The Resource Detection of viable escherichia coli in environmental water using a combined propidium monoazide staining-real-time PCR, By Yuan Yuan

Detection of viable escherichia coli in environmental water using a combined propidium monoazide staining-real-time PCR, By Yuan Yuan

Label
Detection of viable escherichia coli in environmental water using a combined propidium monoazide staining-real-time PCR
Title
Detection of viable escherichia coli in environmental water using a combined propidium monoazide staining-real-time PCR
Statement of responsibility
By Yuan Yuan
Creator
Contributor
Author
Thesis advisor
Subject
Genre
Language
eng
Summary
Escherichia coli, as a principal fecal indicator bacterium, is used to monitor water quality world-wide. Real-time PCR (qPCR) is a promising way to achieve a rapid and sensitive detection of E. coli in water samples. The ability to detect only viable E. coli cells specifically provides a more accurate reflection of water quality and safety. The objective of this study was to test a new self-designed primer set targeting the ycjM gene of E. coli in a propidium monoazide (PMA)-qPCR assay, and to investigate its specificity and efficiency in detecting only viable E. coli in environmental water. Specificity of the ycjM primer set was checked using nineteen different E. coli strains, including E. coli ATCC 25922, E. coli K12, E. coli O157 strains and strains from environmental sources, as well as Shigella. dysenteriae, S. flexneri and S. sonnei. It showed that ycjM primers could detect most of the E. coli strains but did not amplify any DNA from Shigella strains. Four cultures of E. coli Ct 14, Dr 23, H 24, P 24, freshly grown separately, and then mixed together and serially diluted to 102 -106 CFU/mL. Dead cell suspensions were obtained by heating at 80 °C for 20 min. After being spiked with the cell suspension, tap water and other environmental water samples, including water from Lake of the Ozarks, Missouri River and Mississippi River were filtered or centrifuged for the cell collection. Samples were then treated with PMA, followed by DNA isolation and TaqMan® realtime PCR detection. Results showed that in pure culture, 5 æM PMA with a 10-min light exposure was efficient to inhibit the amplification of DNA from 105 CFU /mL dead E. coli cells, with a detection limit of 102 CFU/100mL. In tap and winter environmental water, a higher PMA concentration of 10 æM was required and as low as 103 CFU/100 mL viable cells could be detected, in the presence of 105 CFU/100 mL dead cells; in water samples collected in summer, 102 CFU/10 mL viable cells could be detected after a 20 æM PMA treatment, in the presence of 104 CFU/10 mL dead cell by this optimized PMA-qPCR assay. Significant and strong correlations were found between the PMA-qPCR and EPA Standard Method 1603, without over or underestimation of PMA-qPCR compared with Method 1603. In conclusion, the PMA-qPCR could accurately and effectively differentiate viable E. coli cells from dead cells by suppressing the amplification of DNA from dead cells, but optimization steps to remove suspended solids in environmental water samples were necessary
Cataloging source
MUU
http://library.link/vocab/creatorName
Yuan, Yu'an
Degree
M.S.
Dissertation note
Thesis
Dissertation year
2015.
Government publication
government publication of a state province territory dependency etc
Granting institution
University of Missouri--Columbia
Illustrations
illustrations
Index
no index present
Literary form
non fiction
Nature of contents
  • dictionaries
  • bibliography
  • theses
http://library.link/vocab/relatedWorkOrContributorName
Mustapha, Azlin
http://library.link/vocab/subjectName
  • Escherichia coli
  • Bacterial pollution of water
  • Polymerase chain reaction
  • Water quality
  • Missouri
  • Missouri River
  • Mississippi River
Label
Detection of viable escherichia coli in environmental water using a combined propidium monoazide staining-real-time PCR, By Yuan Yuan
Instantiates
Publication
Note
Dr. Azlin Mustapha, Thesis Supervisor
Bibliography note
Includes bibliographical references (pages 68-80)
Carrier category
online resource
Carrier category code
  • cr
Carrier MARC source
rdacarrier
Content category
text
Content type code
  • txt
Content type MARC source
rdacontent
Control code
986471044
Extent
1 online resource (x, 80 pages)
Form of item
online
Media category
computer
Media MARC source
rdamedia
Media type code
  • c
Other physical details
illustrations (some color)
Specific material designation
remote
System control number
(OCoLC)986471044
Label
Detection of viable escherichia coli in environmental water using a combined propidium monoazide staining-real-time PCR, By Yuan Yuan
Publication
Note
Dr. Azlin Mustapha, Thesis Supervisor
Bibliography note
Includes bibliographical references (pages 68-80)
Carrier category
online resource
Carrier category code
  • cr
Carrier MARC source
rdacarrier
Content category
text
Content type code
  • txt
Content type MARC source
rdacontent
Control code
986471044
Extent
1 online resource (x, 80 pages)
Form of item
online
Media category
computer
Media MARC source
rdamedia
Media type code
  • c
Other physical details
illustrations (some color)
Specific material designation
remote
System control number
(OCoLC)986471044

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