초록 ▼

식중독균 신속검출용 나노바이오센서 기술 연구에서는 대표적인 식중독균인 살모넬라 세균을 신속하게 검출할 수 있도록, 식품시료에서 식중독균을 포획 및 표지하는 나노바이오센서의 검출부를 우선 구명하였다. 형광을 내는 나노양자점과 항체로 이루어진 나노바이오센서의 성능검증은 다른 농도로 준비된 살모넬라균 시료를 이용하여 검출한계를 측정하는 방식으로 수행하였다. 나노바이오센서의 검출 성능을 향상시키기 위하여 검출부 구성물질들의 농도와 부착공정을 최적화 하였으며, 나노바이오센서로부터의 형광 신호를 측정할 수 있는 소형 검출장치를 개발하였다. 나

식중독균 신속검출용 나노바이오센서 기술 연구에서는 대표적인 식중독균인 살모넬라 세균을 신속하게 검출할 수 있도록, 식품시료에서 식중독균을 포획 및 표지하는 나노바이오센서의 검출부를 우선 구명하였다. 형광을 내는 나노양자점과 항체로 이루어진 나노바이오센서의 성능검증은 다른 농도로 준비된 살모넬라균 시료를 이용하여 검출한계를 측정하는 방식으로 수행하였다. 나노바이오센서의 검출 성능을 향상시키기 위하여 검출부 구성물질들의 농도와 부착공정을 최적화 하였으며, 나노바이오센서로부터의 형광 신호를 측정할 수 있는 소형 검출장치를 개발하였다. 나노바이오센서의 살모넬라균 검출 성능을 보다 더 향상시키기 위하여 반도체공정을 이용한 미세유로 기반 마이크로칩을 개발한 다음 표준 버퍼 및 식품 시료에 접종된 살모넬라균의 검출 한계를 평가하였다.
식중독균 검출을 위한 생물학적 검출물질 및 표지기술 개발 연구에서는 항체보다 선택성과 내구성이 식중독균 검출시 측정 가능한 신호를 낼수 있는 물질을 구명하는 방법으로 수행되었다. 이를 위하여, 나노양자점이 부착될 수 있는 바이오틴이 발현되는 대장균용 박테리오파지를 도입하여 증식 및 나노양자점 부착 가능성을 구명하였다. 또한 동일 기술을 살모넬라균을 대상으로 확대 적용할 수 있는 가능성을 조사하였다.

Abstract ▼

Traditional techniques to detect and identify infectious food-borne bacteria are time-consuming due to the steps of isolation and amplification of them. Therefore development of a rapid method for the detection of certain bacteria is essential. It is required to develop rapid methods to identify pat

Traditional techniques to detect and identify infectious food-borne bacteria are time-consuming due to the steps of isolation and amplification of them. Therefore development of a rapid method for the detection of certain bacteria is essential. It is required to develop rapid methods to identify pathogenic Salmonella in food products for protecting and maintaining safety of the public health from Salmonellosis. The objective of the present study was to explore feasibility of the nanotechnology to rapidly detect pathogenic Salmonella. Quantum dot nanoparticle was used to detect Salmonella cells. For selective detection of Salmonella, anti-Salmonella polycolonal antibodies were immobilized onto the quantum dot surface based on streptavidin-biotin binding or covalent binding. To separate and concentrate the cells from the sample, superparamagnetic particles were used. A portable fluorometer was developed to measure fluorescence signal from the quantum dot nanoparticles attached onto Salmonella in the samples. The fluorometer was composed of a UV LED, a bifuricated fiber, optical filters, and a silicon photomultiplier. Sensitivity of the nano-biosensor to detect pathogenic Salmonella was evaluated with serially diluted Salmonella typhimurium in phosphate buffered saline (PBS) and food extracts. Fluorescence response of the nano-biosensor had linear relationship with the concentration of the cells. Detection limit of the sensor was 103 CFU/mL Salmonella spiked in PBS and food extracts. The sensitivity of the sensor could be improved by optimizing reaction procedure of the nano-biosensor components.
As a bacteriophage is a virus which infects host-specific bacteria, it can be a crucial tool to detect and control certain pathogenic bacteria. The purpose of this study is to investigate the application of biotin-tagged bacteriophage and streptavidin-coated quantum dots for rapid detection of bacterial pathogens in agri-foods. We collected 11 E. coli strains and 8 Salmonella strains to investigate host-specificity of T7 and SP6 phages, respectively. T7 phage used in this experiment was previously engineered to express the major capsid protein fused to the biotinylation peptide that so-called T7-bio phage. SP6 phage was also tried to be genetically engineered for the same purpose. The bacterial strains were pre-cultured in LB medium and phages were added into each E. coli or Salmonella to check cell lysis, respectively. Plaque assay was also performed to confirm host-specificity. Afterwards certain strains which can be used for the amplification host of T7-bio phage were genetically modified with pDsRed plasm id showing red fluorescence. The emission spectrum of quantum dot and transformant was tested using fluorescent spectrophotometer.The application of the quantum dot and T7-bio phage for the detection of E. coli were performed. Out of 11 E. coli and 8 Salmonella strains, three strains of each were specifically host to amplify T7 and SP6 phages, respectively. E. coli strains were well-transformed with pDsRed for separation from quantum dot and other bacteria during experiments. The peaks of two quantum dots and transformant were detected around 570nm, 700nm and 620nm, respectively. The quantum dot labelled into the T7-bio phage was clearly detected via fluoresce microscope. However, the trial for genetic engineering with SP6 phage and biotinylation peptide was not successful due to a loss of cell lysis ability of SP6 phage. The data obtained in this experiment will facilitate developing a rapid method for the detection of bacterial pathogens.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 5
• 제1장 서 론 ... 7
• 제2장 국내외 기술개발 현황 ... 9
• 제3장 연구개발수행 내용 및 결과 ... 11
• 제1절 식중독균 신속검출을 위한 나노바이오센서 기술 연구 ... 11
• 제2절 식중독균 검출을 위한 생물학적 검출 물질 및 표지기술 개발 ... 34
• 제3절 식품 부패 측정기 설계 및 제작 (긴급 수행 연구내용으로 추가) ... 42
• 제4장 연구개발목표 달성도 및 대외기여도 ... 46
• 제1절 목표대비 달성도 ... 46
• 제2절 정량적 성과 ... 46
• 제5장 연구개발결과의 활용계획 ... 47
• 제6장 중요 변동사항 ... 48
• 제7장 참고문헌 ... 51