Protocol improvements for low concentration DNA-based bioaerosol sampling and analysis

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

Research output: Contribution to journalArticleResearchpeer-review

29 Citations (Scopus)

Abstract

Introduction
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.
Objectives
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 and Findings
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%.
Original languageEnglish
Article numbere0141158
Number of pages18
JournalPLoS ONE
Volume10
Issue number11
DOIs
Publication statusPublished - 1 Nov 2015
Externally publishedYes

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