title: Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis.
abstract: BACKGROUND
  As bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended.
  
  
  OBJECTIVE
  This study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1) enhanced high temperature sonication during DNA extraction; 2) effect of sampling duration on DNA recoverability; and 3) an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR).
  
  
  RESULTS
  The findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%.
authors.affiliation: SinBerBEST Program, Berkeley Education Alliance for Research in Singapore, Singapore.|School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Irvan
authors.initials: I
authors.lastname: Luhung
authors.affiliation: SinBerBEST Program, Berkeley Education Alliance for Research in Singapore, Singapore.|School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore.|Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore.
authors.forename: Chun Kiat
authors.initials: CK
authors.lastname: Ng
authors.affiliation: Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Dana
authors.initials: D
authors.lastname: Miller
authors.affiliation: School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore.|Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Bin
authors.initials: B
authors.lastname: Cao
authors.affiliation: SinBerBEST Program, Berkeley Education Alliance for Research in Singapore, Singapore.|School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Victor Wei-Chung
authors.initials: VW
authors.lastname: Chang
mesh_terms: D000391:Air Microbiology; D004269:DNA, Bacterial / Q000737:chemistry*; D004271:DNA, Fungal / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013010:Sonication
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal