Development of molecular method for detection of viable Escherichia Coli in environmental waters

Molecular methods are widely used in detection of bacteria in environmental waters. However, most of these methods cannot distinguish between viable and dead cells. Thus, a method for detection of only viable bacteria in environmental waters by DNA based polymerase chain reaction (PCR) was developed...

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Main Author:	Ho, Chin Fong
Format:	Thesis
Language:	English English
Published:	2008
Subjects:	QR75-99.5 Bacteria
Online Access:	https://eprints.ums.edu.my/id/eprint/6802/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/6802/2/FULLTEXT.pdf
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id	my-ums-ep.6802
record_format	uketd_dc
spelling	my-ums-ep.68022024-03-08T06:38:33Z Development of molecular method for detection of viable Escherichia Coli in environmental waters 2008 Ho, Chin Fong QR75-99.5 Bacteria Molecular methods are widely used in detection of bacteria in environmental waters. However, most of these methods cannot distinguish between viable and dead cells. Thus, a method for detection of only viable bacteria in environmental waters by DNA based polymerase chain reaction (PCR) was developed in this study. The developed method consists of DNase treatment to remove dead cells' DNA and 'free' DNA, followed by cell lysis, PCR and agarose gel electrophoresis. A PCR protocol for Escherichia coli (E. coli) as laboratory model, using primers 16El/E2, 16El/E3 or 16El/E2/E3 was established. In removing dead cells' DNA and 'free' DNA, treatment with DNase I was preferred as compared to washing- centrifugation procedure as it was able to completely remove heat-killed cells' DNA and 'free' DNA from water sample without affecting the DNA in viable cells. Ten units of DNase I was used in this study for complete removal of dead cells' DNA and 'free' DNA and PCR analysis was performed within 1 h after DNase treatment. Results also showed that additional DNase inactivation step was unnecessary after DNase treament and equivalent concentration of DNase reaction buffer in samples with DNase was included in the controls to obtain accurate comparison. In application of the developed method in environmental waters, membrane filtration (0.45 Âµm pore size) was used to concentrate water samples and it was found that it could partially remove 'free' DNA. The application of the developed method following membrane filtration in environmental river waters was also feasible. In conclusion, a simple and rapid method to detect viable bacteria in environmental waters was successfully developed in this study. 2008 Thesis https://eprints.ums.edu.my/id/eprint/6802/ https://eprints.ums.edu.my/id/eprint/6802/1/24%20PAGES.pdf text en public https://eprints.ums.edu.my/id/eprint/6802/2/FULLTEXT.pdf text en validuser masters Universiti Malaysia Sabah Sekolah Sains dan Teknologi
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English English
topic	QR75-99.5 Bacteria
spellingShingle	QR75-99.5 Bacteria Ho, Chin Fong Development of molecular method for detection of viable Escherichia Coli in environmental waters
description	Molecular methods are widely used in detection of bacteria in environmental waters. However, most of these methods cannot distinguish between viable and dead cells. Thus, a method for detection of only viable bacteria in environmental waters by DNA based polymerase chain reaction (PCR) was developed in this study. The developed method consists of DNase treatment to remove dead cells' DNA and 'free' DNA, followed by cell lysis, PCR and agarose gel electrophoresis. A PCR protocol for Escherichia coli (E. coli) as laboratory model, using primers 16El/E2, 16El/E3 or 16El/E2/E3 was established. In removing dead cells' DNA and 'free' DNA, treatment with DNase I was preferred as compared to washing- centrifugation procedure as it was able to completely remove heat-killed cells' DNA and 'free' DNA from water sample without affecting the DNA in viable cells. Ten units of DNase I was used in this study for complete removal of dead cells' DNA and 'free' DNA and PCR analysis was performed within 1 h after DNase treatment. Results also showed that additional DNase inactivation step was unnecessary after DNase treament and equivalent concentration of DNase reaction buffer in samples with DNase was included in the controls to obtain accurate comparison. In application of the developed method in environmental waters, membrane filtration (0.45 Âµm pore size) was used to concentrate water samples and it was found that it could partially remove 'free' DNA. The application of the developed method following membrane filtration in environmental river waters was also feasible. In conclusion, a simple and rapid method to detect viable bacteria in environmental waters was successfully developed in this study.
format	Thesis
qualification_level	Master's degree
author	Ho, Chin Fong
author_facet	Ho, Chin Fong
author_sort	Ho, Chin Fong
title	Development of molecular method for detection of viable Escherichia Coli in environmental waters
title_short	Development of molecular method for detection of viable Escherichia Coli in environmental waters
title_full	Development of molecular method for detection of viable Escherichia Coli in environmental waters
title_fullStr	Development of molecular method for detection of viable Escherichia Coli in environmental waters
title_full_unstemmed	Development of molecular method for detection of viable Escherichia Coli in environmental waters
title_sort	development of molecular method for detection of viable escherichia coli in environmental waters
granting_institution	Universiti Malaysia Sabah
granting_department	Sekolah Sains dan Teknologi
publishDate	2008
url	https://eprints.ums.edu.my/id/eprint/6802/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/6802/2/FULLTEXT.pdf
_version_	1794022693469683712

Development of molecular method for detection of viable Escherichia Coli in environmental waters

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