Title Page
Contents
ABSTRACT 9
General Introduction 13
1. Molecular diagnostics 13
2. Molecular diagnostic techniques for detection of pathogens 14
3. Avian influenza virus 16
4. Diagnostic microarray 18
5. Purpose of this study 20
Chapter 1. Design of diagnostic microarray for simultaneous subtyping and pathotyping of avian influenza virus 23
1.1. Introduction 24
1.2. Materials & Methods 27
1.2.1. Viral RNA extraction of avian influenza 27
1.2.2. Design of subtype and pathotype specific probes 27
1.2.3. Fabrication of diagnostic microarray 27
1.2.4. PCR for dye-labeling 28
1.2.5. Hybridization and washing 28
1.2.6. Scanning and image analysis 29
1.3. Results 30
1.3.1. Selection of subtype and pathotype specific probes 30
1.3.2. RT-PCR 30
1.3.3. Hybridization patterns of diagnostic microarray 30
1.4. Discussion 32
Chapter 2. Improvement of diagnostic microarray for subtyping and pathotyping of avian influenza virus 42
2.1. Introduction 43
2.2. Materials & Methods 45
2.2.1. Viral RNA extraction of avian influenza 45
2.2.2. Analysis of AIV HA gene sequence 45
2.2.3. Fabrication of diagnostic microarray 45
2.2.4. PCR for dye-labeling 46
2.2.5. Hybridization and washing 46
2.2.6. Scanning and image analysis 47
2.3. Results 48
2.3.1. Comparative analysis of AIV subtype sequences in Korea and East Asia 48
2.3.2. RT- PCR and pathogenicity primer set test 48
2.3.3. Results of diagnostic microarray 49
2.4. Discussion 51
Chapter 3. Verification of diagnostic microarray for subtyping and pathotyping using avian influenza viruses isolated in overseas (Mongolia) 64
3.1. Introduction 65
3.2. Materials & Methods 68
3.2.1. Viral RNA extraction of avian influenza 68
3.2.2. Analysis of AIV HA gene sequence 68
3.2.3. Fabrication of diagnostic microarray 68
3.2.4. PCR for dye-labeling 69
3.2.5. Hybridization and washing 69
3.2.6. Scanning and image analysis 70
3.3. Results 71
3.3.1. Analysis of AIV HA gene sequence 71
3.3.2. Results of diagnostic microarray 71
3.4. Discussion 73
Conclusion 80
References 82
Appendix 90
Abstract (in Korean) 91
Table 1. Comparison of the characteristics of common molecular diagnostic techniques. 21
Table 2. Primer sets used for PCR amplification. 34
Table 3. The number of AIV samples by subtype used in the microarray experiment. 35
Table 4. Probes for subtyping and pathotyping AIV subtypes H5, H7, and H9. 36
Table 5. Probes for subtyping AIV subtypes N1, N2 and N3. 37
Table 6. Analytical performance results of subtyping and pathotyping. 38
Table 7. Sequences of primer sets (added for pathotyping). 53
Table 8. Result of diagnosis microarray result using 63 samples of previous study. 54
Table 9. Selected probes for subtyping and pathotyping AIV subtypes H5, H7, and H9. 55
Table 10. Result of diagnosis microarray result using 140 samples isolated in 2011-2015. 57
Table 11. Final selected probes for subtyping and pathotyping AIV subtypes H5, H7, and H9 75
Table 12. Verification result of diagnostic microarray using 36 AIV isolates in Mongolia 77
Figure 1. Applications of DNA microarray in disease diagnosis. DNA microarrays have diverse applications in disease diagnosis, including genotyping and identification of... 22
Figure 2. Manufacturing Process of Diagnostic Microarray. 39
Figure 3. Layout of diagnostic microarrays for AIV subtyping and pathotyping. (A) The microarray layout of 55 HA probes, 2 M probes (grey circles), and position markers... 40
Figure 4. Hybridization patterns of diagnostic microarrays for AIV subtyping and pathotyping. (A) H5 type pattern (red box), H7 type pattern (blue box), H9 type pattern... 41
Figure 5. Comparison of HA sequences between Korean and East Asian isolates. Red box is the sequence of the Korean isolates and the rest are the sequence of the East... 60
Figure 6. Comparison of primer reactivity. More samples and specific probes were detected in the reaction of HA + HP/LPAI primers than in the reaction of HA primer... 61
Figure 7. Layout of improved diagnostic microarrays for AIV subtyping and pathotyping. The microarray layout of 79 HA probes, 2 M gene probes (red circles),... 62
Figure 8. Hybridization patterns of improved diagnostic microarrays for AIV subtyping and pathotyping. (A) H5 (red box)/ HPAI (orange box) type pattern, (B) H5(red box)/... 63
Figure 9. Verified hybridization patterns of diagnostic microarrays for AIV subtyping and pathotyping using Mongolian isolates. (A) Verification results of diagnostic... 79