PCR-DGGE profiles of microflora of fish viscera and gills as a tool for traceability | |
| Author | Caspillo, Mary Rosary T. |
| Call Number | AIT Thesis no.FB-05-17 |
| Subject(s) | Aquaculture industry--Thailand Fish trade--*Thailand |
| 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-05-17 |
| Abstract | Traceability is now perceived to be an important aspect of the food industry. Exporters to Canada, US and European Union need to have increasingly strict compliance to a good traceability system. This study provided baseline data that can be developed as a tool to trace the origin of aquaculture products. The molecular technique Polymerase Chain Reaction (PCR) - Denaturing Gradient Gel Electrophoresis (DGGE) that is capable of detecting differences between DNA fragments of the same size but different sequences was employed to profile the commensal microflora found on the gills and viscera of fish samples. Based on the results of Phase I, using normal PCR conditions and the conventional Agarose Gel Electrophoresis, bacterial strains within the same species produced more than one PCR product showing undesirable spurious band formations on the gel matrix. Hence Touchdown PCR was employed and was carried out throughout the study. Furthermore, the V-3 region of 16s rDNA gene of E.coli strains being studied was not variable based on their DGGE band profiles showing similar migration pattern. The addition of a GC-rich region in one of the primers (GC clamp) was shown to be important in order to avoid complete melting of DNA template on the DGGE run. The PCR-DGGE analysis was able to separate twelve different bacterial species predominantly present in aquaculture fish using universal primer amplifying the V3-region of 16s rDNA gene. The DGGE band profiles of fish samples from 8 different locations in Thailand (Pathumthani-Khlong 13, Nakhon Pathom, Samutsakon, Pathumthani-AIT) within the period of March 1 to April 15, 2005 were done in Phase II. Significant differences on the band profiles were observed. This band pattern is unique and can be used as a bar code for that certain location. This method can be develop as a traceability tool wherein exported fish products provided with this unique bar code can be traced back to its original location within the given batch number. When the profiles within the same location were compared throughout the whole sampling period, common bands were noted that can be used as Markers of origin when these bands will be excised and sequenced. Furthermore, when Dice Similarity Coefficient (Sd) values were manually computed and Dendrogram plots were constructed, similarities were clearly noted on the DGGE profiles at different locations and time. |
| Year | 2005 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. FB-05-17 |
| 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 Kumar; |
| Examination Committee(s) | Athapol Noomhom ;Jindal, Vinod Kumar ;Ketudat-Caims, Mariena; |
| Scholarship Donor(s) | Asian Development Bank- Japan Scholarship Program; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2005 |