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

Luhung I, Wu Y, Ng CK, Miller D, Cao B, Chang VW - PLoS ONE (2015)

Bottom Line: In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR).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%.

View Article: PubMed Central - PubMed

Affiliation: SinBerBEST Program, Berkeley Education Alliance for Research in Singapore, Singapore.

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%.

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Related in: MedlinePlus

Comparison of two sampling approaches.Comparison of a sampling approach utilizing a single filter continuously sampled for 24 h (grey bar) and a combined series of three filters, each operated for 8 h (black bar) expressed in terms of total DNA (left bar, left axis) measured by Qubit and in terms of bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA measured by qPCR (N = 3).
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pone.0141158.g005: Comparison of two sampling approaches.Comparison of a sampling approach utilizing a single filter continuously sampled for 24 h (grey bar) and a combined series of three filters, each operated for 8 h (black bar) expressed in terms of total DNA (left bar, left axis) measured by Qubit and in terms of bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA measured by qPCR (N = 3).

Mentions: Fig 5 (left bar) shows that—based on DNA fluorometry—there is no distinct difference in total DNA yield between the 1×24 hour samples (0.14 ng/m3 of air) and the 3×8 hour samples (0.13 ng/m3 of air) (Paired t-test, p value = 0.18). This result led us to further analyze the DNA by performing fungal and bacterial qPCR analysis to investigate whether different yield can be seen for different microbiological targets using these two sampling approaches.


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

Luhung I, Wu Y, Ng CK, Miller D, Cao B, Chang VW - PLoS ONE (2015)

Comparison of two sampling approaches.Comparison of a sampling approach utilizing a single filter continuously sampled for 24 h (grey bar) and a combined series of three filters, each operated for 8 h (black bar) expressed in terms of total DNA (left bar, left axis) measured by Qubit and in terms of bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA measured by qPCR (N = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664469&req=5

pone.0141158.g005: Comparison of two sampling approaches.Comparison of a sampling approach utilizing a single filter continuously sampled for 24 h (grey bar) and a combined series of three filters, each operated for 8 h (black bar) expressed in terms of total DNA (left bar, left axis) measured by Qubit and in terms of bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA measured by qPCR (N = 3).
Mentions: Fig 5 (left bar) shows that—based on DNA fluorometry—there is no distinct difference in total DNA yield between the 1×24 hour samples (0.14 ng/m3 of air) and the 3×8 hour samples (0.13 ng/m3 of air) (Paired t-test, p value = 0.18). This result led us to further analyze the DNA by performing fungal and bacterial qPCR analysis to investigate whether different yield can be seen for different microbiological targets using these two sampling approaches.

Bottom Line: In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR).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%.

View Article: PubMed Central - PubMed

Affiliation: SinBerBEST Program, Berkeley Education Alliance for Research in Singapore, Singapore.

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%.

Show MeSH
Related in: MedlinePlus