Abstract
The aim of this study was to evaluate the applicability of the conventional PCR detection method combined with propidium monoazide (PMA) treatment for the detection of viable but non-culturable (VBNC) state Escherichia coli O157:H7 in ground beef meatballs. Under low temperature, E. coli O157:H7 cells were induced into the VBNC state in ground beef meatballs at − 20 °C after 152 days. The optimal PMA concentration of 5 µg/mL was obtained in beef meatball samples, which could completely inhibit the DNA amplification on dead cells (106 cells/mL) but with no inhibition on viable cells. The established PMA-PCR assay revealed that the VBNC counts exceeded 107 CFU/mL in artificial contamination beef samples, which could be used for semi-quantitative detection of VBNC cells in beef meatball samples. This study indicated that the PMA-PCR assay might be a potential method for detection of VBNC state E. coli O157:H7 cells in food products.
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References
Al-Soud WA, Râdström P (1998) Capacity of nine thermostable DNA polymerases To mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol 64(10):3748–3753
Besnard V, Federighi M, Cappelier JM (2000) Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes. Lett Appl Microbiol 31(1):77–81. https://doi.org/10.1046/j.1472-765x.2000.00771.x
Dinu L-D, Bach S (2013) Detection of viable but non-culturable Escherichia coli O157:H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays. Food Control 31(2):268–273. https://doi.org/10.1016/j.foodcont.2012.10.020
Elizaquível P, Sánchez G, Aznar R (2012) Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR. Food Control 25(2):704–708. https://doi.org/10.1016/j.foodcont.2011.12.003
Kogure K, Simidu U, Taga N (1979) A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 25(3):415–420
Li F, Xie G, Zhou B, Yu P, Yu S, Aguilar ZP, Wei H, Xu H (2016a) Rapid and simultaneous detection of viable Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus in infant food products by PMA-mPCR assay with internal amplification control. LWT-Food Sci Technol 74:176–182. https://doi.org/10.1016/j.lwt.2016.07.044
Li J, Ahn J, Liu D, Chen S, Ye X, Tian D (2016b) Evaluation of ultrasound-induced damage to Escherichia coli and Staphylococcus aureus by flow cytometry and transmission electron microscopy. Appl Environ Microbiol 82(6):1828. https://doi.org/10.1128/AEM.03080-15
Liao X, Liu D, Xiang Q, Ahn J, Chen S, Ye X, Ding T (2016) Inactivation mechanisms of non-thermal plasma on microbes: a review. Food Control 75:83–91. https://doi.org/10.1016/j.foodcont.2016.12.021
Liu Y, Mustapha A (2014) Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR. Int J Food Microbiol 170:48–54. https://doi.org/10.1016/j.ijfoodmicro.2013.10.026
Liu Y, Gilchrist A, Zhang J, Li X-F (2008) Detection of viable but nonculturable Escherichia coli O157:H7 bacteria in drinking water and river water. Appl Environ Microbiol 74(5):1502–1507. https://doi.org/10.1128/AEM.02125-07
Makino S-I, Kii T, Asakura H, Shirahata T, Ikeda T, Takeshi K, Itoh K (2000) Does enterohemorrhagic Escherichia coli O157:H7 enter the viable but nonculturable state in salted Salmon Roe? Appl Environ Microbiol 66(12):5536–5539. https://doi.org/10.1128/AEM.66.12.5536-5539.2000
Masmoudi S, Denis M, Maalej S (2010) Inactivation of the gene katA or sodA affects the transient entry into the viable but non-culturable response of Staphylococcus aureus in natural seawater at low temperature. Mar Pollut Bull 60(12):2209–2214. https://doi.org/10.1016/j.marpolbul.2010.08.017
Murakami T (2012) Filter-based pathogen enrichment technology for detection of multiple viable foodborne pathogens in 1 day. J Food Prot 75(9):1603–1610. https://doi.org/10.4315/0362-028x.jfp-12-039
Nocker A, Cheung C-Y, 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(2):310–320. https://doi.org/10.1016/j.mimet.2006.04.015
Pan Y, Breidt F Jr (2008) Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73(24):8028–8031. https://doi.org/10.1128/AEM.01198-07
Patrone V, Campana R, Vallorani L, Dominici S, Federici S, Casadei L, Gioacchini AM, Stocchi V, Baffone W (2013) CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state. Antonie Van Leeuwenhoek 103(5):979–988. https://doi.org/10.1007/s10482-013-9877-5
Rossen L, Nørskov P, Holmstrøm K, Rasmussen OF (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol 17(1):37–45
Rudi K, Moen B, Drømtorp 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):1018–1024. https://doi.org/10.1128/AEM.71.2.1018-1024.2005
Singh G, Vajpayee P, Bhatti S, Ronnie N, Shah N, McClure P, Shanker R (2013) Determination of viable Salmonellae from potable and source water through PMA assisted qPCR. Ecotoxicol Environ Saf 93:121–127. https://doi.org/10.1016/j.ecoenv.2013.02.017
Vesper S, McKinstry C, Hartmann C, Neace M, Yoder S, Vesper A (2008) Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods 72(2):180–184. https://doi.org/10.1016/j.mimet.2007.11.017
Wang L, Li P, Yang Y, Xu H, Aguilar ZP, Xu H, Yang L, Xu F, Lai W, Xiong Y (2014) Development of an immunomagnetic separation–propidium monoazide–polymerase chain reaction assay with internal amplification control for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk. Int Dairy J 34(2):280–286. https://doi.org/10.1016/j.idairyj.2013.07.006
Zhang S, Ye C, Lin H, Lv L, Yu X (2015) UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa. Environ Sci Technol 49(3):1721–1728. https://doi.org/10.1021/acs.est.5b00769
Zhao F, Bi X, Hao Y, Liao X (2013a) Induction of viable but nonculturable Escherichia coli O157:H7 by high pressure CO2 and its characteristics. PLoS One 8(4):e62388. https://doi.org/10.1371/journal.pone.0062388
Zhao X, Lin CW, Wang J, Oh DH (2013b) Advances in rapid detection methods for foodborne pathogens. J Microbiol Biotechnol 24(3):297–312. https://doi.org/10.4014/jmb.1310.10013
Zhao X, Wang J, Forghani F, Park J-H, Park M-S, Seo K-H, Oh D-H (2013c) Rapid detection of viable Escherichia coli O157 by coupling propidium monoazide with loop-mediated isothermal amplification. J Microbiol Biotechnol 23(12):1708–1716
Zhao X, Zhong J, Wei C, Lin CW, Ding T (2017) Current perspectives on viable but non-culturable state in foodborne pathogens. Front Microbiol 8(703813):580. https://doi.org/10.3389/fmicb.2017.00580
Zhu R-G, Li T-P, Jia Y-F, Song L-F (2012) Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR. J Microbiol Methods 90(3):262–266. https://doi.org/10.1016/j.mimet.2012.05.019
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This work has been supported by the National Natural Science Foundation of China (Grant no. 31501582) and open project program of key laboratory for Green Chemical Process of Ministry of Education in Wuhan Institute of Technology (2017007).
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Junliang Zhong declares that he has no conflict of interest. Xihong Zhao declares that he has no conflict of interest.
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Zhong, J., Zhao, X. Detection of viable but non-culturable Escherichia coli O157:H7 by PCR in combination with propidium monoazide. 3 Biotech 8, 28 (2018). https://doi.org/10.1007/s13205-017-1052-7
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DOI: https://doi.org/10.1007/s13205-017-1052-7