| Author | Nadugala, L.M.N. Sigera |
| Call Number | AIT Thesis no.FB-05-03 |
| Subject(s) | Foodborne diseases
|
| Note | A thesis submitted in partial fulfillment of the requirements for the
degree of Master of Science, School of Environment, Resouces and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. FB-05-03 |
| Abstract | The main objective of this work was to determine methods to overcome the problems associated with rapid detection of food borne pathogens using PCR based techniques. A multiplex PCR method was developed to overcome the problem of testing one organism at a time while DNase I enzyme treatment followed by PCR (DTD PCR) was tried to overcome the problem of false positive results obtained by amplification of DNA from dead cells. Four sets of primers were used for detection of eaeA, hly, invA and gryB genes of frequently occurring food borne pathogens Escherichia coliO157:H7, Listeria monocytogenes, Salmonella enterica and Vibrio parahaemolyticus respectively. An annealing temperature of 45°C and extension temperatures of 74°C were found to be optimum for the multiplex PCR system. The Qiagen PCR master mix kit was found to be suitable for this type of applications. DNase I was experimentally proven for its ability to remove DNA from dead cells without causing any damage to the DNA present inside live cells. DNase I at a level of 10U/100l was found to remove DNA sourced from 5* 107 dead cells in food systems within one hour of incubation period. The PCR protocol used universal culture medium and the same PCR conditions with four pairs of specific primers. In the specificity test no interferences or non-specific amplification was observed when the multiplex protocol was tested with 89 strains of bacteria in these categories, namely, closely related to target organisms, distantly related to target organisms and other food industry related microorganisms. The method developed found to be sensitive to a minimum cell count of 102 cells in both pure cultures and in artificially spiked food systems. There was no interference or inhibitory actions when the protocol was applied on shrimp. Thus, this DTD multiplex PCR assay can be practically applied for identification of viable cells of four important pathogens namely Escherichia coliOl57:H7, Salmonella enterica, Listeria monocytogenes and Vibrio parahaemolyticus simultaneously |
| Year | 2005 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. FB-05-03 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Rakshit, Sudip K.; |
| Examination Committee(s) | Jindal, V. K.;Athapol Noomhorm; |
| Scholarship Donor(s) | Asian Institute of Technology Fellowship ; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2005 |