표제지
목차
Ⅰ. 서론 11
Ⅱ. 실험재료 및 방법 14
1. HCMV의 배양 및 정량 14
2. Real-time PCR primer target 선정 15
2.1. Primer 및 Probe 디자인 15
2.2. Primer 및 Probe 선별 16
3. Virus 유전자 정량 검출을 위한 standard plasmid 제작 19
4. Real-time PCR 조건 최적화 21
4.1. Annealing 온도 최적화 21
4.2. Denaturation 및 Annealing 시간 최적화 24
4.3. Primer 및 Probe 농도 최적화 26
5. Real-time PCR 시험법 신뢰성 검증 29
5.1. 특이성 검증 29
5.2. 검출한계 및 재현성 검증 30
5.3. 정밀성 검증 31
5.4. 완건성 검증 32
Ⅲ. 결과 33
1. Real-time PCR primer target 선정 33
1.1. Primer 디자인 33
1.2. Primer 선별 33
1.3. Probe 디자인 33
2. Real-time PCR 조건 최적화 37
2.1. Annealing 온도 최적화 37
2.2. Denaturation 및 Annealing 시간 최적화 39
2.3. Primer 및 probe 농도 최적화 41
3. Real-time PCR 시험법 신뢰성 검증 43
3.1. 특이성 검증 43
3.2. 검출한계 및 재현성 검증 45
3.3. 정밀성 검증 47
3.4. 완건성 검증 49
3.5. 부정시험 정도관리 기준 설정 및 검증 51
Ⅳ. 고찰 52
Ⅴ. 참고 문헌 54
Ⅵ. Abstract 57
Ⅶ. 적요 59
Table 1. Mixture condition of SYBR Green real-time PCR 17
Table 2. Condition of SYBR Green real-time PCR 18
Table 3. Salient features of HCMV 20
Table 4. Mixture condition of TaqMan probe real-time PCR for annealing temperature test 22
Table 5. Condition of TaqMan probe real-time PCR for annealing temperature test 23
Table 6. Condition of TaqMan probe real-time PCR for annealing and denaturation time test 25
Table 7. Mixture condition of TaqMan probe real-time PCR for primer and probe concentration test 27
Table 8. Condition of TaqMan probe real-time PCR for primer and probe concentration test 28
Table 9. Sequences of oligonucleotide primer sets used in the detection of HCMV gene 34
Table 10. Sequences of oligonucleotide probe used in the detection of HCMV gene 36
Table 11. Ct values obtained during optimization of annealing temperature for quantitative detection of HCMV using TaqMan probe Real-time PCR 38
Table 12. Ct values obtained during optimization of denaturation and annealing time for quantitative detection of HCMV using TaqMan probe Real-time PCR 40
Table 13. Ct values obtained from optimization of primer and probe concentration for quantitative detection of HCMV using TaqMan probe Real-time PCR 42
Table 14. Result of detection limit and repeatability test of TaqMan probe real-time PCR assay for detection of HCMV 46
Table 15. Result of precision test of TaqMan probe real-time PCR assay for detection of HCMV 48
Table 16. Result of robustness test of TaqMan probe real-time PCR assay for detection of HCMV 50
Fig. 1. Selection of primer set for HCMV detection using SYBR Green real-time PCR assay 35
Fig. 2. Specificity of TaqMan probe real-time PCR assay to potential cross-reactive viruses 44