표제지
목차
I. 서론 10
II. 재료 및 방법 15
1. 분석 대상균주 15
1-1. 임상분리 균주의 수집 15
1-2. 균주의 종 동정 15
2. 표현형적 확인시험 16
2-1. 항생제 감수성 시험 16
2-2. Modified Hodge test (MHT) 17
2-3. Carbapenemase inhibition test (CIT) 18
3. 분자진단 방법을 이용한 카바페넴 내성 유전자 검출 20
3-1. 핵산의 추출 및 primer 제작 20
3-2. 중합효소 연쇄반응(Poymerase chain reation, PCR) 22
3-3. 염기서열 분석(Sequencing) 23
3-4. Real time PCR의 primer 제작 및 융해 곡선 분석 24
III. 결과 26
1. 검체의 특성 26
2. 표현형적 시험법 28
2-1. 항생제 감수성 시험 28
2-2. Modified Hodge test 30
2-3. Carbapenemase inhibition test (CIT) 32
2-4. 표현형적 시험결과의 통합 분석 35
3. 분자진단 방법을 이용한 카바페넴 내성 유전자 검출 37
3-1. PCR 방법을 이용한 카바페넴 내성 유전자 검출 37
3-2. 검체별 카바페넴 내성 유전자 분석 41
3-3. 염기서열 분석 43
3-4. Real time PCR 융해곡선 분석(melting curve analysis)을 이용한 카바페넴 내성 유전자 검출 48
IV. 고찰 52
V. 요약 및 결론 58
References 60
ABSTRACT 67
Table 1. The carbapenem resistance criteria of Enterobacteriaceae 16
Table 2. Combined interpretation of modified Hodge test and carbepenemase inhibition test 19
Table 3. PCR primers for the detection of carbapenemase producing bacteria 21
Table 4. Realtime PCR primers for the detection of carbapenemase producing bacteria 25
Table 5. Antibiotic susceptibility test by disk diffusion method of Carbapenem resistance gram-negative rods 29
Table 6. Modified Hodge test positive results for carbapenemase-producing Gram-negative rods test 31
Table 7. The positive result of the carbapenemase inhibition test in Gram-negative rods (Mero+PBA; N=7, Mero+EDTA; N=15) 34
Table 8. Results of phenotypic integration analysis of carbapenem resistance gram negative rods 36
Table 9. PCR result analysis of carbaphenemase-producing gram negative rods and carbapenemase types 40
Table 10. PCR result analysis of carbaphenemase genes by specimen 42
Table 11. Real-time PCR result analysis of carbapenemase gene 51
Figure 1. Reading result of modified Hodge test performed on a MacConkey plate. 17
Figure 2. Reading result of carbepenemase inhibition test. (A) negative, (B) Mero+EDTA positive, (C) Mero+PBA positive. 18
Figure 3. Specimens frequency of analysis strains. 27
Figure 4. Distribution of analysis strains. 27
Figure 5. Result of modified Hodge test performed on a MacConkey plate for analysis strain. 30
Figure 6. Result of carbepenemase inhibition test for analysis strain. 33
Figure 7. PCR amplification profile of the carbapenemase gene from the gram negative rods isolates. . M, 100bp DNA ladder marker; 1.... 38
Figure 8. PCR amplification profile of the blaKPC, blaNDM, blaIMP, blaVIM gene from the gram negative rods isolates. A) blaKPC PCR product size...[이미지참조] 39
Figure 9. Alignment of blaKPC gene sequences.[이미지참조] 44
Figure 10. Alignment of blaNDM gene sequences.[이미지참조] 45
Figure 11. Alignment of blaIMP gene sequences.[이미지참조] 46
Figure 12. Alignment of blaVIM gene sequences.[이미지참조] 47
Figure 13. Melting curve analysis of carbapenemase gene. A) melting curve analysis of blaKPC and blaNDM and B) melting curve analysis of...[이미지참조] 49
Figure 14. Results of carbapenemase gene melting curve analysis in strain. A) melting curve analysis of blaKPC amplicon was generated...[이미지참조] 50