KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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The Early Detection of the Gram Negative Bacteria using Signification and Fluorescent Dye in the Field

현장에서 초음파 파쇄와 형광시약을 이용한 그람 음성균의 조기 탐지

  • Published : 2006.10.30
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Abstract

This study was carried out to establish the optimum condition for cell disruption with a sonificator in the detection of the gram negative bacteria, E. coli for the purpose of developing automatic fluorometer. The efficiency of sonification on the E. coli disruption was greatly dependent on the diameter of sonificator probe tip. The larger sonificator probe diameter showed greater disruption effect. Sonificator probe of 13 mm diameter was the most efficient one for E. coli when sonificated for 20 seconds. The efficiency of the E. coli disruption differed greatly according to the depth of sonificator probe tip sank in the sample solution. The shorter the distance between probe tip end and the bottom of the container, the higher the disruption efficiency. The detection limit of E. coli was $5{\times}10^5CFU/m{\ell}$ when sample was sonificated for 20 seconds with a sonificator probe of 13 mm diameter.

본 연구에서는 sonificator를 장착하여 세포막을 파쇄하고 현장에서 형광을 이용하여 조작이 간편하고 단시간에 DNA를 측정할 수 있는 자동화된 형광기를 개발하기 위하여 그람 음성균인 Escherchia coli를 대상으로 최적의 세포 파쇄조건을 확립하고자 하였다. Incubation time은 형광량에 크게 영향을 주지 못하는 것으로 나타났으며, 가열처리 방법은 현장에서 세포를 파괴하는 방법으로는 파쇄효과가 미약하고 적합하지 않은 것으로 나타났다. Sonificator probe 직경에 따라 세포의 파쇄 효과가 큰 차이를 보였으며 13 mm probe로 20초 동안 sonification시키는 것이 가장 효율적인 것으로 나타났다. 시료에 잠긴 Sonificator probe tip 깊이에 따라서도 세포의 파쇄 효과가 크게 나타났는데 시료에 잠긴 probe tip의 깊이가 깊을수록 큰 파쇄 효과를 발휘하였다. 선정된 최적의 파쇄 조건에서 $5{\times}10^5CFU/m{\ell}$의 Escherchia coli를 탐지 가능한 것으로 나타났다.

Keywords

References

  1. Abolmaaty, A., M. G. El-Shemy, M. F. Khallaf, and R. E. Levin (1998), Effect of lysing methods and their variables on the yield of Escherichia coli O157: H7 DNA its PCR amplification, J. Microbiol. Methods 34, 133-141 https://doi.org/10.1016/S0167-7012(98)00084-0
  2. Leuschner, R. G. K., J. Bew, P. Fourcassier, and G. Bertin (2004), Validation of the official control method based on polymerase chain reaction(PCR) for identification of authorised probiotic yeast in animal feed, System. Appl. Microbiol. 27, 492-500 https://doi.org/10.1078/0723202041438464
  3. Mark, D. L., J. A. Vaughan, B. J. Shiell, G. J. Beddome, and W. P. Michalski (2004), Mycobacterial proteome extraction: Comparison of disruption methods, Proteomics 4, 1094-1100 https://doi.org/10.1002/pmic.200300672
  4. Aguilera, A., F. Gómez, E. Lospitao, and R. Amils (2006), A molecular approach to the characterization of the eukaryotic communities of an extreme acidic environment: Methods for DNA extraction and denaturing gradient gel electrophoresis analysis, System. Appl. Microbiol. In press, 1-13
  5. Rantakokko-Jalava, K. and J. Jalava (2002), Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR, J. Clin. Microbiol. 40(11), 4211-4217 https://doi.org/10.1128/JCM.40.11.4211-4217.2002
  6. Haugland, R. A., M. Varmar, L. J. Wyme, and S. J. Vesper (2004), Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces Species, System. Appl. Microbiol. 27, 198-210 https://doi.org/10.1078/072320204322881826
  7. James, C. J., W. T. Coakley, and D. E. Hughes (1972), Kinetics of protein release from yeast sonicated in batch and flow systems at 20kHz, Biotechnol. Bioeng. 14, 33-42 https://doi.org/10.1002/bit.260140105
  8. Borthwick, K. A. J., W. T. Coakley, M. B. McDonnell, and H. Nowotny (2005), Development of a novel compact sonicator for cell disruption, J. Microbial. Methods 60, 207-216 https://doi.org/10.1016/j.mimet.2004.09.012
  9. Belgrader, P., D. hansford, G. T. A. Kovacs, K. vanketeswaran, R. Mariell, F. Milanovich, S. Nasarabadi, M. Okuzumi, F. Pourahmadi, and M. A. Northrup (1999), A minisonicator to rapidly disrupt bacterial spores for DNA analysis, Anal. Chem. 71, 4232-4236 https://doi.org/10.1021/ac990347o
  10. Furuta, M., M. Yamaguchi, T. Tsukamoto, B. Yim, C. E. Stavarache, K. Hasiba, and Y. Maeda (2004), Inactivation of Escherichia coli by ultrasonic irradiation, Ultrasonics Sonochemistry 11, 57-60 https://doi.org/10.1016/S1350-4177(03)00136-6
  11. Tsukamoto, I., B. Yim, C. E. Stavarache, M. Furuta, K. Hasiba, and Y. Maeda (2004), Inactivation of Saccharomyces cerevisiae by ultrasonic irradiation, Ultrasonics Sonochemistry 11, 61-65 https://doi.org/10.1016/S1350-4177(03)00135-4
  12. Misik, V., N. Miyoshi, and P. Riesz (1995), EPR spin-trapping study of the sonolysis of $H_2O/D_2O$ mixture : probing the temperature of cavitation regions, J. Phys. Chem. 99, 3605-3611 https://doi.org/10.1021/j100011a030
  13. Seghal, C., R. P. Steer, R. G. Sutherland, and R. E. Verrall (1979), Sonoluminescence of argon saturated alkali metal salt solutions as a probe of acoustic cavitation, J. Chem. Phys. 70, 2242-2248 https://doi.org/10.1063/1.437704
  14. Inez, H. and J. E. Thompson (2000), Inactivation of Escherichia coli by sonification at discrete ultrasonic frequencies, Wat. res. 34(15), 3888-3893 https://doi.org/10.1016/S0043-1354(00)00121-4
  15. Blechl, A. E., K. S. Thrasher, W. H. Vensel, and F. C. Greene (1992), Purification and characterization of wheat $\alpha$-gliadin synthesized in the yeast, Saccharomyces cerevisiae, Gene. 116, 119-127 https://doi.org/10.1016/0378-1119(92)90507-L
  16. Calderira, J. C. G. and J. M. S. Cabral (1994), Extraction of a steroid 1,2-dehydrogenase by sonification of Arthrobacter simplex cells, Bioseparation 4, 271-278
  17. Kuboi, R., H. Umakoshi, N. Takagi, and I. Komasawa (1995), Optimal disruption methods for the selective recovery of $\beta$ -galactosidase from Escherichia coli, J. Ferment. Bioeng. 79, 335-341 https://doi.org/10.1016/0922-338X(95)93991-R
  18. Feliu, J. X., R. Cubarsi, and A. Villaverde (1998), Optimized release of recombinant proteins by ultrasonication of E. coli cells, Biotechnology and Bioengineering 58(5), 536-540 https://doi.org/10.1002/(SICI)1097-0290(19980605)58:5<536::AID-BIT10>3.0.CO;2-9
  19. Else, M. F., J. S. Olsen, and G. Skogan (2003), Application of sonification to lease DNA from Bacillus cereus for quantitative detection by real-time PCR, J. Microbiol. Methods 55, 1-10 https://doi.org/10.1016/S0167-7012(03)00091-5