Journal of Korean Society of Water and Wastewater (상하수도학회지)

The Korean Society of Water and Wastewater (대한상하수도학회)
권호 표지

Ethidium monoazide-PCR for the detection of viable Escherichia coli in aquatic environments

수환경에서 살아 있는 대장균의 검출을 위한 ethidium monoazide-중합효소연쇄반응법

  • Received : 2009.02.03
  • Accepted : 2009.03.25
  • Published : 2009.04.15
Download PDF
⟨ Previous Next ⟩

Abstract

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.

Keywords

Acknowledgement

Supported by : 한국수자원공사 K-water 연구원

References

  1. 환경부 (2008) 먹는 물 수질기준 및 검사 등에 관한 규칙. 환경부령 제276호
  2. Cawthorn, D.M. and Witthuhn, R.C. (2008) Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide, J. Appl. Microbiol., 105(4):1178-1185 https://doi.org/10.1111/j.1365-2672.2008.03851.x
  3. Chang, B., Sugiyama, K., Taguri, T., Amemura-Maekawa, J., Kura, F. and Watanabe, H. (2009) Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR, Appl. Environ. Microbiol., 75(1):147-153 https://doi.org/10.1128/AEM.00604-08
  4. Chomvarin, C., Namwat, W., Wongwajana, S., Alam, M., Thaew-Nonngiew, K., Sinchaturus, A. and Engchanil, C. (2007) Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand, J. Gen. Appl. Microbiol., 53(4):229-237 https://doi.org/10.2323/jgam.53.229
  5. Coffman, G.L., Gaubatz, J.W., Yielding, K.L. and Yielding, L,W. (1982) Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with ethidium analog, J. Biol. Chem., 257(22): 13205-13207
  6. DeTraglia, M.C., Brand, J.S. and Tometski, A.M. (1978) Characterization of azidobenzamidines as photoaffinity labeling for trypsin, J. Biol. Chem., 253(6):1846-1852
  7. Flekna, G., Stefanic, P., Wagner, M., Smulders, F.J., Mozina, S.S. and Hein, I. (2007) Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR, Res. Microbiol., 158(5):405-412 https://doi.org/10.1016/j.resmic.2007.02.008
  8. Greisen, K., Loeffelholz, M., Purohit, A. and Leong, D. (1994) PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid, J. Clin. Microbiol., 32(2):335-351
  9. Hixon, S.C., White, W.E. and Yielding, K,L. (1975) Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling, J. Mol. Biol., 92(2):319-329 https://doi.org/10.1016/0022-2836(75)90231-4
  10. Inoue, D., Tsutsui, H., Yamazaki, Y., Sei, K., Soda, S., Fujita, M. and Ike, M. (2008) Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment, Water Sci. Technol., 58(5):1107-1112 https://doi.org/10.2166/wst.2008.474
  11. Josephson, K.L., Gerba, C.P. and Pepper, I.L. (1993) Polymerase chain reaction detection of nonviable bacterial pathogens, Appl. Environ. Microbiol., 59(10):3513-3515
  12. Kong, R.Y., Lee, S.K., Law, T.W., Law, S.H. and Wu, R.S. (2002) Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR, Water Res., 36(11):2802-2812 https://doi.org/10.1016/S0043-1354(01)00503-6
  13. Masters, C.I., Shallcross, J.A. and Mackey, B.M. (1994) Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction. J. Appl. Bacteriol., 77(1):73-79 https://doi.org/10.1111/j.1365-2672.1994.tb03047.x
  14. Mazari-Hiriart, M., Lόpez-Vidal, Y., Castillo-Rojas, G., Ponce de Leόn, S. and Cravioto, A. (2001) Helicobacter pylori and other enteric bacteria in freshwater environments in Mexico City, Arch. Med. Res., 32(5):458-467 https://doi.org/10.1016/S0188-4409(01)00304-6
  15. Nam, S.H., Lee, J.I., Lee, S.T. and Lee, G.C. (2007) Detection of viable Cryptosporidium parvum by real-time RT-PCR, J. Kor. Soc. Environ. Anal., 10(3):134-140
  16. Nebe-von-Caron, G., Stephens, P. and Badley, R.A. (1998) Assessment of bacterial viability status by flow cytometry and single cell sorting, J. Appl. Microbiol., 84(6):988-998 https://doi.org/10.1046/j.1365-2672.1998.00436.x
  17. Nebe-von-Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R. and Badley, R.A. (2000) Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting, J. Microbiol. Methods, 42(1):97-114 https://doi.org/10.1016/S0167-7012(00)00181-0
  18. Nocker, A. and Camper, A.K. (2006) Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide, Appl. Environ. Microbiol., 72(3):1997-2004 https://doi.org/10.1128/AEM.72.3.1997-2004.2006
  19. Nocker, A., Cheung, C.Y. and Camper, A.K. (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods, 67(2):310-320 https://doi.org/10.1016/j.mimet.2006.04.015
  20. Nogva, H.K., Drømtorp, S.M., Nissen, H. and Rudi, K. (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR, Biotechniques, 34(4):804-808, 810, 812-813
  21. Pisz, J.M., Lawrence, J.R., Schafer, A.N. and Siciliano, S.D. (2007) Differentiation of genes extracted from non-viable versus viable micro-organisms in environmental samples using ethidium monoazide bromide, J. Microbiol. Methods, 71(3):312-318 https://doi.org/10.1016/j.mimet.2007.09.015
  22. Rudi, K., Moen, B., Drømtorp, S.M. and Holck, A.L. (2005a) Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples, Appl. Environ. Microbiol., 71(2):1018-1024 https://doi.org/10.1128/AEM.71.2.1018-1024.2005
  23. Rudi, K., Naterstad, K., Drømtorp, S.M. and Holo, H. (2005b) Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR, Lett. Appl. Microbiol., 40(4):301-306 https://doi.org/10.1111/j.1472-765X.2005.01672.x
  24. Soejima, T., Iida, K., Qin, T., Taniai, H., Seki, M. and Yoshida, S. (2008) Method to detect only live bacteria during PCR amplification, J. Clin. Microbiol., 46(7):2305-2313 https://doi.org/10.1128/JCM.02171-07
  25. Wang, S. and Levin, R.E. (2006) Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR, J. Microbiol. Methods, 64(1):1-8 https://doi.org/10.1016/j.mimet.2005.04.023
  26. Yanez, M.A., Carrasco-Serrano, C., Barbera, V.M. and Catalan, V. (2005) Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene, Appl. Environ. Microbiol., 71(7):3433-3441 https://doi.org/10.1128/AEM.71.7.3433-3441.2005