| Author | Do Thi Thu Huong |
| Call Number | AIT Thesis no.FB-06-05 |
| Subject(s) | Foodborne diseases
|
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. FB-06-05 |
| Abstract | The hypothesis for this study was that polymcrase chain reaction (PCR) based bacterial detection without enrichment step could be applicable by concentrating bacteria from food matrix by metal hydroxide. The false positive from exogenous DNA for bacterial detection could be eliminated by DNase I treatment, one step before DNA extraction. DNase I facilitated for removal DNA from dead cells and had no detectable damaging effect signal on the DNA presented in live/intact cells. The desired genes eaeA for Escherichia coli 0157:H7 (384 bp) and E.coli wild type (580 bp), hly gene for Listeria monocytogenes (482 bp) and invA gene for Salmonella typhimurium (685 bp) were successfully amplified for detection by conventional PCR and real-time PCR. In real-time PCR the double-stranded DNA dye SBYR green I was used for quantify the amplicon production. The Ct -values were used to calculate percentage recovery. Bacterial recoveries were calculated based on direct plating of the precipitate from pure culture ranged from 83.57% to 90.30% for titanous hydroxide and from 87.63% to 94.32 % for zirconium hydroxide in immobilization of Escherichia coli 0157:H7, Salmonella typhimurium ATCC 13311 and Staphylococcus aureus. In the case of real-time PCR method, bacterial immobilization with both metal hydroxides at different ratio volume of sample to metal hydroxide (1:1 and 1:2) gave recoveries from 93.57% to 99.3% compared to 31.5% to 54.70% with no immobilization. The ratio 1:1 was suggested for saving metal hydroxide and used for bacterial detection by DTD- real time PCR. Sample volume was reduced 50- fold after immobilization by zirconium hydroxide. The detection limits for E.coli 0157:H7, L.monocytogenes, E.coli wild type was 5x10' cells and of S. typhinuriuin was 5x 102 cells when seeded in 10 ml of whole milk by both conventional and real-time PCR. Low concentrations of bacteria were detected by realtime PCR with high cycles. The most important outcome of these experiments was that all four bacteria strains can be detected at low concentration by simple PCR method not requiring enrichment steps. This was possible with only using immobilization by metal hydroxide. The immobilization step is rapid ( |
| Year | 2006 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. FB-06-05 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Other Field of Studies (No Department) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Rakshit, Sudip Kumar |
| Examination Committee(s) | Athapol Noomhorm;
Jayasuriya, H.P.W. |
| Scholarship Donor(s) | Ministry of Education and Training of Vietnam |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2006 |