보고서 정보
주관연구기관 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2013-11 |
과제시작연도 |
2013 |
주관부처 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs(MAFRA) |
등록번호 |
TRKO201400012420 |
과제고유번호 |
1545006251 |
사업명 |
농림축산검역검사기술개발 |
DB 구축일자 |
2014-07-05
|
키워드 |
나노바이오.살모넬라.Salmonella.chicken.Serotypes.Feces.Nanobiosensor.
|
초록
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4. 최종 연구결과 요약
◦ 세부과제명: 나노바이오센서 이용 살모넬라 신속검출
가. Silver Biopolymer nanocolloid 기질 개발
○ Biopolymer 입자에 silver입자를 코팅하는 방법으로 기질 개발
- Stainless steel plate 위에 Silver biopolymer nanocolloid를 부착시키고 표적세포 (살모넬라균)을 Raman 광학현미경으로 분석가능한 nanobiosensor 개발
ㆍ3종 나노입자(40, 52, 70nm)을 제작하여 TEM상에서 입자 균일도를
4. 최종 연구결과 요약
◦ 세부과제명: 나노바이오센서 이용 살모넬라 신속검출
가. Silver Biopolymer nanocolloid 기질 개발
○ Biopolymer 입자에 silver입자를 코팅하는 방법으로 기질 개발
- Stainless steel plate 위에 Silver biopolymer nanocolloid를 부착시키고 표적세포 (살모넬라균)을 Raman 광학현미경으로 분석가능한 nanobiosensor 개발
ㆍ3종 나노입자(40, 52, 70nm)을 제작하여 TEM상에서 입자 균일도를 조사한 결과 52nm와 70nm 크기의 나노 입자에서 우수한 균질도 확인
○ Silver Bioplymer nanocolloid based nanobiosensor 이용 살모넬라 혈청형 감별
- Biosensor를 이용한 살모넬라(SE, ST) spectra data를 분석한 결과 SE와 ST사이에 각기 다른 15개 spot이 확인됨
ㆍ이들 세균들은 genetic materials 및 균체 proteins에 차이점으로 감별가능
- PCA 통계분석법으로 확인한 결과 SE와 ST 구분 가능성은 98%로 아주 높은 것으로 확인됨
나. Aptamer를 이용한 Salmonella Typhimurium 검출법 구축
- Aptamer-modified silver nanorod array 기질을 작출하여 ST검출이 가능함을 확인함
- Fresh culture에서 직접 ST검출은 가능하였으나 Chicken rinse액을 이용한 검출에서는 양성 89.5%(34/38), 음성 60.7%(17/28)이 확인되어 chicken rinse액을 시료로 직접 사용하는 것은 어려울 것으로 판단되었음
◦ 세부과제명:국내 양계분야의 SE 유행형 분석 가. 국내 SE분리현황
○ 국내 SE분리주 총 188주 분리동정완료
- 닭 임상주 36주, 닭분변 65주, 닭고기 47주, 계사환경 등 20주, 사람유래 20주
○ 국내분리 SE균주에 대한 유전형(MLVA) 비교분석
- 총 188균주(닭156, 사람20)를 대상으로 MLVA법으로 분석한 결과 15종의 MLVA types이 확인되었으며 MLVA10형 MLVA9형이 46.9%, 32%로 출현율이 가장 높았음ㆍMinimum spanning tree법으로 근형 관계 분석 결과 닭, 양계장, 부화장, 닭고기 유래 분리주 상호간 유전형이 매우 유사하였음
○유전형 분석(MLVA, PFGE)결과와 분리 기원을 기본으로 50개 SE균주를 선발한 다음 Microarray법을 이용한 병원성 유전자 발현율 조사를 실시함
- 병원성관련 유전자 180sets와 Housekeeping 유전자 및 항생제 내성유전자 80여개에 대한 probe 제작하고 Microplate에 유전자를 심은 다음 GenePix Scanner로 분석하였음
- 2종류의 Genotypes이 확인되었으며 닭 실질장기, 닭분변, 닭고기 작업장, 시판 닭고기등에서 분리된 SE균주들이 매우 밀접한 관련성이 있는 것으로 확인되었으며, 특히 사람에서 분리된 임상 SE균주가 닭 생산단계에서 분리된 균주들과 유전적 상관이 아주 높은 것으로 확인됨
- Type 4 secretion system 관련 병원성 유전자 발현은 실질장기에서만 관찰되었으나, 편모발현 유전자 발현율은 분변유래 SE균에서 더욱 높았다. 하지만 Fimbria 병원성 유전자들 중 pefI, stdB, stfC, stfD, stfE 발현율은 실질장기 유래 균에서 유의성 있게 높았다.
○병원성 유전자 발현율이 높은 35균주(닭 실질장기14주, 닭분변11주, 환경8주, 계육4주, 사람3주)를 선발하여 2종의 세포주(Caco-2, Hd-11)를 대상으로 Cell Invasion Assay를 실시함
- 2개 세포주에 가장 침습력이 좋은 5개 균주(SE-050, SE-063, SE-072, SE-112, SE-136)를 선발하였으며, 병원성과 가장 밀접한 관련성을 보이는 15개 병원성 유전자의 발현율을 조사한 결과 5개 균주 모두가 100% 발현율을 보였음
Abstract
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Surface Enhanced Raman Scattering (SERS) can detect the pathogen in rapid and accurate. In SERS weak Raman scattering signals are enhanced by many orders of magnitude. In this study silver metal with biopolymer was used. Silver encapsulated biopolymer polyvinyl alcohol nano-colloid was prepared and
Surface Enhanced Raman Scattering (SERS) can detect the pathogen in rapid and accurate. In SERS weak Raman scattering signals are enhanced by many orders of magnitude. In this study silver metal with biopolymer was used. Silver encapsulated biopolymer polyvinyl alcohol nano-colloid was prepared and deposited on stainless steel plate. This was used as metal substrate for SERS. Salmonella Typhimurium common food pathogen was selected for this study. Salmonella Typhimurium bacteria cells were prepared in different concentrations in cfu/㎖. Small amount of these cells were loaded on the metal substrate individually, scanned and spectra were recorded using confocal Raman microscope. The cells were exposed to laser diode at 785 nm excitation and object 50x was used to focus the laser light on the sample. Raman shifts were obtained from 400 to 2400 cm-1. Multivariate data analysis was carried out to predict the concentration of unknown sample using its spectra. Concentration prediction gave an R2 of 0.93 and standard error of prediction of 0.21. The results showed that it could be possible to find out the Salmonella cells present in a low concentration in food samples using SERS
For the detection and differentiation of Salmonella serotypes using Surface Enhanced Raman Scattering (SERS) technique, the optical detection method was developed using with biopolymer silver nano colloidal substrate. A nano-colloid was prepared by adding 100 mg of silver nitrate to a 2% polyvinyl alcohol (PVA) solution, then adding 2% trisodium citrate to the solution to reduce silver nitrate into silver ions. The silver nano-colloid was deposited on a stainless steel plate. Fresh cultures of Salmonella Typhimurium and Salmonella Enteritidis were washed and suspended in 10 ㎖ of sterile
deionized water. Approximately 5 ㎕ of the bacterial suspensions were placed on the nano-colloidal substrate individually and exposed to 785 nm laser excitation. SERS spectral data was recorded between 400 and 1800 cm-1. SERS signals were collected from 15 different spots on the substrate for each sample. Principal component analysis (PCA) of spectral data was used to classify Salmonella serotypes. PC1 identified 92% of the variation between the Salmonella serotypes, and PC2 identified 6% of variation and in total 98% between the isolates. Soft Independent Modeling of Class Analogies (SIMCA) classification of serotypes in validation set gave a correct classification of 89% for one set of sample and 95 for another set of sample. Comparison of the SERS spectra of Salmonella serotypes indicated that both isolates have similar cell walls and cell membrane structures, which were identified by spectral regions between 520 and 1050 cm-1. However, major differences were detected in cellular genetic material and proteins between 1200 and 1700 cm-1. SERS with biopolymer silver nano-colloid may potentially be used to detect and classify foodborne pathogens. Also, PCA variation in calibration and validation models may be promising, application of this method in pathogen detection. Besides, aptamer-based SERS detection for ST with biopolymer encapsulate silver nanoparticles (BeSN) developed and 98.3% detection accuracy can be obtained to identify Salmonella from background (E. coli was used as a negative control) when aptamers are used as binding agent. Also, hyperspectral microscope imaging to validate biopolymer encapsulated with silver nanoparticle (BeSN) conjugated with anti-Salmonella Typhimurium (ST) were done for specific detection of ST from poultry carcass rinse. In this study biopolymer was used to prepare stable silver nano-colloids. Findings from this study indicate that this technology may be used for rapid pathogen detection and classification of bacteria.
We isolated and identified 188 Salmonella Enteritidis (SE) isolates from chicken feces, chicken carcass, visceral organs (liver, joint, etc) from dead chickens, and human who had clinical signs such as abdominal pains and diarrhea. One hundred and eleven SE isolates were tested to antibiotic susceptibility. The high resistant rates of antibiotics were streptomycin, ampicillin, nalidixic acid and sulfisoxazole. To estimate the relationship between different origins of SE isolated from poultry and human, we did molecular epidemiological analysis using PFGE, MLVA and phage typing. From the results of both PFGE and MLVA, we found that the isolates from both poultry industries and human's stool were very close similarity based on the genetic properties, exhibiting the product such as chicken meat and eggs contaminated with SE could transfer pathogens including SE and ST to human. In addition, from the phage typing, the major phage types were PT1 (15.3%), PT35 (12.6%), PT21 (8.1%) and PT4 (2.7%).
To estimate the virulence of Korean SE isolates, we tested cellular invasion assay (In-vitro) and inoculated the bacteria to the mouse through intraperitoneal route. In cellular invasion assay, several SE isolates had higher virulence than ST and E. coli, In-vivo test, SE isolates caused 66.6% to 100% mortality of the mouse after 6-10 days post inoculation. The dead animal also showed whitish and multiple necrotic foci in the liver with severe enteritis. Furthermore, we analysed the frequency of virulence genes and antibiotic resistant genes by using microarray method. The specific results showed that the virulence genes directly associated with type IV secretion systems and flagella were more expressed to clinical isolates from visceral organs than healthy chicken's feces.
After cellular invasion tests, two different SE isolates (SE-050, SE-112) were selected to attenuate by using Lambda Red Mutagenesis. The mutant in which rfbA and rfaH genes were deleted showed much lower virulence than wild isolates. It is needed that the genetic mutant will be showed less pathogenicity to chicken and mouse after intramuscular injection.
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