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Rapid and sensitive detection of Salmonella spp. in raw minced meat samples using droplet digital PCR

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Abstract

With its ability to easily infect each step of food production chain, Salmonella spp. stands as one of the most important foodborne pathogens which impose threats against economy and human health by creating high morbidity and mortality, worldwide. Although sensitive screening and detection methodologies based on common conventional techniques have been developed for Salmonella spp., their long turnaround time and strict legislative demands directs the researchers to develop more sensitive and rapid methods. Digital PCR has many potential advantages over its closest competitor, Real-Time PCR. It gives more accurate, sensitive, and precise results, especially for samples that contain low pathogen concentrations and shows more resistance against PCR inhibitors. In this study, we developed a droplet digital PCR screening methodology with a short pre-enrichment step. For this purpose, 25 g raw minced meat was subjected to Salmonella contamination, and following a very short 3 h enrichment step, bacterial isolation from 5 mL of sample was achieved. For the specific detection of the Salmonella spp., bipA gene region was selected as a better candidate. Subsequently, droplet digital PCR setup was performed and detection sensitivity of the test was determined as 1.39 cfu/mL. Furthermore, parallel experiments were also conducted using Real-Time PCR, for comparative and validative purposes. Conclusively, data gathered in this study have shown that droplet digital PCR with our proposed setup can sensitively detect Salmonella spp. in raw minced meat samples while generating the same-day results.

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References

  1. Besser JM (2018) Salmonella epidemiology: a whirlwind of change. Food Microbiol 71:55–59. https://doi.org/10.1016/j.fm.2017.08.018

    Article  PubMed  Google Scholar 

  2. Havelaar AH, Kirk MD, Torgerson PR, Gibb HJ, Hald T, Lake RJ, Praet N, Bellinger DC, Silva NR, Gargouri N, Speybroeck N, Cawthorne A, Mathers C, Stein C, Angulo FJ, Devleesschauwer B (2015) World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med 12(12):e1001923. https://doi.org/10.1371/journal.pmed.1001923

    Article  PubMed  PubMed Central  Google Scholar 

  3. Blackburn WC, McClure PJ (eds) (2009) Foodborne pathogens: hazards, risk analysis and control. Elsevier, Amsterdam

    Google Scholar 

  4. Mughini-Gras L, Franz E, van Pelt W (2018) New paradigms for Salmonella source attribution based on microbial subtyping. Food Microbiol 71:60–67. https://doi.org/10.1016/j.fm.2017.03.002

    Article  PubMed  Google Scholar 

  5. McEntire J, Acheson D, Siemens A, Eilert S, Robach M (2014) The public health value of reducing Salmonella levels in raw meat and poultry. Food Protect Trends 34(6):386–392

    Google Scholar 

  6. de Freitas Neto OC, Penha Filho RAC, Barrow P, Berchieri Junior A (2010) Sources of human non-typhoid salmonellosis: a review. Braz J Poultry Sci 12(1):01–11. https://doi.org/10.1590/S1516-635X2010000100001

    Article  Google Scholar 

  7. Gill AO, Gill CO (2015) Developments in sampling and test methods for pathogens in fresh meat. Advances in Microbial Food Safety. Woodhead Publishing, Cambridge, pp 257–280

    Book  Google Scholar 

  8. Carli KT, Unal CB, Caner V, Eyigor A (2001) Detection of Salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis. J Clin Microbiol 39(5):1871–1876. https://doi.org/10.1128/JCM.39.5.1871-1876.2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. McGuinness S, McCabe E, O’Regan E, Dolan A, Duffy G, Burgess C, Fanning S, Barry T, O’Grady J (2009) Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat. Meat Sci 83(3):555–562. https://doi.org/10.1016/j.meatsci.2009.07.004

    Article  CAS  PubMed  Google Scholar 

  10. Hagren V, von Lode P, Syrjälä A, Korpimäki T, Tuomola M, Kauko O, Nurmi J (2008) An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR. Int J Food Microbiol 125(2):158–161. https://doi.org/10.1016/j.ijfoodmicro.2008.03.037

    Article  CAS  PubMed  Google Scholar 

  11. Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M (2005) Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol Lett 253(1):155–161. https://doi.org/10.1016/j.femsle.2005.09.032

    Article  CAS  PubMed  Google Scholar 

  12. Wolffs PF, Glencross K, Thibaudeau R, Griffiths MW (2006) Direct quantitation and detection of salmonellae in biological samples without enrichment, using two-step filtration and real-time PCR. Appl Environ Microbiol 72(6):3896–3900. https://doi.org/10.1128/AEM.02112-05

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Law JWF, Ab Mutalib NS, Chan KG, Lee LH (2015) Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Front Microbiol 5:770. https://doi.org/10.3389/fmicb.2014.00770

    Article  PubMed  PubMed Central  Google Scholar 

  14. Garrido A, Chapela MJ, Román B, Fajardo P, Lago J, Vieites JM, Cabado AG (2013) A new multiplex real-time PCR developed method for Salmonella spp. and Listeria monocytogenes detection in food and environmental samples. Food Control 30(1):76–85. https://doi.org/10.1016/j.foodcont.2012.06.029

    Article  CAS  Google Scholar 

  15. Mangal M, Bansal S, Sharma SK, Gupta RK (2016) Molecular detection of foodborne pathogens: a rapid and accurate answer to food safety. Crit Rev Food Sci Nutr 56(9):1568–1584. https://doi.org/10.1080/10408398.2013.782483

    Article  CAS  PubMed  Google Scholar 

  16. Chapela MJ, Garrido-Maestu A, Cabado AG (2015) Detection of foodborne pathogens by qPCR: a practical approach for food industry applications. Cogent Food Agric 1(1):1013771. https://doi.org/10.1080/23311932.2015.1013771

    Article  CAS  Google Scholar 

  17. Hoshino T, Inagaki F (2012) Molecular quantification of environmental DNA using microfluidics and digital PCR. Syst Appl Microbiol 35(6):390–395. https://doi.org/10.1016/j.syapm.2012.06.006

    Article  CAS  PubMed  Google Scholar 

  18. Nixon G, Garson JA, Grant P, Nastouli E, Foy CA, Huggett JF (2014) Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. Anal Chem 86(9):4387–4394

    Article  CAS  Google Scholar 

  19. Rački N, Dreo T, Gutierrez-Aguirre I, Blejec A, Ravnikar M (2014) Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples. Plant methods 10(1):42

    Article  Google Scholar 

  20. 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. https://doi.org/10.1016/0168-1605(92)90017-W

    Article  CAS  PubMed  Google Scholar 

  21. Zhao Y, Xia Q, Yin Y, Wang Z (2016) Comparison of droplet digital PCR and quantitative PCR assays for quantitative detection of Xanthomonas citri. subsp citri. PLoS ONE 11:7. https://doi.org/10.1371/journal.pone.0159004

    Article  CAS  Google Scholar 

  22. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Kitano TK et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22):8604–8610. https://doi.org/10.1021/ac202028g

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Huggett JF, Cowen S, Foy CA (2015) Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 61(1):79–88. https://doi.org/10.1373/clinchem.2014.221366

    Article  CAS  PubMed  Google Scholar 

  24. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84(2):1003–1011. https://doi.org/10.1021/ac202578x

    Article  CAS  PubMed  Google Scholar 

  25. Bhunia AK (2014) One day to one hour: how quickly can foodborne pathogens be detected? Future Microbiol 9(8):935–946. https://doi.org/10.2217/fmb.14.61

    Article  CAS  PubMed  Google Scholar 

  26. Calvó L, Martínez-Planells A, Pardos-Bosch J, Garcia-Gil LJ (2008) A new real-time PCR assay for the specific detection of Salmonella spp. targeting the bipA gene. Food Anal Methods 1(4):236–242. https://doi.org/10.1007/s12161-007-9008-x

    Article  Google Scholar 

  27. Salehi TZ, Mahzounieh M, Saeedzadeh A (2005) Detection of invA gene in isolated Salmonella from broilers by PCR method. Int J Poult Sci 4(8):557–559. https://doi.org/10.3923/ijps.2005.557.559

    Article  Google Scholar 

  28. Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Pfaffl MW et al (2013) The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem 59(6):892–902. https://doi.org/10.1373/clinchem.2013.206375

    Article  CAS  PubMed  Google Scholar 

  29. Demeke T, Dobnik D (2018) Critical assessment of digital PCR for the detection and quantification of genetically modified organisms. Anal Bioanal Chem 410(17):4039–4050. https://doi.org/10.1007/s00216-018-1010-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63(10):3741

    Article  CAS  Google Scholar 

  31. Dreo T, Pirc M, Ramšak Ž, Pavšič J, Milavec M, Žel J, Gruden K (2014) Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot. Anal Bioanal Chem 406(26):6513–6528. https://doi.org/10.1007/s00216-014-8084-1

    Article  CAS  PubMed  Google Scholar 

  32. Pecoraro S, Berben G, Burns M, Corbisier P, De Giacomo M, De Loose M, Dagand E, Dobnik D, Eriksson R, Holst-Jensen A, Kagkli DM, Kreysa J, Lievens A, Mäde D, Mazzara M, Paternò A, Peterseil V, Savini C, Sovová T, Sowa S, Spilsberg B (2019) Overview and recommendations for the application of digital PCR. EUR 29673 EN. Publications Office of the European Union, Luxembourg. https://doi.org/10.2760/192883. ISBN 978-92-7600180-5

  33. Malorny B, Löfström C, Wagner M, Krämer N, Hoorfar J (2008) Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Appl Environ Microbiol 74(5):1299–1304. https://doi.org/10.1128/AEM.02489-07

    Article  CAS  Google Scholar 

  34. European Commission (2005) Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Off J Eur Union L338:1–26

    Google Scholar 

  35. Fachmann MSR, Löfström C, Hoorfar J, Hansen F, Christensen J, Mansdal S, Josefsen MH (2017) Detection of Salmonella enterica in meat in less than 5 hours by a low-cost and noncomplex sample preparation method. Appl Environ Microbiol 83(5):e03151–e3216. https://doi.org/10.1128/AEM.03151-16

    Article  PubMed  PubMed Central  Google Scholar 

  36. Sedlak RH, Jerome KR (2013) Viral diagnostics in the era of digital polymerase chain reaction. Diagn Microbiol Infect Dis 75(1):1–4. https://doi.org/10.1016/j.diagmicrobio.2012.10.009

    Article  CAS  PubMed  Google Scholar 

  37. Shanmugasamy M, Velayutham T, Rajeswar J (2011) Inv A gene specific PCR for detection of Salmonella from broilers. Veterinary World 4(12):562. https://doi.org/10.5455/vetworld.2011.562-564

    Article  Google Scholar 

  38. Cremonesi P, Cortimiglia C, Picozzi C, Minozzi G, Malvisi M, Luini M, Castiglioni B (2016) Development of a droplet digital polymerase chain reaction for rapid and simultaneous identification of common foodborne pathogens in soft cheese. Front Microbiol 7:1725. https://doi.org/10.3389/fmicb.2016.01725

    Article  PubMed  PubMed Central  Google Scholar 

  39. Porcellato D, Narvhus J, Skeie SB (2016) Detection and quantification of Bacillus cereus group in milk by droplet digital PCR. J Microbiol Methods 127:1–6. https://doi.org/10.1016/j.mimet.2016.05.012

    Article  CAS  PubMed  Google Scholar 

  40. Wang M, Yang J, Gai Z, Huo S, Zhu J, Li J, Zhang L et al (2018) Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk. Int J Food Microbiol 266:251–256. https://doi.org/10.1016/j.ijfoodmicro.2017.12.011

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Prof. Fikrettin Şahin from the Yeditepe University, Prof. Işın Akyar from the Acıbadem Labmed, and Prof. Gülay Özcengiz from the Middle East Technical Univeristy for providing strains used in this study.

Funding

This study was funded by The Scientific and Technological Research Council of Turkey (Grant no. 1120262).

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Authors and Affiliations

  1. Molecular Biology-Biotechnology and Genetics Research Center (MOBGAM) and Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey

    Yeliz Yücel Öz & Ayten Yazgan Karataş

  2. Department of Research and Development, AYA Inc., Istanbul, Turkey

    Öykü İrigül Sönmez & Can Bora Unal

  3. Department of Research and Development, Iontek Inc., Istanbul, Turkey

    Yeliz Yücel Öz & Sibel Karaman

  4. Department of Statistics, Yildiz Technical University, Istanbul, Turkey

    Ersoy Öz

  5. Department of Molecular Biology Genetic and Biotechnology, İstanbul Technical University, 34469, Maslak-Istanbul, Turkey

    Yeliz Yücel Öz

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  1. Yeliz Yücel Öz
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  2. Öykü İrigül Sönmez
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  3. Sibel Karaman
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  4. Ersoy Öz
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Contributions

All authors have contributed to the study conception and design. Material preparation, data collection, and analysis were performed by YYÖ, ÖİS, SK, EÖ, CBU, and AYK. The first draft of the manuscript was written by YYÖ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yeliz Yücel Öz.

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Öz, Y.Y., Sönmez, Ö.İ., Karaman, S. et al. Rapid and sensitive detection of Salmonella spp. in raw minced meat samples using droplet digital PCR. Eur Food Res Technol 246, 1895–1907 (2020). https://doi.org/10.1007/s00217-020-03531-x

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  • DOI: https://doi.org/10.1007/s00217-020-03531-x

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