Determination of viability in foodborne bacteria with inter-calating dyes: ethidium monoazide (EMA) and propidium monoazide (PMA)

  • Authors

    • Arife Ezgi Telli University of Selcuk, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology
    • Yusuf DoÄŸruer University of Selcuk, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology
    2017-11-29
    https://doi.org/10.14419/ijbr.v5i2.8561
  • Ethidium Monoazide (EMA), Foodborne Bacterial Pathogens, Intercalating Dyes, Propidium Monoazide (PMA), Viability.

  • Abstract

    The ability to distinguish between living and dead cells is considered to be very important for biological researches. It is an important problem that the technology used up to day does not allow the quantitative differentiation of specific cells in a mixed cell community. Determination of whether the microorganisms present in the foods are in a viable form is an important phenomenon in determining the disease-forming potential.

    It is a fact that DNA, which is found in cells that lose their viability, can maintain its activity for a long time. Discrimination of live-dead cell occurs when the intercalating dye is covalently bound to DNA that is cleaved in the dead cell where membrane integrity is impaired. The formation of the covalent bond is activated by photoactivation. Inter-collating dyes only affect dead cells that are damaged by cell wall or membrane integrity. Due to the covalent binding of the inter-collating dye, DNA amplification cannot occur in PCR and other molecular techniques based on PCR.

    Among the non-permeable stains, it is accepted that PI is the most commonly used. PMA is identical to PI and additionally contains azide groups. Azide groups allow PMA to cross-covalently bond with DNA in bright light. Another inter-collating dyes with an azide group is ethidium mono azide (EMA).The The PMA molecule provides a higher selectivity on discrimination of live-dead cells by virtue of its’ higher charge when compared to EMA. Many researchers have combined EMA and PMA with PCR, Real-time PCR and LAMP in order to differentiate the live population of bacterial, viral, fungal and parasitic food-borne pathogens because they are claimed to be more successful in complex samples than in fluorescence based techniques.

  • References

    1. [1] Banihashemi A1, Van Dyke MI, Huck PM (2012). Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella. J Appl Microbiol, 113:4, 863-73. https://doi.org/10.1111/j.1365-2672.2012.05382.x.

      [2] Biebricher AS, Heller I, Roijmans RFH, Hoekstra TP, Peterman EJG, Wuite GJL (2015). The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics. Nat Commun, 6:7304, 1-12. https://doi.org/10.1038/ncomms8304.

      [3] Bolton PH, Kearns DR (1978). Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids. Nucleic Acids Res, 5:12, 4891-903. https://doi.org/10.1093/nar/5.12.4891.

      [4] Cawthorn DM, Witthuhn RC (2008). Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide. J Appl Microbiol, 105:4, 1178-85. https://doi.org/10.1111/j.1365-2672.2008.03851.x.

      [5] Chen S, Wang F, Beaulieu JC, Stein RE, Ge B (2011). Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification, Appl Environ Microbiol, 77:12, 4008.

      [6] Elizaquivel P, Sánchez G, Selma MV, Aznar R (2012). Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water. Food Microbiol, 30:1, 316-20. https://doi.org/10.1016/j.fm.2011.10.008.

      [7] Flekna G, Stefanic P, Wagner M, Smulders FJM, Mozina SS, 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, 405-12. https://doi.org/10.1016/j.resmic.2007.02.008.

      [8] Fujimoto J, Watanabe K (2013). Quantitative detection of viable Bifidobacterium bifidum BF-1 cells in human feces by using propidium monoazide and strain-specific primers. Appl Environ Microbiol, 79:7, 2182-8. https://doi.org/10.1128/AEM.03294-12.

      [9] Garcia-Cayuela T, Tabasco R, Peláez C, Requena T (2009). Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR. Int Dairy J, 19:6-7, 405-9. https://doi.org/10.1016/j.idairyj.2009.02.001.

      [10] Gensberger ET, Sessitsch A, Kostic T (2013) Propidium monoazide–quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora. Anal Biochem, 441:1, 69-72. https://doi.org/10.1016/j.ab.2013.05.033.

      [11] Hixon SC, White Jr WE, Yielding KL (1975). Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labelling. J Mol Biol, 92:2, 319-29. https://doi.org/10.1016/0022-2836(75)90231-4.

      [12] Josefsen MH, Löfström C, Hansen TB, Christensen LS, Olsen JE, Hoorfar J (2010). Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Appl Environ Microbiol, 76:15, 5097-104. https://doi.org/10.1128/AEM.00411-10.

      [13] Kobayashi H, Oethinger M, Tuohy MJ, Hall GS, Bauer TW (2009). Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide. Lett Appl Microbiol, 48:5, 633-8. https://doi.org/10.1111/j.1472-765X.2009.02585.x.

      [14] Kralik P, Nocker A, Pavlik I (2010). Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int J Food Microbiol, 141, 80-6. https://doi.org/10.1016/j.ijfoodmicro.2010.03.018.

      [15] Liu YH, Wang CH, Wu JJ, Lee GB (2012). Rapid detection of live methicillin-resistant Staphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis. Biomicrofluidics, 6:3, 034119.

      [16] Lovdal T, Hovda MB, Björkblom B, Møller SG (2011). Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua. J Microbiol Methods, 85:2, 164-9. https://doi.org/10.1016/j.mimet.2011.01.027.

      [17] Lu Y, Yang W, Shi L, Li L, Alam MJ, Guo S, Miyoshi S (2009). Specific detection of viable Salmonella cells by an ethidium monoazide-loop mediated isothermal amplification (EMA-LAMP) method, J Health Sci, 55, 820-4. https://doi.org/10.1248/jhs.55.820.

      [18] Myron TLD, Venkateswaran KJ, Mohapatra B (2012). US20120231961 A1.

      [19] Nam S, Kwon S, Kim MJ, Chae JC, Jae Maeng P, Park JG, Lee GC (2011). Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction. Microbiol Immunol, 55:12, 841-6. https://doi.org/10.1111/j.1348-0421.2011.00388.x.

      [20] Nocker A, Camper AK (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.

      [21] Nocker A, Cheung CY, Camper AK (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, 310-20 https://doi.org/10.1016/j.mimet.2006.04.015.

      [22] Nogva HK, Dromtorp SM, Nissen H, Rudi K (2003). Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR. Biotechniques, 34:4, 804-13.

      [23] Rawsthorne H, Dock CN, Jaykus LA (2009). PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores. Appl Environ Microbiol, 75:9, 2936-9. https://doi.org/10.1128/AEM.02524-08.

      [24] Regan JM, Oldenburg PS, Park HD, Harrington GW, Noguera DR (2003). Simultaneous determination of bacterial viability and identity in biofilms using ethidium monoazide and fluorescent in situ hybridization. Water Sci Technol, 47:5, 123-8.

      [25] Rudi K, Moen B, Dromtorp SM, Holck AL (2005). Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environ Microbiol, 71:2, 1018-24. https://doi.org/10.1128/AEM.71.2.1018-1024.2005.

      [26] Soejima Takashi, Iida K, Qin T, Taniai H, Seki M, Yoshida S (2008). Method to detect only live bacteria during PCR amplification. J. Clin. Microbiol. 46:2305–2313 https://doi.org/10.1128/JCM.02171-07.

      [27] Taskin B, Gozen AG, Duran M (2011). Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification. Appl Environ Microbiol, 77:13, 4329-35. https://doi.org/10.1128/AEM.02895-10.

      [28] Vladescu ID, McCauley MJ, Nuñez ME, Rouzina I, Williams MC (2007). Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching. Nat Methods, 4, 517-22. https://doi.org/10.1038/nmeth1044.

      [29] Wang L, Mustapha Y, Li A (2009). Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR. J Appl Microbiol, 107:5, 1719-28. https://doi.org/10.1111/j.1365-2672.2009.04358.x.

      [30] Wang L, Zhong Q, Li Y (2012). Ethidium monoazide-loop mediated isothermal amplification for rapid detection of Vibrio parahaemolyticus in viable but non-culturable state. Energy Procedia, 17, 1858-63. https://doi.org/10.1016/j.egypro.2012.02.323.

      [31] Wang S, Levin RE (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.

      [32] Zhang Z, Wang L, Xu H, Aguilar ZP, Liu C, GanB, Xiong Y, Lai W, Xu F, Wei H(2014). Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control. Food Cont, 35:1, 401-6. https://doi.org/10.1016/j.foodcont.2013.07.035.

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  • How to Cite

    Telli, A. E., & DoÄŸruer, Y. (2017). Determination of viability in foodborne bacteria with inter-calating dyes: ethidium monoazide (EMA) and propidium monoazide (PMA). International Journal of Biological Research, 5(2), 59-62. https://doi.org/10.14419/ijbr.v5i2.8561

    Received date: 2017-10-27

    Accepted date: 2017-11-23

    Published date: 2017-11-29