Title Page
Contents
Abstract 9
Introduction 11
Materials and Methods 15
1. Collection of samples 15
2. DNA Extraction 18
3. Confirmation of HPV 19
4. DNA Cloning - Gel preparation 19
5. Ligation and transformation 21
6. Sequencing 22
7. Sequence analysis 23
8. Quantitative Real-time PCR 23
9. Primer designing 24
Results and Discussion 26
1. Confirmation of HPV infection by PCR 28
2. Sequence and analysis of the viral genome 34
3. Quantification of viral loads 39
4. Multiple sequencing and phylogenetic tree 41
References 48
Table 1. Summary of sample collection 16
Table 2. Primers used for PCR assays 25
Table 3. Summary of PCR results 30
Table 4. Summary of similarity of DNA sequence of samples to the FcDNV 38
Fig. 1. Sample groups of shrimps used for the experiment. Sample 1, 2, 3 and 4 respectively 17
Fig. 2. Electrophoresis gel for detection of HPV of shrimps collected from a local market in Busan. Lane 1. 1K plus DNA ladder, Lane 2: negative... 31
Fig. 3. Electrophoresis gel for HPV of shrimps from Geojedo: Lane 1. 1K bp DNA ladder, Lane 2: negative control, Lanes 3-10: PCR products from... 31
Fig. 4. Electrophoresis gel for HPV of shrimps from Seosan: Lane 1. 1K plus DNA ladder, Lane 2: negative control, Lanes 3-10: PCR... 32
Fig. 5. Electrophoresis gel for HPV of shrimps from Malaysia: Lane 1. 1K plus bp DNA ladder, Lane 2: negative control, Lanes 3-12: PCR... 32
Fig. 6. Electrophoresis gel for HPV of shrimps from Gyeonggi: Lane 1. 1K bp DNA ladder, Lane 2: negative control, Lanes 3-11: HPV negative PCR... 33
Fig. 7. Sample electrophoresis gel for HPV colonies of shrimp, Lane 1. 1K bp DNA ladder, Lane 17: negative control, Lanes 2-16: PCR... 35
Fig. 8. Sample electrophoresis gel for HPV on DNA plasmid of shrimp, Lane 1. 1K bp DNA ladder, Lane 3: negative control, Lanes 2-9: PCR... 35
Fig. 9. The blast result of shrimp from a local market in Busan 36
Fig. 10. Blast result of sample shrimp from shrimp Geojedo 36
Fig. 11. Blast result of a shrimp sample from Malaysia 37
Fig. 12. Blast result of a shrimp from Seosan 37
Fig. 13. Standard curve of HPV Taqman real-time PCR. Threshold cycle (CT) plotted against HPV concentration. 40
Fig. 14. The genome map of typical HPV with the location of primers 43
Fig. 15. Multiple sequence alignment of four shrimp samples from different geographical origins compared to the Korean isolate of HPV strain (FcDNV) 45
Fig. 16. Phylogenetic tree of the relationship between samples of shrimp from four different geographical origins compared to the Korean isolate of HPV strain (FcDNV) 46