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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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DNA measurements of AHU filter samples for seven treatment pathways of sonication at varying temperature.(a)—Total DNA yields (Qubit). (b)–Results from bacterial and fungal qPCR. N = 4 for each case. * denotes statistically significant difference to mean of DNA yield extracted with 65°C incubation.
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pone.0141158.g003: DNA measurements of AHU filter samples for seven treatment pathways of sonication at varying temperature.(a)—Total DNA yields (Qubit). (b)–Results from bacterial and fungal qPCR. N = 4 for each case. * denotes statistically significant difference to mean of DNA yield extracted with 65°C incubation.

Mentions: The effects of sonication and thermal incubation on the final DNA yield are reported in terms of total DNA per cm2 area of filter or per m3 of air (as determined by Qubit), and as targeted DNA sequence per cm2 area of filter or per m3 of air (as determined by qPCR). Fig 3 shows that the DNA yields of the tested AHU filter samples increases gradually as the incubation temperature rises. The total DNA yield shown in Fig 3A was 1.48 ng/cm2 with no treatment, increasing to 4.56 ng/cm2 and 5.22 ng/cm2 with incubation treatment at 65°C and 70°C, respectively. The qPCR result on the same set of samples (Fig 3B) shows a similar trend. The bacterial DNA concentration rose from 51 pg/cm2 to 320 pg/cm2 with incubation at 65°C. The bacterial DNA yield then declined to 262 pg/cm2 with incubation at 70°C and 168 pg/cm2 with incubation at 75°C. Similarly, the highest concentration of fungal DNA (1290 pg/cm2) was also obtained from samples incubated at 65°C. The concentration of fungal DNA remained relatively constant for samples incubated at 70°C before decreasing to 914 pg/cm2 at 75°C.


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)

DNA measurements of AHU filter samples for seven treatment pathways of sonication at varying temperature.(a)—Total DNA yields (Qubit). (b)–Results from bacterial and fungal qPCR. N = 4 for each case. * denotes statistically significant difference to mean of DNA yield extracted with 65°C incubation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141158.g003: DNA measurements of AHU filter samples for seven treatment pathways of sonication at varying temperature.(a)—Total DNA yields (Qubit). (b)–Results from bacterial and fungal qPCR. N = 4 for each case. * denotes statistically significant difference to mean of DNA yield extracted with 65°C incubation.
Mentions: The effects of sonication and thermal incubation on the final DNA yield are reported in terms of total DNA per cm2 area of filter or per m3 of air (as determined by Qubit), and as targeted DNA sequence per cm2 area of filter or per m3 of air (as determined by qPCR). Fig 3 shows that the DNA yields of the tested AHU filter samples increases gradually as the incubation temperature rises. The total DNA yield shown in Fig 3A was 1.48 ng/cm2 with no treatment, increasing to 4.56 ng/cm2 and 5.22 ng/cm2 with incubation treatment at 65°C and 70°C, respectively. The qPCR result on the same set of samples (Fig 3B) shows a similar trend. The bacterial DNA concentration rose from 51 pg/cm2 to 320 pg/cm2 with incubation at 65°C. The bacterial DNA yield then declined to 262 pg/cm2 with incubation at 70°C and 168 pg/cm2 with incubation at 75°C. Similarly, the highest concentration of fungal DNA (1290 pg/cm2) was also obtained from samples incubated at 65°C. The concentration of fungal DNA remained relatively constant for samples incubated at 70°C before decreasing to 914 pg/cm2 at 75°C.

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