Advertisement

Food Analytical Methods

, Volume 11, Issue 5, pp 1257–1266 | Cite as

Quantification and Discrimination of Viable and Dead Escherichia coli O157:H7 Cells from Chicken Without Enrichment by Ethidium Bromide Monoazide Real-time Loop-Mediated Isothermal Amplification

  • Yuan Yang Zhao
  • Kai Jie Tang
  • Tian Tian Zhang
  • Yan Yan Gao
  • Li Ping Lin
  • Guo Ping Wu
Article

Abstract

In this study, a rapid and sensitive method of real-time loop-mediated isothermal amplification (Rti-LAMP) assays was developed for quantification and discrimination of viable and heat-killed E. coli O157:H7 cells treated with low concentration of ethidium bromide monoazide (EMA). Four micrograms per milliliter of EMA was chosen as the optimal concentration which did not inhibit DNA amplification derived from viable cells, but significantly increased the Tt values of dead cells in Rti-LAMP assays. When the DNA from 2.0 × 103 viable CFU of E. coli O157:H7 was subjected to EMA-Rti-LAMP, the resulting Tt value was 17.73 min. In contrast, the DNA from 2.0 × 103 CFU completely heat destroyed CFU of E. coli O157:H7 did not yield a positive amplification which Tt value was regarded as 60 min. When the DNA from viable plus heat-killed CFU at a ratio of 5:2995 was subjected to EMA-Rti-LAMP, the resulting Tt value was 23.06 min, which was statistically identical (P < 0.05) to the Tt value of 24.07 min obtained with the DNA from only 5 viable CFU. The results indicate that even though 3.0 × 103 dead cells yielded a negative amplification setting the Tt value as 60 min, low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tt values. Detection of E. coli O157:H7 derived from contaminated chicken samples, the EMA-Rti-LAMP could notably distinguish viable and heat-killed cells from 5.0 × 101 to 1.0 × 104 CFU/g without enrichment.

Keywords

Escherichia coli O157:H7 Ethidium bromide monoazide (EMA) Rti-LAMP Viable cells Heat-killed cells Chicken Without enrichment 

Notes

Funding Information

This study was funded by the National Natural Science Foundation of China (No. 31560480, 31760483), Jiangxi Natural Science Foundation of China (No. 20171ACB20013), and the special funds for collaborative innovation of modern agricultural science and research in Jiangxi, China (No. JXXTCX201703).

Compliance with Ethical Standards

Conflict of Interest

Yuan Yang Zhao declares that he has no conflict of interest. Kai Jie Tang declares that she has no conflict of interest. Tian Tian Zhang declares that she has no conflict of interest. Yan Yan Gao declares that she has no conflict of interest. Li Ping Lin declares that she has no conflict of interest. Guo Ping Wu declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Not applicable.

References

  1. Abolmaaty A, Vu C, Oliver J, Levin RE (2000) Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction. Microbios 101:181–189Google Scholar
  2. Barbau-Piednoir E, Mahillon J, Pillyser J, Coucke W, Roosens NH, Botteldoorn N (2014) Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis. J Microbiol Methods 103:131–137CrossRefGoogle Scholar
  3. Caprioli J, Peng L, Remuzzi G (2005) The hemolytic uremic syndromes. Curr Opin Crit Care 11:487–492CrossRefGoogle Scholar
  4. Cocolin L, Rajkovic A, Rantsiou K, Uyttendaele M (2011) The challenge of merging food safety diagnostic needs with quantitative PCR platforms. Trends Food Sci Technol 22:S30–S38CrossRefGoogle Scholar
  5. Communities CotE (2013) Commission Regulation (EU) No. 209/2013 of 11 March 2013 amending regulation (EC) No 2073/2005 as regards microbiological criteria for sprouts and the sampling rules for poultry carcasses and fresh poultry meatGoogle Scholar
  6. Dong HJ, Cho AR, Hahn TW, Cho S (2014) Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples. J Food Prot 77:1593–1598CrossRefGoogle Scholar
  7. Fittipaldi M, Codony F, Adrados B, Camper AK, Morato J (2011) Viable real-time PCR in environmental samples: can all data be interpreted directly? Microb Ecol 61:7–12CrossRefGoogle Scholar
  8. Guo PW, HC S, Levin RE (2015) Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification. J Microbiol Methods 117:41–48CrossRefGoogle Scholar
  9. Hara-Kudo Y, Nemoto J, Ohtsuka K, Segawa Y, Takatori K, Kojima T, Ikedo M (2007) Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J Med Microbiol 56:398–406CrossRefGoogle Scholar
  10. Josefsen MH, Lofstrom 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 76:5097–5104CrossRefGoogle Scholar
  11. Kibbee RJ, Ormeci B (2017) Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent. J Microbiol Methods 132:139–147CrossRefGoogle Scholar
  12. Kokkinos PA, Ziros PG, Bellou M, Vantarakis A (2014) Loop-mediated isothermal amplification (LAMP) for the detection of salmonella in food. Food Anal Methods 7:512–526CrossRefGoogle Scholar
  13. Lee JL, Levin RE (2006) Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction. J Microbiol Methods 67:456–462CrossRefGoogle Scholar
  14. Lee JL, Levin RE (2009) A comparative study of the ability of EMA and PMA to distinguish viable from heat killed mixed bacterial flora from fish fillets. J Microbiol Methods 76:93CrossRefGoogle Scholar
  15. Li B, Chen J-Q (2013) Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food. BMC Microbiol 13:273CrossRefGoogle Scholar
  16. 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 170:48–54CrossRefGoogle Scholar
  17. Martin B, Raurich S, Garriga M, Aymerich T (2013) Effect of amplicon length in propidium monoazide quantitative PCR for the enumeration of viable cells of salmonella in cooked ham. Food Anal Methods 6:683–690CrossRefGoogle Scholar
  18. Nocker A, Camper AK (2006) Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol 72:1997–2004CrossRefGoogle Scholar
  19. Nogva HK, Drømtorp SM, Nissen H, Rudi K (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. BioTechniques 34:804–813Google Scholar
  20. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63CrossRefGoogle Scholar
  21. Novak JS, Juneja VK (2001) Detection of heat injury in Listeria monocytogenes Scott A. J Food Prot 64:1739–1743CrossRefGoogle Scholar
  22. Techathuvanan C, D’Souza DH (2012) Reverse-transcriptase loop-mediated isothermal amplification as a rapid screening/monitoring tool for Salmonella enterica detection in liquid whole eggs. J Food Sci 77:200–205CrossRefGoogle Scholar
  23. Wang DG, Huo GC (2011) Rapid detection viable Escherichia coli O157 in raw milk using loop-mediated isothermal amplification with aid of ethidium monoazide. Adv Mater Res 343-344:1217–1221CrossRefGoogle Scholar
  24. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63:3741–3751Google Scholar
  25. Wong CS, Jelacic S, Habeeb RL, Watkins SL, Tarr PI (2000) The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med 342:1930–1936CrossRefGoogle Scholar
  26. Wu GP, Chen SH, Levin RE (2015a) Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification. J Microbiol Methods 117:41–48CrossRefGoogle Scholar
  27. Wu GP, Chen SH, Levin RE (2015b) Rapid real-time loop-mediated isothermal amplification combined with coated activated carbon for detection of low numbers of Salmonella enterica from lettuce without enrichment. Food Control 56:47–52CrossRefGoogle Scholar
  28. Yan M, Xu L, Jiang H, Zhou Z, Zhou S, Zhang L (2017) PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state. Microb Pathog 105:245–250CrossRefGoogle Scholar
  29. Yanez MA, Nocker A, Soria-Soria E, Murtula R, Martinez L, Catalan V (2011) Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR. J Microbiol Methods 85:124–130CrossRefGoogle Scholar
  30. Yang Y, Wan C, Xu H, Lai W, Xiong Y, Xu F, You X, Xu H, Aguilar ZP, Sun J et al (2012) Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars. Food Control 28:456–462CrossRefGoogle Scholar
  31. Zhang S, Zhu X, Wu Q, Zhang J, Xu X, Li H (2015) Prevalence and characterization of Escherichia coli O157 and O157:H7 in retail fresh raw meat in South China. Ann Microbiol 65:1993–1999CrossRefGoogle Scholar
  32. Zhao X, Wang J, Forghani F, Park JH, Park MS, Seo KH, DH O (2013) Rapid detection of viable Escherichia coli O157 by coupling propidium monoazide with loop-mediated isothermal amplification. J Microbiol Biotechnol 23:1708–1716CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Yuan Yang Zhao
    • 1
  • Kai Jie Tang
    • 1
  • Tian Tian Zhang
    • 1
  • Yan Yan Gao
    • 1
  • Li Ping Lin
    • 1
  • Guo Ping Wu
    • 1
  1. 1.College of Food Science and EngineeringJiangxi Agricultural UniversityNanchangChina

Personalised recommendations