Detection of pathogenic bacteria that pose a great risk to human health requires a rapid, convenient, reliable, and sensitive detection method. In this study, we developed a selective filtration method using monoclonal antibody (MAb)-magnetic nanoparticle (MNP) nanocomposites for the rapid and sensitive colorimetric detection of Salmonella typhimurium. The method contains two key steps: the immunomagnetic separation of the bacteria using MAb-MNP nanocomposites and the filtration of the nanocomposite-bound bacteria. Color signals from the nanocomposites remaining on the membrane were measured, which reflected the amount of bacteria in test samples. Immunomagnetic capture efficiencies of 8 to 90 % for various concentrations of the pathogen (2 × 10(4)-2 × 10(1) cells) were obtained. After optimization of the method, 2 × 10(1) cells of S. typhimurium in pure culture solution was detectable as well as in artificially inoculated vegetables (100 cells/g). The method was confirmed to be highly specific to S. typhimurium without cross-reaction to other pathogenic bacteria and could be concluded within 45 min, yielding results in a shorter or similar time period as compared with recently reported antibody immobilized on magnetic-particle-based methods. This study also demonstrated direct application of MAb-MNP nanocomposites without a dissociation step of bacteria from magnetic beads in colorimetric assays in practice.
The purpose of this study was to analyze microbiological hazards for plants, cultivation environments and personal hygiene of perilla leaf farms at the harvesting stage. Samples were collected from three perilla leaf farms(A, B, C) located in Gyeongnam, Korea and tested for sanitary indications, fungi and pathogenic bacteria(Escherichia coli O157:H7, Listeria monocytogens, Salmonella spp., Staphylococcus aureus and Bacillus cereus). As a result, total bacteria and coliform in perilla leaf were detected at the levels of 4.4~5.2 and 3.4~4.3 log CFU/g, respectively, but E. coli was not detected in all samples. Among the pathogenic bacteria, B. cereus(perilla leaf: 2.0~2.4 log CFU/g, stem: 1.4~2.1 log CFU/g, water: 0.7 log CFU/ml, soil: 4.2~5.0 log CFU/g, hands: 3.0 log CFU/ hand, gloves: 2.1~2.4 log CFU/100 cm 2 , glothes: 1.5~2.8 log CFU/100 cm 2) and S. aureus(3.4 log CFU/hand) were detected in all samples and worker's hand from farm A, respectively. However, other pathogenic bacteria were not detected. This study demonstrates that perilla leaf at the harvesting stage was significantly contaminated with microbial hazards.
To analyze aflatoxin M 1 (AFM 1 ), we dissolved infant formula in warm water and cleaned it by using an immunoaffinity column (IAC). The amount of AFM 1 was determined by high-performance liquid chromatography coupled with fluorescence detection. AFM 1 was detected in 281 of 439 samples. Thus, the detection rate of AFM 1 was 64.0%. The average concentration of AFM 1 in positive samples was 2.6 ng/kg (of prepared formula). The estimated daily intake (EDI) of AFM 1 through infant formula was 0.087-0.646 ng/kg body weight/day and the additional number of cases of liver cancer associated with exposure to AFM 1 would be 0.003-0.020 cancer cases/1,000,000. Because there is less than 1 cancer case/ 1,000,000 per year, the exposure to AFM 1 through infant formula in Korea is considered to be an unlikely human health concern.Keywords: aflatoxin M 1 , risk assessment, contamination, infant formula 재료 및 방법 대상시료국내에 유통되고 있는 분유 및 조제분유 내의 아플라톡신 M 1 오염현황을 파악하기 위해 조제분유(modified milk powder) 142 건, 성장기용 조제분유(modified milk powder for follow-up) 68건, 성장기용 조제식(follow-up formula) 96건, 영유아용 곡류조제식 (cereal based food for infants and young children) 51건, 영유아 용 특수조제식(special medical purpose for infants and young children) 17건, 영아용 조제식(infant formula) 6건, 기타 영유아식 (other foods for infants and young children) 59건을 유통기한이 다른 제품으로 총 439건 수집하여 아플라톡신 M 1 을 분석하였다. 시약 및 기구아플라톡신 M 1 표준물질 10 µg/mL (Supelco, Bellefonte, PA, USA; purity 99%)을 구입하여 HPLC grade용 acetonitrile을 이용 하여 1 µg/mL 농도로 제조하여 냉장보관하면서 stock solution으 로 사용하였다. 이 표준원액을 acetonitrile으로 희석하여 사용하였 다. 본 연구에서는 acetonitrile (Merck, Darmstadt, Germany), methanol (Merck)을 사용하였고, 정제를 위한 면역친화성칼럼 (immunoaffinity column)은 AflaM 1 Test (Vicam, Milford, DE, USA)를 사용하였다. 또한, 시험법 검증을 위해 인증표준물질(CRM, certified reference material)인 분유제품(ERM-BD283, Munich,
Herein, we have developed a label-free and homogeneous fluorescence resonance energy transfer (FRET) immunoassay for the detection of neopterin (NPT), which is an early and valuable biochemical marker of cellular immunity. Owing to intrinsic fluorescence properties of antibody and NPT, anti-NPT antibody (anti-NPT) and analyte played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of NPT increases, the fluorescence intensity at ~350 nm decreases owing to the formation of increasing amounts of the anti-NPT/NPT complex in which FRET takes place. The assay system was found to display a high specificity and a low detection limit (0.14 ng mL -1 ) for NPT. A practical application of the FRET immunoassay system was demonstrated by its use in the detection of NPT in spiked human serum samples. The observations made in these efforts show that the homogeneous FRET immunoassay strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of NPT.
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