Details

Autor(en) / Beteiligte

• Luhung, Irvan
• Wu, Yan
• Ng, Chun Kiat
• Miller, Dana
• Cao, Bin
• Chang, Victor Wei-Chung
• Yao, Maosheng

Titel

Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis

Ist Teil von

• PloS one, 2015-11, Vol.10 (11), p.e0141158-e0141158

Ort / Verlag

United States: Public Library of Science

Erscheinungsjahr

2015

Link zum Volltext

Quelle

EZB Free E-Journals

Beschreibungen/Notizen

• 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. 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). 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%.