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

• 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; https://eprints.ums.edu.my/id/eprint/6802/

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 µm 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.