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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

Sampling duration experiments for microorganisms collected on filters.(a)–Comparison of 1×24 hour versus 3×8 hour parallel sampling. Two sets of sampling trains collected air at the same time and location for 24 hours. The first set sampled the air continuously for 24 hours on a single filter, while the other set’s filter was replaced every 8 hours. DNA extracted from the three 8-h filters was then pooled and the result is compared with the first set which was extracted alone. (b)–Testing loss of GFP-tagged bacteria during sampling. A set of filters sampled air at the same time and location for a total duration of 20 hours. Known quanta of GFP-tagged S. oneidensis cells were spiked onto the filters at staggered timepoints, exposing them to different durations of air sampling.
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pone.0141158.g001: Sampling duration experiments for microorganisms collected on filters.(a)–Comparison of 1×24 hour versus 3×8 hour parallel sampling. Two sets of sampling trains collected air at the same time and location for 24 hours. The first set sampled the air continuously for 24 hours on a single filter, while the other set’s filter was replaced every 8 hours. DNA extracted from the three 8-h filters was then pooled and the result is compared with the first set which was extracted alone. (b)–Testing loss of GFP-tagged bacteria during sampling. A set of filters sampled air at the same time and location for a total duration of 20 hours. Known quanta of GFP-tagged S. oneidensis cells were spiked onto the filters at staggered timepoints, exposing them to different durations of air sampling.

Mentions: Two experiments shown in Fig 1 were carried out to assess the effect of sampling duration on extracted DNA results. Fig 1A illustrates the first experiment, which employed a pair of identical sampling trains operated in parallel. With one train, the air was sampled continuously for 24 hours using a single filter. The second train also operated for 24 hours, but the filters were replaced with new ones at eight-hour intervals so that three sequential filters were collected in all. All four filters were then processed. The one 24-hour filter was extracted alone, while the DNA extracted from three 8-hour filters was pooled into a single DNA solution. Qubit measurements and qPCR were then applied to the final DNA solutions. The purpose of this investigation was to assess whether there is any DNA loss associated with long-duration filter-based sampling. If DNA degrades with extended sampling, then the yield recovered from three separate filters would be higher in sum than the single 24-h filter, since these sequential filters are each exposed to a lesser duration of air sampling.


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)

Sampling duration experiments for microorganisms collected on filters.(a)–Comparison of 1×24 hour versus 3×8 hour parallel sampling. Two sets of sampling trains collected air at the same time and location for 24 hours. The first set sampled the air continuously for 24 hours on a single filter, while the other set’s filter was replaced every 8 hours. DNA extracted from the three 8-h filters was then pooled and the result is compared with the first set which was extracted alone. (b)–Testing loss of GFP-tagged bacteria during sampling. A set of filters sampled air at the same time and location for a total duration of 20 hours. Known quanta of GFP-tagged S. oneidensis cells were spiked onto the filters at staggered timepoints, exposing them to different durations of air sampling.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141158.g001: Sampling duration experiments for microorganisms collected on filters.(a)–Comparison of 1×24 hour versus 3×8 hour parallel sampling. Two sets of sampling trains collected air at the same time and location for 24 hours. The first set sampled the air continuously for 24 hours on a single filter, while the other set’s filter was replaced every 8 hours. DNA extracted from the three 8-h filters was then pooled and the result is compared with the first set which was extracted alone. (b)–Testing loss of GFP-tagged bacteria during sampling. A set of filters sampled air at the same time and location for a total duration of 20 hours. Known quanta of GFP-tagged S. oneidensis cells were spiked onto the filters at staggered timepoints, exposing them to different durations of air sampling.
Mentions: Two experiments shown in Fig 1 were carried out to assess the effect of sampling duration on extracted DNA results. Fig 1A illustrates the first experiment, which employed a pair of identical sampling trains operated in parallel. With one train, the air was sampled continuously for 24 hours using a single filter. The second train also operated for 24 hours, but the filters were replaced with new ones at eight-hour intervals so that three sequential filters were collected in all. All four filters were then processed. The one 24-hour filter was extracted alone, while the DNA extracted from three 8-hour filters was pooled into a single DNA solution. Qubit measurements and qPCR were then applied to the final DNA solutions. The purpose of this investigation was to assess whether there is any DNA loss associated with long-duration filter-based sampling. If DNA degrades with extended sampling, then the yield recovered from three separate filters would be higher in sum than the single 24-h filter, since these sequential filters are each exposed to a lesser duration of air sampling.

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