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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

Improving DNA yield with additional heat and sonication lysis.Additional sonication and thermal lysis show improved DNA yield for (a)—AHU filter samples and (b)–ambient air samples as measured by the Qubit fluorometer for total DNA (left bar, left axis) and by qPCR for bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA (N = 4).
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pone.0141158.g004: Improving DNA yield with additional heat and sonication lysis.Additional sonication and thermal lysis show improved DNA yield for (a)—AHU filter samples and (b)–ambient air samples as measured by the Qubit fluorometer for total DNA (left bar, left axis) and by qPCR for bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA (N = 4).

Mentions: The total DNA extracted from both sets of environmental samples increased substantially with the addition of sonication at 65°C, as shown in Fig 4A and 4B. For the AHU filter samples, the additional sonication and heat incubation boosts the mean of total DNA yield from 14 to 31 ng/cm2, more than a two-fold increase (Fig 4A, left bar). The DNA yield of the ambient air samples increases from 0.03 ng/m3 to 0.44 ng/m3 (Fig 4B, left bar), more than a ten-fold increase.


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)

Improving DNA yield with additional heat and sonication lysis.Additional sonication and thermal lysis show improved DNA yield for (a)—AHU filter samples and (b)–ambient air samples as measured by the Qubit fluorometer for total DNA (left bar, left axis) and by qPCR for bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA (N = 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141158.g004: Improving DNA yield with additional heat and sonication lysis.Additional sonication and thermal lysis show improved DNA yield for (a)—AHU filter samples and (b)–ambient air samples as measured by the Qubit fluorometer for total DNA (left bar, left axis) and by qPCR for bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA (N = 4).
Mentions: The total DNA extracted from both sets of environmental samples increased substantially with the addition of sonication at 65°C, as shown in Fig 4A and 4B. For the AHU filter samples, the additional sonication and heat incubation boosts the mean of total DNA yield from 14 to 31 ng/cm2, more than a two-fold increase (Fig 4A, left bar). The DNA yield of the ambient air samples increases from 0.03 ng/m3 to 0.44 ng/m3 (Fig 4B, left bar), more than a ten-fold increase.

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