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Bope Master's Thesis ESGP FINAL.pdf (534.74 KB)

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Using Digital PCR to Improve Quantitative Measurement of Bacterial and Fungal DNA from Carpet Dusts in the Built Environment

Bope, Ashleigh M
http://orcid.org/0000-0001-5671-4801
http://rave.ohiolink.edu/etdc/view?acc_num=osu1524229362168898

Abstract Details

2018, Master of Science, Ohio State University, Environmental Science.
Genetic quantification methods used to analyze microbial communities in house dust can help elucidate potential human health exposures. However, quality assurance measures associated with this method are mostly undefined, especially for more recent technologies. Quantifying and including extraction efficiencies when analyzing environmental samples is of the upmost importance because microbial abundances could be grossly underestimated if these are excluded. The goal of this study is to determine the accuracy, precision, and method detection limits (MDLs) of PCR-based methods in quantitative analysis of microbes in house dust samples. Real-time, or quantitative, polymerase chain reaction (qPCR) is a well-established analytical method for enumeration of microbial populations in environmental samples. Digital PCR (dPCR) is a newer technology that offers increased precision at lower concentration and is comparatively less prone to inhibition. There are advantages and limitations associated with both technologies but combining these methods when analyzing indoor microbial concentrations offers more precise and accurate quantification. To fully define this analysis pipeline, dust spiked with bacterial cells of Escherichia coli, and Bacillus atrophaeus and fungal spores of Aspergillus fumigatus were embedded into nylon carpet of varying pile heights and recovered by vacuuming. Efficiencies associated with recovery of the dust from the carpet after embedment ranged from 61% to 97% and varied based on pile height. Efficiencies associated with recovery of DNA from the samples ranged from 18% for A. fumigatus spores to 77% for B. atrophaeus cells. MDL calculations using A. fumigatus spores indicated the smallest amount of detectable DNA maintaining accuracy and precision corresponded to 134 spores/µL of environmental sample for qPCR and 60 spores/µL of environmental sample for dPCR. As PCR technologies advance, accuracy, precision and MDLs associated with analysis need to be updated to ensure results enable mechanistic investigation of the fate and sources of indoor microbial communities.
Karen Dannemiller (Advisor)
Andrew May (Committee Member)
Jiyoung Lee (Committee Member)
49 p.
Environmental Science
microbial exposures; digital PCR, indoor environment

Recommended Citations

Citations

  • Bope, A. M. (2018). Using Digital PCR to Improve Quantitative Measurement of Bacterial and Fungal DNA from Carpet Dusts in the Built Environment [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524229362168898

    APA Style (7th edition)

  • Bope, Ashleigh. Using Digital PCR to Improve Quantitative Measurement of Bacterial and Fungal DNA from Carpet Dusts in the Built Environment . 2018. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1524229362168898.

    MLA Style (8th edition)

  • Bope, Ashleigh. "Using Digital PCR to Improve Quantitative Measurement of Bacterial and Fungal DNA from Carpet Dusts in the Built Environment ." Master's thesis, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524229362168898

    Chicago Manual of Style (17th edition)

osu1524229362168898
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This open access ETD is published by The Ohio State University and OhioLINK.