Application of a quadruplex PCR for the simultaneous detection of fraud and the presence of Salmonella sp. and Listeria monocytogenes in commercial samples of Marajó Cheese
DOI:
https://doi.org/10.5433/1679-0359.2025v46n4p1039Keywords:
Food fraud, Hygienic-sanitary control, Microbiological contamination, Molecular methods, Pathogenic microorganisms.Abstract
Bubaline cheese from the Marajó Archipelago has gained prominence for its nutritional value and unique flavor. However, due to the seasonal scarcity of buffalo milk, cases of fraud with cow's milk have been reported. Additionally, the cheese's rich nutrient content and the potential lack of hygienic-sanitary controls in its production make it a favorable substrate for pathogenic microorganisms, such as Salmonella sp. and Listeria monocytogenes. The objective of this study was to develop a multiplex PCR for the simultaneous detection and differentiation of fraud by bovine milk, through identification of buffalo and bovine DNA, as well as the detection of Salmonella sp. and Listeria monocytogenes in commercial samples of Marajó Cheese, aiming to support its application as a routine tool in sanitary inspection. To conduct the experiment, 25 samples of buffalo cheese were collected at different time points. DNA was extracted from the samples and subjected to the proposed quadruplex PCR. Fraud was detected in 20% of the samples (5/25), and Salmonella in 8% (2/25), while Listeria monocytogenes was not detected. These findings confirm the presence of bovine milk in buffalo cheeses from the Marajó Archipelago and demonstrate that the developed quadruplex PCR may serve as an important tool for use by inspection agencies.
Downloads
References
Aabo, S., Rasmussen, O. F., Roseen, L., Sørensen, P. D., & Olsen, J. E. (1993). Salmonella identification by the polymerase chain reaction. Molecular and Cellular Probes, 7(3), 171-178. doi: 10.1006/mcpr.1993.1026
Abedini, A., Salimi, M., Mazaheri, Y., Sadighara, P., Sani, M. A., Assadpour, E., & Jafari, S. M. (2023). Assessment of cheese frauds, and relevant detection methods: a systematic review. Food Chemistry: X, 19, 100825. doi: 10.1016/j.fochx.2023.100825
Agostini, C., Kreling, C. S., Bustamante, I. C., Fº., Souza, C. F. V. de, Biolchi, V., & Pozzobon, A. (2012). Detecção de Listeria monocytogenes pela técnica da reação em cadeia da polimerase (PCR) em amostras de leite bovino in natura. Revista do Instituto de Laticínios Cândido Tostes, 67(389), 15-20. doi: 10.5935/2238-6416.20120073
Allaion, J. R., Barrionuevo, K. G., & Franco, B. D. G. de M. (2021). Assessing the microbiological safety parameters of minas artisanal cheese samples in retail environments in São Paulo, Brazil. Applied Sciences, 11(19), 9331. doi: 10.3390/app11199331
Azevedo, B. T., Vercesi, A. E., Fº., Gutmanis, G., Verissimo, C. J., Katiki, L. M., Okino, C. H., Sena Oliveira, M. C. de, & Giglioti, R. (2021). New sensitive methods for fraud detection in buffalo dairy products. International Dairy Journal, 117, 105013. doi: 10.1016/j.idairyj.2021.105013
Bittencourt, R. H. F. P. de M., Cortez, M. A. S., Mársico, E. T., Rosa, R. M. S. S., Taxi, C. M. A. D., Faturi, C., & Ermita, P. A. N. (2013). Caracterização de Requeijão Marajoara e Minas Frescal produzidos com leite de búfalas no Estado do Pará, Brasil. Ciência Rural, 43(9), 1687-1692. doi: 10.1590/S0103-84782013005000110
Border, P. M., Howard, J. J., Plastow, G. S., & Siggens, K. W. (1990). Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Letters in Applied Microbiology, 11(3), 158-162. doi: 10.1111/j.1472-765X.1990.tb00149.x
Cardoso, G. V. F., Oliveira, A. C. do S. de, Silva, A. S. da, Araújo, W. S. C., Dantas, V. V., Silva, J. B. da, Lima, J. S., Nunes, E. do S. C. de L., Guimarães, R. J. de P. S., & Roos, T. B. (2019). Detection of fraud by addition cow's milk in cheese buffalo and its connection with seasonality. Revista Brasileira de Ciência Veterinária, 26(4), 152-157. doi: 10.4322/rbcv.2019.027
Darwish, S. F.; Allam, H. A.; Amin, A. S. (2009). Evaluation of PCR assay for detection of cow's milk in water buffalo's milk. World Applied Sciences Journal, 7(4), 461-467. https://www.researchgate.net/publication/237796736_Evaluation_of_PCR_Assay_for_Detection_of_Cow's_Milk_in_Water_Buffalo's_Milk
Decree No. 10.468, of August 18, 2020. Amends Decree No. 9.013, of March 29, 2017, which regulates Law No. 1.283, of December 18, 1950, and Law No. 7.889, of November 23, 1989, on the regulation of industrial and sanitary inspection of products of animal origin. Diário Oficial da União, Brasília, 2020. https://www.planalto.gov.br/ccivil_03/_ato2019-2022/2020/decreto/d10468.htm
Figueiredo, E. L., Lourenço Júnior, J. de B. L., Toro, M. U., & Lima, S. C. G. de. (2011). Marajó cream-type cheese: physicochemical and sensory parameters. Revista do Instituto de Laticínios Cândido Tostes, 66(378), 26-33. Available at: https://www.revistadoilct.com.br/rilct/article/view/152
Gérard, A., Elâ€Hajjaji, S., Niyonzima, E., Daube, G., & Sindic, M. (2018). Prevalence and survival of Listeria monocytogenes in various types of cheese - a review. International Journal of Dairy Technology, 71(4), 825-843. doi: 10.1111/1471-0307.12552
Guzman-Hernandez, R., Contreras-Rodriguez, A., Hernandez-Velez, R., Perez-Martinez, I., Lopez-Merino, A., Zaidi, M. B., & Estrada-Garcia, T. (2016). Mexican unpasteurised fresh cheeses are contaminated with Salmonella spp., non-O157 Shiga toxin producing Escherichia coli and potential uropathogenic E. coli strains: a public health risk. International Journal of Food Microbiology, 237, 10-16. doi: 10.1016/j.ijfoodmicro.2016.08.018 105013
Instrução Normativa No. 60, of December 23, 2019. Establishes microbiological standards for food. Ministério da Agricultura, Pecuária e Abastecimento. Diário Oficial da União, Brasília, DF, 2019. https://cvs.saude.sp.gov.br/zip/U_IN-MS-ANVISA-60_231219.pdf
Kamimura, B. A., Magnani, M., Luciano, W. A., Campagnollo, F. B., Pimentel, T. C., Alvarenga, V. O., Pelegrino, B. O., Cruz, A. G., & Sant'Ana, A. S. (2019). Brazilian artisanal cheeses: an overview of their characteristics, main types and regulatory aspects. Comprehensive Reviews in Food Science and Food Safety, 18(5), 1636-1657. doi: 10.1111/1541-4337.12486
Li, B., Yu, M., Xu, W., Chen, L., & Han, J. (2023). Comparison of PCR techniques in adulteration identification of dairy products. Agriculture, 13(7), 1450. doi: 10.3390/agriculture13071450
Lima, J. S., Sampaio, A., Dufossé, M. C. S., Rosa, A., Sousa, P. F. M., Silva, J. B., Cardoso, G. V. F., Moraes, C. M., & Roos, T. B. (2021). Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 73, 781-790. doi: 10.1590/1678-4162-12218 105013
López-Calleja, I., Alonso, I. G., Fajardo, V., Rodríguez, M. A., Hernández, P. E., García, T., & Martín, R. (2005). PCR detection of cows' milk in water buffalo milk and mozzarella cheese. International Dairy Journal, 15(11), 1122-1129. doi: 10.1016/j.idairyj.2004.12.003
Maia, D. S. V., Haubert, L., Würfel, S. de F. R., Kroning, I. S., Cardoso, M. R. de I., Lopes, G. V., Fiorentini, Â. M., & Silva, W. P. da. (2019). Listeria monocytogenes in sliced cheese and ham from retail markets in southern Brazil. FEMS Microbiology Letters, 366(22), fnz249. doi: 10.1093/femsle/fnz249
Ministério da Agricultura, Pecuária e Abastecimento (2017). Regulamento de Inspeção Industrial e Sanitária de Produtos de Origem Animal - RIISPOA (Approved by Decree No. 9.013). Diário Oficial da República Federativa do Brasil. https://www.gov.br/agricultura/pt-br/assuntos/mpa/legislacao/legislacao-geral-da-pesca/decreto-no-9-013-de-29-03-2017.pdf/view
Montgomery, H., Haughey, S. A., & Elliott, C. T. (2020). Recent food safety and fraud issues within the dairy supply chain (2015-2019). Global Food Security, 26, 100447. doi: 10.1016/j.gfs.2020.100447 105013
Mudadu, A. G., Piras, G., Melillo, R., Salza, S., Cau, S., Virgilio, S., Meloni, D., & Mele, P. (2022). Survival of naturally contaminating Listeria monocytogenes in commercial mediterranean-style dry fermented sausages during Storage. Journal of Food Protection, 85(11), 1576-1583. doi: 10.4315/JFP-22-023
Nesterova, E., Morozova, P., Gladkikh, M., Kazemzadeh, S., & Syromyatnikov, M. (2024). Molecular methods for detecting microorganisms in beverages. Beverages, 10(2), 46. doi: 10.3390/beverages10020046
Portaria No. 0418/2013 - ADEPARÁ. (2013). Technical regulation for the production of Queijo do Marajó and other provisions. Agência de Defesa Agropecuária do Estado do Pará (ADEPARÁ). Available at: https://www.legisweb.com.br/legislacao/?id=252036
Silva, C. L., Sales, G. A., Santos Neto, J. G., Silva, J. da, Lima, S. C. G. de, Leite, F. P. L., Lima, E. do S. C. de, Moraes, C. C. G. de, Roos, T. B., & Moraes, C. M. de. (2015). Detecção de fraude em amostras comerciais de queijo bubalino por adição de leite bovino por meio da técnica de Reação em Cadeia da Polimerase (PCR) multiplex. Revista do Instituto Adolfo Lutz, 74(1), 21-29. https://docs.bvsalud.org/biblioref/ses-sp/2015/ses-32090/ses-32090-5948.pdf
Silva, J. A. R., Garcia, A. R., Almeida, A. M. de, Bezerra, A. S., & Brito Lourenço, J. de., Jr. (2021). Water buffalo production in the Brazilian Amazon Basin: a review. Tropical Animal Health and Production, 53(3), 343. doi: 10.1007/s11250-021-02744-w
Visciano, P., & Schirone, M. (2021). Food frauds: global incidents and misleading situations. Trends in Food Science & Technology, 114, 424-442. doi: 10.1016/j.tifs.2021.06.010
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Adrianne Maria Brito Pinheiro da Rosa, Matheus Martins Ramos, Ana Paula Presley Oliveira Sampaio, Mylla Christy da Silva Dufossé, Joelson Sousa Lima, Talita Bandeira Roos, Carina Martins de Moraes
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Semina: Ciências Agrárias adopts the CC-BY-NC license for its publications, the copyright being held by the author, in cases of republication we recommend that authors indicate first publication in this journal.
This license allows you to copy and redistribute the material in any medium or format, remix, transform and develop the material, as long as it is not for commercial purposes. And due credit must be given to the creator.
The opinions expressed by the authors of the articles are their sole responsibility.
The magazine reserves the right to make normative, orthographic and grammatical changes to the originals in order to maintain the cultured standard of the language and the credibility of the vehicle. However, it will respect the writing style of the authors. Changes, corrections or suggestions of a conceptual nature will be sent to the authors when necessary.
