Title Page
ABSTRACT
Contents
1. Introduction 14
2. Materials and Methods 21
2.1. Bacterial strains, media, and growth conditions 21
2.2. Isolation of foodborne pathogens 24
2.3. DNA extraction 24
2.4. 16S rRNA gene sequencing 25
2.5. Pathogenic identification of E. coli using PCR 26
2.6. Genome sequencing and analysis 26
2.7. NGS panel primer design and optimization 27
2.8. Singleplex PCR and crosscheck PCR 28
2.9. Multiplex PCR 29
2.10. Collection of agricultural water samples and the preparation of contaminated water samples with selected pathogens 30
2.11. Collection of fermented food samples and the preparation of contaminated fermented food samples with selected pathogens 31
2.12. NGS panel analysis 33
2.13. Quantitative real-time PCR (qPCR) 34
2.14. Statistical analysis 35
3. Results 36
3.1. NGS panel set 1: multiple detection and identification of foodborne pathogens in agricultural water 36
3.1.1. Isolation and identification of foodborne pathogens 36
3.1.2. General genome features of selected foodborne pathogens and the design of primer sets 37
3.1.3. Validation of designed primer sets 42
3.1.4. NGS panel analysis 50
3.1.5. qPCR analysis 64
3.1.6. Comparative evaluation of NGS panel analysis and qPCR 70
3.2. NGS panel set 2: multiple detection and identification of foodborne pathogens in fermented foods 74
3.2.1. Isolation and identification of foodborne pathogens 74
3.2.2. General genome features of selected foodborne pathogens and the design of primer sets 76
3.2.3. Validation of designed primer sets 81
3.2.4. NGS panel analysis 89
3.2.5. qPCR analysis 102
3.2.6. Comparative evaluation of NGS panel analysis and qPCR 109
4. Discussion 112
5. References 115
국문초록 125
Table 1. Bacterial strains, culture medium, samples, and sampling locations 22
Table 2. General genome features of selected foodborne pathogens isolated from seafood samples 38
Table 3. Selected pathogen species-specific genes, their functions, and the associated designed primer sets in NGS panel set 1 39
Table 4. In silico prediction of primer binding sites in selected foodborne pathogens isolated from seafood samples 41
Table 5. Summary of NGS panel set 1 output in six agricultural water samples with or without target pathogen 53
Table 6. General genome features of selected foodborne pathogens isolated from animal samples 77
Table 7. Selected pathogen species-specific genes, their functions, and the associated designed primer sets in NGS panel set 2 78
Table 8. In silico prediction of primer binding sites in selected foodborne pathogens isolated from animal samples 80
Table 9. Summary of NGS panel set 2 outputs in six fermented food samples with or without target pathogen 91
Fig. 1. Gel electrophoresis results of singleplex PCR using NGS panel set 1 primer sets. 43
Fig. 2. Gel electrophoresis results of the first crosscheck PCR using NGS panel set 1 primer sets. 46
Fig. 3. Gel electrophoresis results of the second crosscheck PCR using NGS panel set 1 primer sets. 47
Fig. 4. Gel electrophoresis results of multiplex PCR using NGS panel set 1 primer sets. 49
Fig. 5. Dot plot of NGS panel analysis in six agricultural water samples with a mixture of target pathogens. 56
Fig. 6. Heat map of NGS panel analysis result in six agricultural water samples with a mixture of target pathogens. 57
Fig. 7. Target pathogens-specific genes mapped read in six agricultural water samples with or without target pathogens using NGS panel. 58
Fig. 8. NGS panel analysis average read summary of target pathogens-specific genes in six agricultural water samples with a mixture of target pathogens. 63
Fig. 9. Dot plot of qPCR results in six agricultural water samples with a mixture of target pathogens. 65
Fig. 10. Heat map of qPCR result in six agricultural water samples with a mixture of target pathogens. 66
Fig. 11. Ct values of target pathogens in six agricultural water samples with or without target pathogens using qPCR. 67
Fig. 12. Linear regression of NGS panel analysis and qPCR results in six agricultural water samples with a mixture of target pathogens. 72
Fig. 13. Spearman correlation between NGS panel analysis and multiplex qPCR in six agricultural water with a mixture of target pathogens. 73
Fig. 14. Gel electrophoresis result of pathogenic type-specific PCR of E. coli. 75
Fig. 15. Gel electrophoresis results of singleplex PCR using NGS panel set 2 primer sets. 82
Fig. 16. Gel electrophoresis results of the first crosscheck PCR using NGS panel set 2 primer sets. 85
Fig. 17. Gel electrophoresis results of the second crosscheck PCR using NGS panel set 2 primer sets. 86
Fig. 18. Gel electrophoresis results of the multiplex PCR using NGS panel set 2 primer sets. 88
Fig. 19. Dot plot of NGS panel analysis in six fermented food samples with a mixture of target pathogens. 94
Fig. 20. Heat map of NGS panel analysis result in six fermented food samples with a mixture of target pathogens. 95
Fig. 21. Target pathogens-specific genes mapped read in six fermented food samples with or without a mixture of target pathogens. 96
Fig. 22. NGS panel analysis average read summary of target pathogens-specific genes in six fermented food samples with a mixture of target pathogens. 101
Fig. 23. Dot plot of qPCR results in six fermented food samples with a mixture of target pathogens. 104
Fig. 24. Heat map of qPCR result in six fermented food samples with a mixture of target pathogens. 105
Fig. 25. Ct values of target pathogens in six fermented food samples with or without a mixture of target pathogens. 106
Fig. 26. Linear regression of NGS panel analysis and qPCR results in six fermented food samples with a mixture of target pathogens. 110
Fig. 27. Spearman correlation between NGS panel analysis and multiplex qPCR results in six fermented food samples with a mixture of target pathogens. 111