices to narrow the gaps in bioaerosol research, paving the ways for future. These advancements can help scientists, public health planners, policymakers, and regulators to improve the air quality, and protecting the environment, and also provide suggestions and warnings to the public on self-protection, ultimately contributing to creating healthier conditions on our habitable planet together from both sides