Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Detection of Pseudomonas aeruginosa with a Label-free Immunosensor from Various Cold Storage Foods

비표지 면역센서에 의한 냉장유통 식품 중 Pseudomonas aeruhinosa의 간이검출

  • Published : 2003.09.01
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Abstract

The aim of this study is to develop a label-free immunosensor for microbial detection and to evaluate its applicability to Pseudomonas aeruginosa detection in various food samples. The antibodies used were a polyclonal antiserum from rabbit (polyvalent type) and a monoclonal antibody raised against the flagella of P. aeruginosa. Antibody immobilization was done by a thiolated antibody chemisorption onto one gold electrode of a piezoelectric quartz crystal with a thiol-cleavable, heterobifunctional cross-linker, sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate. To the Stomacher-treated samples from various raw and processed foods under cold storage, comprising sirloin, cod and pettitoes, spiking and enrichment culture were done to prepare the model samples, followed by the measurements of the frequency shifts after sample injections. The frequency shifts obtained by the sample matrices themselves were in the range of 52~89 Hz. The injections of the spiked samples caused the frequency shifts of 108~200 Hz, whereas the enriched samples decreased the steady-state resonant frequencies by 162~222 Hz. All sample measurements including baseline stabilization, sample injection and acquisition of the steady-state response were accomplished within 30 min.

냉장식품의 주요한 변패원일균으로서 저온세균인 Pseudomonas aeruhinosa를 최소 전처리한 후 신속히 검출할 수 있는 비표지 면역센서 시스템을 개발하였다. 수정결정전극상으로의 생물요소인 항체의 고정화는 이형이기능성 가교화제인 sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate를 사용하여 항체를 티올화시킨 후 티올화된 항체를 화학흡착하여 행하였고, P. aeruginosa flagella에 대한 단클론항체를 사용하였을 때 다클론항체를 사용한 경우보다 센서감응이 우수한 것으로 나타났다. 항체가 고정화된 센서 chip과 flow형 quart crystal microbalance 계측 시스템을 이용하여 균 첨가 및 증균을 행한 10종의 모델시료에 대한 계측을 행하였다. 이 때, 시료자체에 의한 진동수변화가 52~89 Hz 범위인 반면 균 첨가 시에 나타난 진동수변화는 108~200 Hz이었고 증균시료에 의한 진동수변화는 162~222 Hz 범위로 나타났다. 시스템 안정화, 시료주입 및 정상상태이 센서반응 획득, 시스템 세척으로 이루어지는 한 주기의 센서계측에 소요된 시간은 모든 시료에 있어 30분 이내였다.

Keywords

References

  1. Stanier, R.Y., Ingraham, J.L., Wheelis, M.L. and Painter, P.R.: Chapter 8. Effect of the environment on microbial growth. In The Microbial World, Fifth Ed. Prentice-Hall, New Jersey, pp.196-212 (1986)
  2. Kristiansen, K.: Evaluation of two selective media for rapid isolation of Pseudomonas strains. Dansk Veterinaertidsskrift, 66, 83-91 (1983)
  3. Venkitanarayanan, K., Khan, M.L, Faustman, C. and Berry, B.W.: Detection of meat spoilage bacteria by a single polymerase chain reaction(PCR) and DNA probe. IFT Annual Meeting, p. 36 (1995)
  4. Stewart, G.S.A.B., Jassim, S.A.A., Denyer, S.P., Newby, P., Ninley, K. and Dhir, V.K.: The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification. J Appl. Microbial., 84, 777-783 (1998) https://doi.org/10.1046/j.1365-2672.1998.00408.x
  5. Labadie, J. and Desnier, I.: Selection of cell wall antigens for the rapid detection of bacteria by immunological methods. J Appl. Bacterial., 72, 220-226 (1992) https://doi.org/10.1111/j.1365-2672.1992.tb01827.x
  6. Jabbar, H. and Joishy, K.N. Rapid detection of Pseudomonas in seafoods using protease indicator. J Food Sci., 64,547-549 (1999) https://doi.org/10.1111/j.1365-2621.1999.tb15082.x
  7. Tanaka, H., Shinji, T., Sawada, K., Monji, Y., Seto, S., Yajima, M. and Vagi, O.: Development and application of a bioluminescent ATP assay method for rapid detection of coliform bacteria. Water Res., 31, 1913-1918 (1997) https://doi.org/10.1016/S0043-1354(97)00032-8
  8. Rowley, D.B. and Previte, J.J.: Radiometry, a rapid screening technique for estimating the level of foodborne bacteria. Activities Report, 27, 114-121 (1975)
  9. Byrne, R.D.: Capture filtration for concentration and detection of selected microorganisms in milk. Dissertation Abstracts Intl. B, 55, 1240 (1994)
  10. Weaver,J.C., Williams, G.B., Klibanov, A. and Demain, A.L.: Gel microdroplets, rapid detection and enumeration of individual microorganisms by their metabolic activity. Bio/Technology, 6, 1084-1089 (1988) https://doi.org/10.1038/nbt0988-1084
  11. Sauerbrey, G.: Verwendungvon Schwingquarzen zur W$\ddot a$gung d$\ddot u$nner Schichten und zur Mikrow$\ddot a$gung. Z. Phys., 155, 206-222 (1959) https://doi.org/10.1007/BF01337937
  12. Kim, N. and Park, I.-S.: Application of a flow-type antibody sensor to the detection of Escherichia coli in various foods. Biosens. Bioelectron., 18, 1101-1107 (2003) https://doi.org/10.1016/S0956-5663(02)00240-3
  13. Park, I.-S. and Kim, N.: Thiolated Salmonella antibody immobilization onto the gold surface of piezoelectric quartz crystal. Biosens. Bioelectron., 13, 1091-1097 (1998) https://doi.org/10.1016/S0956-5663(98)00067-0
  14. Park, I.-S., Kim, w.-Y., and Kim, N.: Operational characteristics of an antibody-immobilized QCM system detecting Salmonella spp. Biosens. Bioelectron., 15, 167-172 (2000) https://doi.org/10.1016/S0956-5663(00)00053-1
  15. Shana, Z.A., Zong, H., Jesse, F. and Jeutter, D.C.: Analysis of electrical equivalent circuit of quartz crystal resonator loaded with viscous conductive liquids. J. Electroanal. Chem., 379, 21-33 (1994) https://doi.org/10.1016/0022-0728(94)87121-3