Skip to main content
SpringerLink
Log in

Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples

  • Food Safety and Quality - Research Paper
  • Published:
Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290–1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290–1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Availability of data and material

The data and material are available.

Code availability

Not applicable.

References

  1. Allerberger F, Wagner M (2010) Listeriosis: A resurgent foodborne infection. Clin Microbiol Infect 16:16–23. https://doi.org/10.1111/j.1469-0691.2009.03109.x

    Article  CAS  PubMed  Google Scholar 

  2. Hamidiyan N, Salehi-Abargouei A, Rezaei Z et al (2018) The prevalence of Listeria spp. food contamination in Iran: A systematic review and meta-analysis. Food Res Int 107:437–450. https://doi.org/10.1016/j.foodres.2018.02.038

    Article  PubMed  Google Scholar 

  3. Farber J, Peterkin PI (1991) Listeria monocytogenes, a food-borne pathogen. Microbiol Rev 55:476–511. https://doi.org/10.1128/mr.55.3.476-511

  4. Jackson KA, Gould LH, Hunter JC et al (2018) Listeriosis Outbreaks Associated with Soft Cheeses, United States, 1998–2014. Centers Dis Control Prev 24:1116–1118

    Google Scholar 

  5. International Organization for Standardization (2017) Microbiology of food and animal feeding stuffs. Horizontal method for the detection and enumeration of Listeria monocytogenes and Listeria spp. Part 1. Detection method. ISO 11290–1:2017. International Organization for Standardization, Geneva

    Google Scholar 

  6. Law JWF, Ab Mutalib NS, Chan KG, Lee LH (2015) An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food. Front Microbiol 6:1–15. https://doi.org/10.3389/fmicb.2015.01227

    Article  Google Scholar 

  7. Liu D (2006) Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen. J Med Microbiol 55:645–659. https://doi.org/10.1099/jmm.0.46495-0

    Article  PubMed  Google Scholar 

  8. De Oliveira Souza SM, Brasileiro IS, De Carvalho AF et al (2012) Using nested PCR to detect the hlyA gene of Listeria monocytogenes in minas frescal cow’s milk cheese. J Food Prot 75:1324–1327. https://doi.org/10.4315/0362-028X.JFP-11-393

    Article  CAS  Google Scholar 

  9. Di PA, Forte VT, Guastadisegni MC et al (2007) A comparison of DNA extraction methods for food analysis. Food Control 18:76–80. https://doi.org/10.1016/j.foodcont.2005.08.011

    Article  CAS  Google Scholar 

  10. Heo EJ, Kim HY, Suh SH, Moon JS (2022) Comparison of DNA extraction methods for the quantification of Listeria monocytogenes in dairy products by Real-Time Quantitative PCR. J Food Prot 85:1531–1537. https://doi.org/10.4315/JFP-22-117

    Article  CAS  PubMed  Google Scholar 

  11. Norton D (2002) Polymerase chain reaction-based methods for detection of Listeria monocytogenes: toward real-time screening for food and environmental samples. J AOAC Int 85:505–515

    Article  CAS  PubMed  Google Scholar 

  12. Gianfranceschi MV, Rodriguez-Lazaro D, Hernandez M et al (2014) European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese. Int J Food Microbiol 184:128–133. https://doi.org/10.1016/j.ijfoodmicro.2013.12.021

    Article  CAS  PubMed  Google Scholar 

  13. O’Grady J, Sedano-Balbás S, Maher M et al (2008) Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target. Food Microbiol 25:75–84. https://doi.org/10.1016/j.fm.2007.07.007

    Article  CAS  PubMed  Google Scholar 

  14. Hyong SK, Kim DM, Neupane GP et al (2008) Comparison of conventional, nested, and real-time PCR assays for rapid and accurate detection of Vibrio vulnificus. J Clin Microbiol 46:2992–2998. https://doi.org/10.1128/JCM.00027-08

    Article  CAS  Google Scholar 

  15. Khanaliha K, Bokharaei-Salim F, Hedayatfar A et al (2021) Comparison of real-time PCR and nested PCR for toxoplasmosis diagnosis in toxoplasmic retinochoroiditis patients. BMC Infect Dis 21:1–7. https://doi.org/10.1186/s12879-021-06873-3

    Article  CAS  Google Scholar 

  16. Montoya A, Miró G, Blanco MA, Fuentes I (2010) Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats. Res Vet Sci 89:212–213. https://doi.org/10.1016/j.rvsc.2010.02.020

    Article  PubMed  Google Scholar 

  17. Sharifdini M, Mirhendi H, Ashrafi K et al (2015) Comparison of nested polymerase chain reaction and real-time polymerase chain reaction with parasitological methods for detection of Strongyloides stercoralis in human fecal samples. Am J Trop Med Hyg 93:1285–1291. https://doi.org/10.4269/ajtmh.15-0309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Longhi C, Maffeo A, Penta M et al (2003) Detection of Listeria monocytogenes in Italian-style soft cheeses. J Appl Microbiol 94:879–885. https://doi.org/10.1046/j.1365-2672.2003.01921.x

    Article  CAS  PubMed  Google Scholar 

  19. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. https://doi.org/10.1373/clinchem.2008.112797

    Article  CAS  PubMed  Google Scholar 

  20. Aladhadh M (2023) A review of modern methods for the detection of foodborne pathogens. Microorganisms 11(5):1111. https://doi.org/10.3390/microorganisms11051111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gupta P, Adhikari A (2022) Novel approaches to environmental monitoring and control of Listeria monocytogenes in food production facilities. Foods 11(12):1760. https://doi.org/10.3390/foods11121760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Labrador M, Giménez-Rota C, Rota C (2021) Real-time pcr method combined with a matrix lysis procedure for the quantification of Listeria monocytogenes in meat products. Foods 10(4):735. https://doi.org/10.3390/foods10040735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. D’Agostino M, Wagner M, Vazquez-Boland JA et al (2004) A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model - Towards an international standard. J Food Prot 67:1646–1655. https://doi.org/10.4315/0362-028X-67.8.1646

    Article  PubMed  Google Scholar 

  24. Malorny B, Tassios PT, Rådström P et al (2003) Standardization of diagnostic PCR for the detection of foodborne pathogens. Int J Food Microbiol 83:39–48. https://doi.org/10.1016/S0168-1605(02)00322-7

    Article  CAS  PubMed  Google Scholar 

  25. Cox T, Frazier C, Tuttle J et al (1998) Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5’ nuclease assay. J Ind Microbiol Biotechnol 21:167–174. https://doi.org/10.1038/sj.jim.2900578

    Article  CAS  Google Scholar 

  26. Rossmanith P, Krassnig M, Wagner M, Hein I (2006) Detection of Listeria monocytogenes in food using a combined enrichment/real-time PCR method targeting the prfA gene. Res Microbiol 157:763–771. https://doi.org/10.1016/j.resmic.2006.03.003

    Article  CAS  PubMed  Google Scholar 

  27. Chen Y, Ross WH, Scott VN, Gombas DE (2003) Listeria monocytogenes: Low levels equal low risk. J Food Prot 66:570–577. https://doi.org/10.4315/0362-028X-66.4.570

    Article  PubMed  Google Scholar 

  28. Roberts TA, Baird-Parker AC, Tompkin RB (1996) Listeria monocytogenes. In: Microorganisms in foods 5: characteristics of microbial pathogens. Blackie Academic and Professional, London, pp 141–182

  29. Abubakar I, Irvine L, Aldus CF, et al (2007) A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health Technol Assess (Rockv) 11. https://doi.org/10.3310/hta11360

  30. Wideman NE, Oliver JD, Crandall PG, Jarvis NA (2021) Detection and potential virulence of viable but non-culturable (VBNC) Listeria monocytogenes: A review. Microorganisms 9:1–11. https://doi.org/10.3390/microorganisms9010194

    Article  CAS  Google Scholar 

  31. Cordano AM, Rocourt J (2001) Occurrence of Listeria monocytogenes in Food in Chile. Int J Food Microbiol 70:175–178

    Article  CAS  PubMed  Google Scholar 

  32. Marzocca MA, Marucci PL, Sica MG, Alvarez EE (2004) Detección de Listeria monocytogenes en distintos productos alimenticios y en muestras ambientales de una amplia cadena de supermercados de la ciudad de Bahía Blanca (Argentina). Rev Argent Microbiol 36:179–181

    CAS  PubMed  Google Scholar 

  33. Gonzales Gutiérrez ML, Córdoba Ramos JS, Alberto G, Lozano Zanelly GA (2023) Prevalencia de Listeria monocytogenes asociados a factores de riesgo en quesos expendidos en el Valle del Mantaro – Junín. Cátedra Villarreal Posgrado Lima, Perú 2:2

    Google Scholar 

Download references

Funding

This project has received funding from National University of Rafaela (UNRAf), INTA, and Asociación Cooperadora INTA Rafaela (TCP #426100).

Author information

Author notes

  1. María V. Zbrun and Nadia Moreno contributed equally to this work.

Authors and Affiliations

  1. Instituto de Investigación de La Cadena Láctea (IdICaL) (INTA- CONICET), Ruta 34 Km 227, (2300), Rafaela, Santa Fe, Argentina

    María V. Zbrun, Cecilia M. Camussone & Marcelo L. Signorini

  2. Department of Public Health, Faculty of Veterinary Science, Litoral National University, Kreder 2805, (3080), Esperanza, Santa Fe, Argentina

    María V. Zbrun & Marcelo L. Signorini

  3. Faculty of Technology and Innovation for Development, Food Sciences Area, National University of Rafaela (UNRAf), Bv. Roca 989, (2300), Rafaela, Santa Fe, Argentina

    Nadia Moreno

  4. Instituto de Investigación de La Cadena Láctea (IdICaL) (INTA- CONICET), Ruta 34 Km 227, Faculty of Technology and Innovation for Development, Food Sciences Area, National University of Rafaela (UNRAf), Bv. Roca 989, (2300), Rafaela, Santa Fe, Argentina

    María E. Primo

Authors
  1. María V. Zbrun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nadia Moreno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cecilia M. Camussone
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcelo L. Signorini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. María E. Primo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MVZ participated in conceptualization, investigation, formal analysis, methodology, validation, visualization, writing – review & editing. NM and CC participated in conceptualization, methodology, writing – review & editing. MLS participated in conceptualization, writing – review & editing. MEP participated in conceptualization, project administration, supervision, investigation, formal analysis, methodology, validation, visualization, writing – original draft, writing – review & editing.

Corresponding author

Correspondence to María E. Primo.

Ethics declarations

Competing Interests

The authors have no conflicts of interest associated with this research work.

Ethics statement

This work does not contain studies with human participants or live animals, or hazardous or sensitive material.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Responsible Editor: Luis Augusto Nero

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 10830 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zbrun, M.V., Moreno, N., Camussone, C.M. et al. Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples. Braz J Microbiol (2024). https://doi.org/10.1007/s42770-024-01353-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s42770-024-01353-7

Keywords

Search

Navigation