Multiplex PCR for the detection of three pathogenic species of Vibrio in shrimp and crab | |
| Author | Subedi, Parang Kush |
| Call Number | AIT Thesis no.FB-05-02 |
| Subject(s) | Pathogenic bacteria Pathogenic microorganisms--Type specimens Shrimps--Pathogens Crabs--Pathogens |
| 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-05-02 |
| Abstract | This study describes the development of a multiplex PCR system for the simultaneous detection of three pathogenic Vibrio species that are found in crustaceans particularly shrimp and crab. Multiplex PCR amplification of vvh gene of Vibrio vulnificus, 16sRDNA gene of Vibrio harveyi and gyrB gene of Vibrio parahaemolvticus were used for the detection of these pathogenic strains. For simultaneous detection of these Vibrio species, an annealing temperature of 60°C, DNA template concentration and volume of 0.3 pg/50pL and 0.4 l respectively and the primers concentration and volume of 1 mM and 0.3 l respectively gave good results. The specificity studies showed that the protocol developed was specific for the three species studies and did not amplify other Vibrio and non-Vibrio species of aquaculture significance and each other. The sensitivity detection for pure culture in haemolymph with and without enrichment showed that the lower detection limit were 10 cells/ml for Vibrio harvevi and Vibrio parahaemolyticus and I x 10; cells/ml for Vibrio vulnificus. However, after enrichment in haemolymph mixed with TSBYE broth, the detection limit was 10 cells /ml for each Vibrio species. Applications of multiplex PCR system in pure culture, spiked haemolymph of crab and shrimp and contaminated samples from aquaculture ponds gave good results. Thus the multiplex PCR system developed would help to ensure rapid and accurate detection of these three Vibrio species pathogenic to crustaceans simultaneously. This would help to improve the safety of Vibrio infection to crustaceans and associated food for consumers |
| Year | 2005 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. FB-05-02 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Energy and Climate Change (Former title: Department of Energy, Environment, and Climate Change (DEECC)) |
| Academic Program/FoS | Environmental Engineering and Management (EV) |
| Chairperson(s) | Rakshit, Sudip K.; |
| Examination Committee(s) | Athapol Noomhorm;Jindal, Vinod. K. ;Praparsiri Barnette ; |
| Scholarship Donor(s) | Asian Institute of Technology Fellowship; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2005 |