Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis
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 res...
| Main Authors: | , , , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
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2016
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| Online Access: | https://hdl.handle.net/10356/82698 http://hdl.handle.net/10220/40195 |
| _version_ | 1811679676439986176 |
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| author | Luhung, Irvan Wu, Yan Ng, Chun Kiat Miller, Dana Cao, Bin Chang, Victor Wei-Chung |
| author2 | Yao, Maosheng |
| author_facet | Yao, Maosheng Luhung, Irvan Wu, Yan Ng, Chun Kiat Miller, Dana Cao, Bin Chang, Victor Wei-Chung |
| author_sort | Luhung, Irvan |
| collection | NTU |
| description | 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%. |
| first_indexed | 2024-10-01T03:12:56Z |
| format | Journal Article |
| id | ntu-10356/82698 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:12:56Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | ntu-10356/826982022-02-16T16:30:10Z Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis Luhung, Irvan Wu, Yan Ng, Chun Kiat Miller, Dana Cao, Bin Chang, Victor Wei-Chung Yao, Maosheng School of Civil and Environmental Engineering Interdisciplinary Graduate School (IGS) Singapore Centre for Environmental Life Sciences Engineering Civil and Environmental Engineering 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%. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version 2016-02-26T07:33:13Z 2019-12-06T15:00:35Z 2016-02-26T07:33:13Z 2019-12-06T15:00:35Z 2015 Journal Article Luhung, I., Wu, Y., Ng, C. K., Miller, D., Cao, B.,& Chang, V. W. C. (2015). Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis. PLOS ONE, 10(11), e0141158-. 1932-6203 https://hdl.handle.net/10356/82698 http://hdl.handle.net/10220/40195 10.1371/journal.pone.0141158 26619279 en PLOS ONE © 2015 Luhung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 18 p. application/pdf |
| spellingShingle | Civil and Environmental Engineering Luhung, Irvan Wu, Yan Ng, Chun Kiat Miller, Dana Cao, Bin Chang, Victor Wei-Chung Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis |
| title | Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis |
| title_full | Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis |
| title_fullStr | Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis |
| title_full_unstemmed | Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis |
| title_short | Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis |
| title_sort | protocol improvements for low concentration dna based bioaerosol sampling and analysis |
| topic | Civil and Environmental Engineering |
| url | https://hdl.handle.net/10356/82698 http://hdl.handle.net/10220/40195 |
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