표제지

제출문

목차

보고서 요약서 5

요약문 6

SUMMARY 8

연구 성과 실적 및 향후 계획 10

참여연구원 현황표 23

I/II. 총괄연구과제 연구결과 25

1. 연구개발과제의 배경 및 필요성 27

2. 국내외 기술개발 현황 32

3. 연구개발과제의 추진체계 44

4. 연구개발수행 내용 및 결과 47

5. 목표달성도 및 관련분야 기여도 115

6. 향후 연구성과 추진 계획 (* 해당되는 경우만 작성) 120

7. 연구개발결과의 파급효과 121

8. 연구개발결과의 활용계획 122

9. 연구개발과정에서 수집한 해외과학기술정보 125

II/III. 127

제 1세부 연구과제 연구결과 127

1. 연구개발과제의 목표 129

2. 연구개발과제의 추진체계 132

3. 연구개발수행 내용 및 결과(1세부과제) 133

4. 참고문헌 182

제 2세부 연구과제 연구결과 185

1. 연구개발과제의 목표 187

2. 연구개발과제의 추진체계 192

3. 연구개발수행 내용 및 결과 194

4. 참고문헌 246

제 3세부 연구과제 연구결과 251

1. 연구개발과제의 목표 253

2. 연구개발과제의 추진체계(추젠체계) 253

3. 연구개발수행 내용 및 결과 254

4. 참고문헌 282

제 4세부 연구과제 연구결과 285

1. 연구개발과제의 목표 287

2. 연구개발과제의 추진체계 295

3. 연구개발 수행 내용 및 결과 296

4. 참고문헌 342

III/IV. 첨부서류 347

1. 자체평가의견서 349

2. 연구성과 실적 증빙서류 355

Table 1. Summary of the characteristics of the three PCR-based typing methods 34

Table 2. The summary of indirect ELISA procedures for the detection of L. monocytogenes. 50

Table 3. The summary of indirect ELISA procedures for the detection of C. perfringens. 51

Table 4. The summary of sandwich ELISA procedures for the detection of food-borne pathogens. 51

Table 5. Optimal combination between gold-antibody conjugates in conjugate pad and antibodies in test line. 55

Table 6. Confirmation of antibody-magnetic bead conjugate with BSA-magnetic bead as control 57

Table 7. Results of spiked bacteria to beef and lettuce samples by three diagnostic methods. 58

Table 8. Comparison between immunomagnetic bead system and immuno-nanomagnetic system with two kinds samples. 59

Table 9. Detection of Listeria monocytogenes by Selective agar, ELISA and ICG 60

Table 10. Comparison of the detection efficiency among detection methods of KFDA, FDA and USDA for L. monocytogenes. 62

Table 11. Comparison of the detection efficiency among detection methods of KFDA, FDA and USDA for Salmonella spp. isolated from each sample. 63

Table 12. Verification of the detection efficiency among detection methods of KFDA, FDA and USDA using TSI, SS and PCR, respectively. 63

Table 13. The Changes of number of Listeria spp. and other bacteria cells after addition of immuomagnetic bead (IMB). 64

Table 14. Restriction endonucleases used in MLRFT 80

Table 15. Possession frequency of the 5 VNTR loci among foodborne isolates 82

Table 16. Oligonucleotide sequences of the probes used for 16s rDNA microarray in food borne pathogenic bacteria 89

Table 17. 깻잎 중 존재하는 L. monocytogenes의 온도별 성장 측정 (2차 실험) 111

제 1세부 연구과제 연구결과 127

Table 1. List of food-borne pathogenic bacteria used in this study 133

Table 2. Preparation of immunogens and coating antigens for L. monocytogenes and C. perfringens 133

Table 3. Available hybridoma cell line from fused cell 134

Table 4. The obtatined lgY from immunized chickens. 136

Table 5. The summary of indirect ELISA procedures for the detection of L. monocytogenes. 138

Table 6. The summary of indirect ELISA procedures for the detection of C. perfringens. 139

Table 7. The summary of sandwich ELISA procedures for the detection of food-borne pathogens. 140

Table 8. Optimal combination between gold-antibody conjugates in conjugate pad and antibodies in test line. 145

Table 9. Confirmation of antibody-magnetic bead conjugate with BSA-magnetic bead as control 150

Table 10. Recovery ratio for various cell count by immunomagnetic bead separation system. 151

Table 11. The results of spiked L. monocytogenes to beef and lettuce samples by three diagnostic methods. 151

Table 12. The results of spiked C. perfringens to beef and lettuce samples by two diagnostic methods. 152

Table 13. Comparison between immunomagnetic bead system and immuno- nanomagnetic system with two kinds samples. 153

Table 14. Results of recovery ratio for pathogenic bacteria by various isolation methods. 154

Table 15. Effects of washing times with PBS and 0.85%NaCl for recovery ratioin IMS combined percoll system. 154

Table 16. Determination of L. monocytogenes by Selective agar, ELISA and ICG with or without enrichment after percoll-IMS. 155

Table 17. List of commercial ELISA kit in korea. 156

Table 18. Detection limit of L. monocytogenes by two type of ELISA kit 156

Table 19. Cross reactivity of other pathogenic bacteria by two type of ELISA kit 157

Table 20. Detection of spiked L. moncytogenes and C. perfringens in beef samples by ELISA kit developed in this study and commercial kit. 158

Table 21. Comparison of the detection efficiency among detection methods of KFDA, FDA and USDA for L. monocytogenes. 159

Table 22. Comparison of the detection efficiency among detection methods of KFDA, FDA and USDA for Salmonella spp. isolated from each sample. 160

Table 23. Verification of the detection efficiency among detection methods of KFDA, FDA and USDA using TSI, SS and PCR, respectively. 160

Table 24. The Changes of number of Listeria spp. and other bacteria cells after addition of immuomagnetic bead (IMB) 161

Table 25. Captured efficiency of dynabeads anti-Listeria 167

Table 26. Comparison of the capture efficiencies of pre-culture temperatures using dynabeads anti-Listeria 167

Table 27. Recovery of L. monocytogenes at different concentrations of PBS(pH7.4) from cell suspension using dynabeads anti-Listeria 168

Table 28. Recovery of L. monocytogenes at different pHs of 0.01M PBS from cell suspensions using DynabeadsR(이미지참조) anti-Listeria(Dynal, Norway) 168

Table 29. The separation efficiency of DynabeadsR(이미지참조) anti-Listeria for each pathogens after mixed culture of Listeria spp., Staphylococcus spp. and Salmonella spp.. 170

Table 30. Recovery of L. monocytogenes from 25g beef using enrichment procedure and Immunomagenetic separation 170

Table 31. Recovery of L. monocytogenes from 25g lettuce using enrichment procedure and Immunomagnetic separation 170

Table 32. The efficiency of affinity between 6×107(이미지참조)beads/100㎕ Clostridium antibody beads and C. perfringens. 171

Table 33. Comparison of separation efficiencies between immuno-magnetic separation (IMS) and immuno-nano magnetic separation(INMS) for L. monocytogenes in lettuce and beef after artificial inoculation 172

Table 34. Detections of L. monocytogenes using various methods at different artificial inoculation levels 174

Table 35. Detections of C. perfrigens using various methods at different artificial inoculation levels 174

Table 36. Results of detection for L. monocytogenes using Percoll 176

Table 37. Detection of L. monocytogenes using various methods at different artificial inoculation levels 179

Table 38. Detections of C. perfrigens using various methods at different artificial inoculation levels 180

제 2세부 연구과제 연구결과 185

Table 1. Listeria reference strains used for this study 229

Table 2. Primers used for this study and their expected amplicon sizes 230

Table 3. PCR conditons optimized to apply each gene of prfA virulence gene cluster 231

Table 4. Restriction endonucleases used in MLRFT 233

Table 5. Primers and amplicon sizes of 16S rDNA nested PCR 236

Table 6. Sequences and relative position of 16S rDNA PCR primers 236

Table 7. GeneBank내의 C. perfringens와 L. monocyogenes의 16s-rDNA 유전자의 alignment결과... 237

Table 8. Possession frequency of the 5 VNTR loci among foodborne isolates 241

Table 9. spa gene characteristics 242

Table 10. clfA gene characteristics 243

Table 11. clfB gene characteristics 244

Table 12. coa gene characteristics 244

Table 13. ssp gene characteristics 245

제 3세부 연구과제 연구결과 251

Table 1. Oligonucleotide sequences of the probes used for 16s rDNA PCR amplification and DNA microarray in food borne pathogenic bacteria 266

Table 2. Oligonucleotide sequences of the probes used for 16s rDNA microarray in food borne pathogenic bacteria 268

Table 3. Bacterial strains and condition of cultivation 269

Table 4. Composition of PCR Reaction mixture 270

Table 5. PCR Reaction condition 270

제 4세부 연구과제 연구결과 285

Table 1. Total aerobic bacteria and coliforms in lettuces, sesame leafs and cucumbers distributed in markets〈1차 모니터링〉 310

Table 2. Escherichia coli in lettuces, sesame leafs and cucumbers〈1차 모니터링〉 310

Table 3. Total aerobic bacteria and coliforms in lettuces, sesame leafs and cucumbers distributed in markets 310

Table 4. Escherichia coli in lettuces, sesame leafs and cucumbers 310

Table 5. Staphylococcus aureus in lettuces, sesame leafs and cucumbers 311

Table 6. Total aerobic bacteria and coliforms in sandwich marketed in the convenience stores 311

Table 7. Escherichia coli in sandwich marketed in the convenience stores 311

Table 8. Staphylococcus aureus in sandwich marketed in the convenience stores 311

Table 9. Bacillus cereus in sandwich marketed in the convenience stores 311

Table 10. 여름철/오전에 대형매장과 재래시장에서 유통 중인 상추, 깻잎, 오이의 식품유통의 영향 요인 312

Table 11. 겨울철/오후에 대형매장과 재래시장에서 유통 중인 상추, 깻잎, 오이의 식품유통의 영향 요인 312

Table 10. SGR에 대한 유도된 변수값과 이차모델식 316

Table 11. LT에 대한 유도된 변수값과 이차모델식 317

Table 12. Statistical summary of the secondary modeling step for specific growth rate(SGR) and Lag time(LT) 318

Table 13. Value of lag time and specific growth rate obtained from Gompertz model in the sesame leaf 319

Table 14. Secondary model and dependent variable for SGR 321

Table 15. Secondary model and dependent variable for LT 321

Table 16. Square root models for the effects of temperature on specific growth rate (SGR) and lag time (LT) of L. monocytogenes in sesame leaf 321

Table 17. Statistical indices of the secondary modeling step for lag time (LT) and specific growth rate (SGR) 323

Table 18. Dose-response models of Listeria monocytogenes 326

Table 19. Description and distribution of variables for L. monocytogenes of the Exposure Model for at the consumption of raw vegetables 328

Table 20. 깻잎 중 L. monocytogens의 잔존율 331

Table 21. 깻잎 중 존재하는 L. monocytogenes의 온도별 성장 측정 (1차 실험) 332

Table 22. 깻잎 중 존재하는 L. monocytogenes의 온도별 성장 측정 (2차 실험) 334

Table 23. The expected level of Listeriosis through vegetables consumption by various conditions and controls 338

Table 24. The expected level of contamination of L. monocytogenes at vegetable by various conditions and controls 340

III/IV. 첨부서류 347

Table 1. List of bacterial strains used in this study. 369

Table 2. Specificity of ICG strip test for L. monocytogenes, Listeria species, and other pathogenic bacteria. 370

Table 3. Analysis of L. monocytogenes in inoculated pork, beef, chicken, fish, and sausage samples by ICG strip test. 371

Table 4. Results for screening of L. monocytogenes in meat and processed meat samples by ICG strip test and PCR. 372

Fig. 1. 국내 식중독 발생추이 (1996-2005)(The Outbreak Pattern of Food Poisoning in Korea) 27

Fig. 2. Simple illustration of strategies to detect of microbial pathogens using biochips and biosensors 36

Fig. 3. PCR-DNA 마이크로어레이 기술을 활용한 식중독균 검출 방법 38

Fig. 4. DNA 마이크로어레이를 이용한 16s rRNA 직접 분석법 40

Fig. 5. Standard curves for Listeria monocytogenes using monoclonal antibody in different stabilizing solutions on (6 months storage, room temperature). 52

Fig. 6. Standard curves for Clostridium perfringens using monoclonal antibody in different stabilizing solutions on (6 months storage, room temperature). 52

Fig. 7. Standard curves for L. monocytogenes by terasaki plate kit. 53

Fig. 8. Standard curves for C. perfringens by terasaki plate kit. 53

Fig. 9. The schematic diagram of test strip for detection of foodborne pathogens. 54

Fig. 10. Sensitive of test strip for the detection of L. monocytogenes. NC : negative control 55

Fig. 11. Sensitive of test strip for the detection of C. perfringens. NC : negative control 55

Fig. 12. Cross-reactivity of L. monocytogenes test strip for other foodborne pahtogens.... 56

Fig. 13. Cross-reactivity of C. perfringens test strip for other foodborne pahtogens.... 56

Fig. 14. The schematic diagram of simultaneous detection kit for L. monocytogenes and C. perfringens. 56

Fig. 15. Results of simultaneous detection test strip for L. monocytogenes and C. perfringens. 57

Fig. 16. PCR images for ten fold dilutions of L. monocytogenes ATCC 19115 before IMS(a) and after IMS(b).... 65

Fig. 18. 올리고 고정화 최적화.... 84

Fig. 19. 직접 rRNA 측정에 의한 E. coli 검출 85

Fig. 20. 직접 rRNA 측정에 의한 C. perfringens 검출 85

Fig. 21. PNA와 DNA의 다른 backbone 특성을 이용한 새로운 신호증폭 방법 86

Fig. 22. 새로운 신호증폭 방법을 이용한 고감도 rRNA 측정 87

Fig. 23. Organization of the 16s rDNA in Listeria monocytogenes M58822 89

Fig. 24. PCR amplification of 16s rDNA in Food borne bacteria using Cy5-Uni4(forward) and Biotin-Uni 1392(reward) primer set (deduced PCR product size, 359 base pair) 90

Fig. 25. Schematic representation of 16s rDNA PCR based microarray for food borne bacteria detection 90

Fig. 26. Optimization of oligonucleotide microarray aldehyde surface 91

Fig. 27. Optimization of microarray hybridization condition 92

Fig. 28. Microarray analysis of 16s rDNA PCR fragments (Cy5-rU4 and B-U1392) 93

Fig. 29. A sensorgram showing the procedure of testing antibody affinity with L. monocytogenes cells on the CM-Dex surface.... 94

Fig. 30. SPR angle shift for binding of mouse monoclonal antibodies on protein L (a) and binding of 109(이미지참조) cells/mL L. monocytogenes on the antibody (b). 94

Fig. 31. SPR assay of Listeria monocytogenes cells using A18 antibody as a capture probe. 95

Fig. 32. 최종 제품의 분석 모식도 96

제 1세부 연구과제 연구결과 127

Fig. 1. Titration of antisera against L. monocytogenes using HKLM as coating antigen. 134

Fig. 2. Titration of antisera against C. perfringens using α-toxin as coating antigen. 134

Fig. 3. Comparison of four purification methods by SDS-PAGE.... 135

Fig. 4. Reaction pattern of FKLM-3B12-21 to various pathogenic bacteria by western blot... 136

Fig. 5. Reaction pattern of HKCP-2G8-5 to various pathogenic bacteria by western bolt... 136

Fig. 6. Reaction pattern of anti-HKLM lgY to various pathogenic bacteria by western blot... 137

Fig. 7. Reaction pattern of anti-HKCP lgY to various pathogenic bacteria by western blot... 137

Fig. 8. Standard curve for the detection of L. monocytogenes using MAb 138

Fig. 9. Standard curve for the detection of L. monocytogenes using lgY 138

Fig. 10. Standard curve for the detection of C. perfringens using MAb 139

Fig. 11. Standard curve for the detection of C. perfringens using lgY 139

Fig. 12. Standard curve for detection of L. monocytogenes by sandwich ELISA 140

Fig. 13. Standard curve for detection of C. perfringens by sandwich ELISA 140

Fig. 14. Standard curve for L. monocytogenes using monoclonal antibody in different stabilizing solution on (6 months storage, room temperature). 141

Fig. 15. Standard curve for C. perfringens using monoclonal antibody in different stabilizing solutions on (6 months storage, room temperature). 141

Fig. 16. Standard curves for L. monocytogenes by terasaki plate kit. 143

Fig. 17. Standard curves for C. perfringens by terasaki plate kit. 143

Fig. 18. The schematic diagram of test strip for detection of foodborne pathogens 143

Fig. 19. The photograph of produced colloidal gold by TEM (Magnification 110K).... 144

Fig. 20. Determination of optimal antibodies amount for stabilization of colloidal gold solution. 144

Fig. 21. Checking of gold particles-antibodies conjugate by test strip... 145

Fig. 22. Determination of each amount of antibodies in test line.... 146

Fig. 23. Determination for amount of gold-antibody conjugate in conjugate pad on each test strip.... 146

Fig. 24. Development of test strips for the detection of foodborne pathogens using SHF 09004 membrane.... 147

Fig. 25. Results of test strips with PBS as working solution for sample preparation.... 147

Fig. 26. Sensitive of test strip for the detection of L. monocytogenes. NC : nagative control 148

Fig. 27. Sensitive of test strip for the detection of C. perfringens. NC : negative control 148

Fig. 28. Cross-reactivity of L. monocytogenes test strip for other food-borne pahtogens.... 148

Fig. 29. Cross-reactivity of C. perfringens test strip for other food-borne pahtogens.... 148

Fig. 30. The schematic diagram of simultaneous detection kit for L. monocytogenes and C. perfringens. 149

Fig. 31. Results of simultaneous detection test strip for L. monocytogenes and C. perfringens. 149

Fig. 32. Procedures of IMS and percoll-IMS for the rapid isolation of food-borne pathogenic bacteria 153

Fig. 33. Confocal images of Listraia monocytogenes bound on anti-listeria immunomagnetic bead... 161

Fig. 34. Growth curves of Listeria monocytogene in BHI, UVM and Fraser broth 162

Fig. 35. Growth curves of Listeria monocytogene, Salmonella enteritidis, Staphylococcus aureus in BHI broth 162

Fig. 36. Growth curves of Listeria monocytogene, Salmonella enteritidis, Staphylococcus aureus in UVM broth 162

Fig. 37. Growth curves of Listeria monocytogene, Salmonella enteritidis, Staphylococcus aureus in Fraser broth 162

Fig. 38. PCR images for ten fold dilutions of L monocytogenes ATCC 19115 before IMS(a) and after IMS(b). 163

Fig. 39. Schematic representation of rapid sampling for Listeria spp. using immuno-magnetic separation and cultivation in UVM and BHI broth. 164

Fig. 40. PCR images for different concentration steps of Listeria spp. from ground beef... 165

Fig. 41. Growth curves of L. monocytogenes, S. enteritis and S. aureus for 0hr, 2hr, 4hr and 6hr... 166

Fig. 42. Recovery rates of L. monocytogenes using Dynabeads anti-Listeria at different temperature of pre-cultures (A), concentrations of PBS used in IMS(B), pHs of PBS used in IMS(C) 169

Fig. 43. The change of O.D. value of C. perfringens at 560nm. 171

Fig. 44. Comparison of efficiency between BDC method and BE method on Lettuce at various concentration of L. monocytogenes... 175

Fig. 45. Comparison of detection efficiency between BDC and BE method on beef at various concentration of L. monocytogenes... 176

Fig. 46. Results of PCR detection image of L. monocytogenes using Percoll.... 177

Fig. 47. Comparison of detection efficiency between IMS, BDC and IMS+BDC method on lettuce and beef at various concentration of L. monocytogenes... 181

Fig. 48. Comparison of detection efficiency between IMS, BDC and IMS+BDC method on lettuce and beef at various concentration of C. perfringens... 181

제 2세부 연구과제 연구결과 185

Fig. 1. Phylogenetic relationship among 16S rDNA genes derived from V. cholerae, V. parahaemolyticus, and V. vulnificus 195

Fig. 2. Agarose gel electrophoresis of PCR products - Pathogenic genes 199

Fig. 3. Agarose gel electrophoresis of PCR products - 16S rRNA gene(s) 200

Fig. 4. RFLP of PCR products - rpoS-mutS from various V. vulnificus strains 200

Fig. 5. Separation of 16S rRNA PCR fragments on a 1.5% agarose gel... 222

Fig. 6. Separation of rpoB gene PCR fragments for DGGE on a 1.5% agarose gel... 223

Fig. 7. DGGE analysis of 16S rRNA V3 region... 224

Fig. 8. DNA sequences of amplicons (194bps) derived from amplification of 16S rRNA V3 region. The sequence of the Escherichia amplicon is given as a reference. 225

Fig. 9. Negative image of ethidium bromide-stained DGGE (A) and TGGE (B) separation patterns of nested-PCR-generated 16S rDNA V3 region fragments for five genera.... 226

Fig. 10. Restriction fragment length polymorphisms of PCR-ampified 16S-rDNA products digested by Bbv 1.... 227

Fig. 11. Band patterns of 16S-23S intergenic space region PCR fragments separated on a 2% agarose gel.... 228

Fig. 12. Genetic structure of the chromosomal region of the prfA virulence gene cluster in L. monocytogenes.... 229

Fig. 13. PCR amplification products using prfA virulence gene primer sets against Listeria species.... 232

Fig. 14. A. Representative possible MLRFT types of 7 loci for all MLST date B. MLRFT types of 7 loci for Kimbap isolates 235

Fig. 15. Bar table showing the distribution of alleles at spa gene.... 242

Fig. 16. Bar table showing the distribution of alleles at clfA gene.... 243

Fig. 17. Bar table showing the distribution of alleles at clfB gene.... 243

Fig. 18. Bar table showing the distribution of alleles at coa gene.... 244

Fig. 19. Bar table showing the distribution of alleles at ssp gene.... 245

제 3세부 연구과제 연구결과 251

Fig. 1. 올리고 고정화 최적화.... 255

Fig. 2. L. monocygenes와 C. perfringens의 2nd structure와 probe 위취 258

Fig. 3. 직접 rRNA 측정에 의한 E. coli 검출 260

〈그림 4〉 직접 rRNA 측정에 의한 C. perfringens 검출 261

Fig. 5. DNA와 PNA의 비교 263

Fig. 6. PNA와 DNA의 다른 backbone 특성을 이용한 새로운 신호증폭 방법 264

Fig. 7. 새로운 신호증폭 방법을 이용한 고감도 rRNA 측정 265

Fig. 8. Multiple alignments of 16s rDNA in food borne bacteria using ClustalW 1.8 program 267

Fig. 9. Organization of the 16s rDNA in Listeria monocytogenes M58822 268

Fig. 10. PCR amplification of 16s rDNA in food borne bacteria using Cy5-Uni4(forward) and Biotin-Uni1392(reward) primer set (deduced PCR product size, 359 base pair) 270

Fig. 11. Schematic representation of 16s rDNA PCR based microarray for food borne bacteria detection 271

Fig. 12. Optimization of oligonucleotide microarray aldehyde surface 272

〈그림13〉 Optimization of microarray hybridization condition 273

Fig. 14. Microarray analysis of 16s rDNA PCR fragments (Cy5-rU4 and B-U1392) 273

Fig. 15. 2769-bp DNA염기배열 (Salmonella typhimurium Glutamate synthase) 276

Fig. 16. Salmonella typhimurium와 E.coli glutamate synthase비교. 278

Fig. 17. A sensorgram showing the procedure of testing antibody affinity with L. monocytogenes cells on the CM-Dex surface.... 278

Fig. 18/17. SPR angle shift for binding of mouse monoclonal antibodies on protein L (a) and binding of 109 cells/mL L. monocytogenes on the antibody (b). 279

Fig. 19. SPR assay of Listeria monocytogenes cells using A18 antibody as a capture probe. 279

Fig. 20. 최종 제품의 분석 모식도 280

제 4세부 연구과제 연구결과 285

Fig. 1. Predicted of distribution for prevalence of Listeria monocytogenes from storaged lettuce in market 307

Fig. 2. The probability of Listeriosis outbreak per one per day 307

Fig. 3. Predicted of distribution for the case of Listeriosis from lettuce per year in Korea 308

Fig. 4. The results of sensitivity of distribution for the case of Listeriosis from lettuce per year in Korea 308

Fig. 5. Growth of L. monocytogenes in the TSB containing different concentrations of NaCl adjusted to different pH levels at 37℃ 313

Fig. 6. Growth of L. monocytogenes in the TSB containing different concentrations of NaCl adjusted to different pH levels at 25℃ 314

Fig. 7. Growth of L. monocytogenes in the TSB containing different concentrations of NaCl adjusted to different pH levels at 10℃ 314

Fig. 8. Value of specific growth rate obtained from Gompertz model in the TSB NG : no growth 315

Fig. 9. Value of lag time obtained from Gompertz model in the TSB TLTC : Too Long Too Count 316

Fig. 10. Response surface model for the effects of temperature in combination with acidic to basic pH and NaCl on lag time (LT) in tryptic soy broth 318

Fig. 11. Response surface model for the effects of temperature in combination with acidic to basic pH and NaCl on specific growth rate (SGR) of L. monocytogenes in tryptic soy broth. 318

Fig. 12. Growth of L. monocytogenes in the sesame leaf at 4, 10, 25℃. 320

Fig. 13. Relationship between values of lag time obtained from predicted equation and values of lag time obtained from Gompertz model in the sesame leaf. 322

Fig. 14. Relationship between values of specific growth rate obtained from predicted equation and values of specific growth rate obtained from Gompertz model in the sesame leaf. 322

Fig. 15. FAO/WHO의 위해평가에서 사용한 Listeria monocytogenes의 dose-response model. 326

Fig. 16. Design of model simulating the probability of contamination level for L. monocytogenes at the consumption of raw vegetables. 327

Fig. 17. The relative frequency of estimated initial contamination level for L. monocytogenes at vegetable sold in markets. 329

Fig. 18. The relative frequency of estimated contamination level for L. monocytogenes at vegetable after stored in markets. 330

Fig. 19. The relative frequency of estimated contamination level for L. monocytogenes at vegetable to carry home from purchase at markets. 330

Fig. 20. The relative frequency of estimated contamination level for L. monocytogenes at vegetable after washing in home 331

Fig. 21. 온도별(5, 10, 20, 30) 깻잎 중 Listeria monocytogenes의 성장 특성(1차 실험) 333

Fig. 22. 온도별(5, 10, 20, 30) 깻잎 중 Listeria monocytogenes의 성장 특성(2차 실험) 335

Fig. 23. The relative frequency of estimated contamination level for L. monocytogenes at vegetable after stored at home 336

Fig. 24. Comparing of cumulative probability for estimated contamination level for L. monocytogenes at vegetable to home storage from markets.... 336

Fig. 25. The relative frequency of estimated consumption level for L. monocytogenes through vegetables consumption 337

Fig. 26. Correlation coefficient for sensitivity risk factors affecting the final contamination level of L. monocytogenes at vegetable 338

Fig. 27. The comparing of relative frequency of estimated contamination level for L. monocytogenes at vegetable before(A) and after(B) stored at home 339

III/IV. 첨부서류 347

Fig. 1. Schematic diagram of immunochromatography strip test. 374

Fig. 2. Reactivities of MAb to Listeria species (A) and other pathogenic bacteria (B) by indirect ELISA. 375

Fig. 3. Confirmation of colloidal gold-MAb conjugate. To determine non-specific binding of colloidal gold and immunoreagents in test and control line, colloidal gold-BSA conjugate was treated in conjugate pad... 376

Fig. 4. Sensitivity of ICG strip test for the detection of L. monocytogenes.... 377

I. 연구개발의 목적 및 필요성

- 식품의 안전성 확보를 위하여 주요 식중독 원인균을 정성, 정량적으로 신속히 동시에 분석할 수 있는 microarray, 면역분석기법, PCR기법 등 진단 신기술을 개발하고 아울러 이들 기술을 복합한 현장 동시진단 시스템을 연구하여 이를 위해평가에 활용.

II. 연구개발의 내용 및 범위

- Listeria monocytogenes 및 Clostridium perfringens를 대상으로 항체를 개발하여 ELISA kit, 간이검사 kit를 개발함.

- PCR 기법 (SNP-dependent 및 SNP-independent PCR)을 사용하여 주요 식중독 원인세균들을 동시에 진단할 수 있는 분자생물학적 기술의 개발.

- L. monocytogenes, C. perfringens를 포함한 10종 이상의 식중독균 검출용 마이크로어레이 측정시스템을 개발, 식중독균의 현장 동시진단에 활용.

- 유통 중인 식품 내에 존재하는 식중독균의 생장특성과 위해성 평가 및 위해관리방안을 제안.

III. 연구개발결과

- Listeria속, Clostridium속에 대한 특이항체 개발 및 대량생산과 정제를 실시한 후 ELISA 최적화 및 ELISA kit 개발.

- Listeria 및 Clostridium속 균에 대한 Terasaki plate kit 개발, 간이 Test-strip 개발, 동시 진단 kit 개발, 식품 내 식중독 균 고효율 분리용 나노입자 immunomagnetic 정제 시스템개발을 개발하여 신속한 식중독 원인균 검사시스템 확보.

- 개발된 각종 분석법을 현재 시판되고 있는 각종 검사 kit와 비교 실험하여 본 과제에서 개발된 분석법의 우수성을 확인, 또한 첨단면역진단기법에 의한 현장에서의 식품 중 식중독원인균의 진단.

- 식품에 존재하는 식중독균의 샘플링 방법 개발.

- 국내 유통 병원성 미생물 검출 kit 분석.

- 식품에 오염된 식중독균의 샘플링을 위한 전배양 조건 분석, 분리단계에서 영향을 미치 는 영향요인 분석을 통한 분리 효율 향상 조건 분석, IMS(immuno-magnetic bead)법을 이용한 식중독균의 분리 조건 분석, Percoll을 이용한 전처리과정을 통한 IMS의 효율 증대.

- 개발된 미생물위해평가방법과 식중독 원인균 동시진단 신기술간의 상호검증.

- L. monocytogenes 등의 주요 식중독 원인 세균 별 PCR 반응 조건의 최적화 및 PCR 반응생성물의 특징 파악.

- 속간, 종간, 또는 종내 식중독 세균의 동시 진단용 PCR 반응의 최적화.

- 최적화된 동시진단용 PCR 기법을 이용한 모델시스템에서 식중독 세균의 동시 동정 실험.

- 국내 및 해외 PCR 기법들과의 비교평가.

- 타 세부과제에서 개발된 신기술과의 비교분석.

- Listeria monocytogense, Clostridium perfringens 등 총 10종의 식중독균 동시 검출용 올리고 마이크로어레이 칩을 제작.

- 이 중 7종 이상을 검출 하였으며, Clostridium perfringens과 E. coli는 지금까지 보고된 것 중 가장 고감도의 검출을 실현하였다. 또한 기존 형광 태깅이나 샌드위치법을 탈피한 간단한 검출법을 개발하였음.

- 1, 2세부와의 공동연구로 항체, PCR을 이용한 바이오센서/칩 연구를 성공적으로 수행하였음.

- 식중독균의 미생물위해평가(MRA) 모델 개발, 식중독균의 위험성 확인, 대상식품 선정, L. monocytogenes균의 노출량 평가에 필요한 야채류, 샌드위치류 중 초기 오염수준 조사, 식품 유통 및 소비 단계에서의 영향 요인을 분석하여 broth와 야채류에서의 예측미생물모델(predictive microbial mode) 개발.

- 온도(5-30℃)와 세척 여부, 보관 시간에 따른 감소 정도인 전이율 예측을 통한 노출량 예측, 식중독균의 용량-반응평가 등을 통한 L. monocytogens균의 미생물위해평가(MRA)를 정밀하게 실시한 결과 보관 온도관리 철저 등 위해관리방안을 마련하였음.

IV. 연구개발결과의 활용계획

- 단크론성 항체 및 계란 IgY 항체들의 다양한 항체 개발기술을 축적하고, 개발된 항체를 이용하여 ELISA kit를 생산할 수 있는 안정화 조건 예측함으로서 식중독원인균의 면역분석 kit의 국산화에 활용함.

- 식품에 오염된 식중독 세균을 확인하는 데 있어서 우수한 방법으로 인정되고 있는 PCR을 단독으로 사용 혹은 다른 세부과제의 방법들과 병용하여 국내 주요 식중독 원인균의 동시진단에 사용.

- 식중독균 동시 검출에 활용.

- 면역기법과 PCR기법을 결합한 고기능 식중독균 동시 검출에 활용.

- 우리나라 식품유통 및 위생실정에 적합하고 실질적 활용이 가능한 미생물위해평가(MRA) 프로그램을 개발하여 문제시되고 있는 식품 중 L. monocytogenes균의 MRA를 수행함으로써 우리나라에서도 명실상부한 식중독균 기준 규격의 정량적 관리를 시행할 수 있도록 기초자료를 제공, 식품위생정책의 선진화에 기여함.