PCR Techniques as An Alternative for Rapid Detection of Listeria Monocytogenes in Cheese

Milk industry has a global market projected to reach more than 1.2 billion USD by 2028 [1]. In Mexico, more than 13 billion liters of milk were produced with a value greater than 112 billion Mexican pesos in 2022, highlighting the states of Coahuila, Chihuahua, Durango, Guanajuato, Jalisco and Veracruz as principal milk producers [2]. Non-fermented or aged dairy products, such as fluid milk and fresh cheeses (e.g. Panela, Ranchero and Oaxaca), provide good conditions for the growth of microorganisms (low acidity, high moisture, low salt content), including Listeria monocytogenes [3]. L. monocytogenes causes a disease known as listeriosis; a disease transmitted to humans by food consumption often associated with the consumption of fresh cheeses [4]. In Mexico, L. monocytogenes has been detected in raw milk (at farms), and manufactured cheeses made either with pasteurized milk (commercial) or with raw milk (artisanal), including Fresh, Ranchero, Chihuahua, Adobera and Panela cheeses [3]. Several reports of detection of Listeria monocytogenes in cheese using PCR techniques have been carried out and many reviews in molecular methods to detect this pathogen in dairy products. This paper aimed to provide a narrative minireview of PCR techniques to detect L. monocytogenes in cheese, we will focus on giving an overview of the potential of PCR molecular methods to be implemented in protocols of detection of this pathogen in fresh cheeses in Mexico.

Listeria monocytogenes detection methods for fresh cheese in Mexico

Rapid and accurate detection of L. monocytogenes in raw materials (raw milk), processing areas and finished product, is important for Mexican diary producers to ensure the quality and safety of fresh cheeses and dairy products [5]. In Mexico, the official standard NOM-243- SSA1-2010 establishes zero tolerance for L. monocytogenes (absence in 25g or mL) in milk and dairy products, such as fresh cheeses. The standard also establishes the test method for detection of Listeria in foods, mainly based on traditional culture methods, which involve enrichment steps (2 days), plating in selective culture media (2 days of incubation) and finally confirmatory and identification tests (1-5 days), which are based on morphological and biochemical characteristics of this pathogenic bacteria [6]. Although the enrichment culture method is a standard in the food industry in Mexico, due to lower costs, results obtention require around 5 to 10 days, causing it to be not efficient enough for the dairy industry to make quick decisions.

Molecular detection methods

Implementation of molecular techniques based by in the Polymerase Chain Reaction (PCR) is an alternative to rutinary analysis in cheeses and general food, which can reduce the overall detection time of L. monocytogenes (e.g. enrichment about 18h, and detection 4-6h), even when is present in small quantities in cheese, obtaining results (detection, characterization and confirmation of the microbial species) in approximately 1-2 days [7,8], compared to 5-10 days of traditional culture methods [6], allowing Mexican dairy producers to improve production processes and routine practices and the ability for short-term decision making.

In general, molecular methods for the detection of L. monocytogenes, such as PCR and biosensors, are mainly based on the detection of nucleic acids such as DNA or RNA, as well as the detection of surface antigens, to identify at the species level, as well as serovar [9-12]. Among the PCR-based molecular methods that have been used for the detection of Listeria monocytogenes in cheeses and other foods, there are endpoint PCR, real-time PCR; Droplet digital PCR that detects different pathogenicity genes of the microorganism (PrFA, PRS, Hyla, etc.) Some examples of these methods are briefly described in Table 1; [13-20]. These molecular methods based in PCR allow a rapid detection and they do not present limitations to be used in samples of foods with high fat content such as cheeses [21], but specialized equipment is required (e.g. thermocycler), which increases the initial cost for the implementation of these methods, however the precision, specificity, sensitivity and response time in the analysis not only of Listeria spp. but other pathogenic or food spoilage microorganisms, are the alternative trend for microorganisms detection in the short term.

Table 1:Detection of Listeria monocytogenes with molecular methods based by PCR.

Related to price of the analysis, traditional culture methods, as established by the official Mexican standard NOM-243-SSA1-2010 and depending on the analysis laboratory, have an average price per sample of $800.00 Mexican pesos [22], and using molecular methods based on PCR, already established in a specialized analysis laboratory, this price can be reduced by up to 60% [23]. Therefore, they can be an attractive alternative due to lower costs and time involved for cheese and food producers in general, who send their samples to be analyzed in specialized external laboratories. Nevertheless, there are few laboratories in Mexico that currently perform this type of analysis for the detection of L. monocytogenes, and other pathogenic microorganisms, by molecular methods, due to initial investment in the equipment and trained personnel required in this area. But the implementation of these molecular techniques is common at research settings such as University graduate projects, allowing technology transfer to large analysis laboratories and eventually becoming more available to support food producers.

Some molecular methods using real-time PCR are currently valid commercial alternatives for the detection of L. monocytogenes in foods within the legislation of countries such as the United States [24] and Canada [25]: BAX® Automated System Assurance, GDS® Listeria spp. Tq Genetic Detection System and iQ-Check Listeria monocytogenes PCR Detection Kit. In particular, BAX® Automated System method is used to detect L. monocytogenes in food, and it was validated by AOAC International (official method AOAC 2003.12), however molecular methods based by PCR are not part of the official methods to detect L. monocytogenes in food, including dairy products in Mexico [6]. Therefore, cheese and other dairy producers in Mexico still cannot resort to non-traditional methods for routine quality and safety control analysis that they must comply with by standard.

Detection of L. monocytogenes by molecular methods arises as a need for companies producing fresh cheeses, since they need to know the safety of their cheeses produced, as well as validate the cleaning and disinfection processes within the plant and be able to make decisions about corrective actions, such as the frequency of cleaning and disinfection of the plant, replacement of equipment that does not have a sanitary design, the use of food additives to inhibit L. monocytogenes in the finished cheese, among others. The establishment of protocols for the rapid detection of L. monocytogenes in fresh cheeses would allow producing companies in Mexico to obtain data faster for making decisions more effectively, at convenient costs and times, directly impacting the improvement of safety and a lower risk of infection by L. monocytogenes for consumers of fresh cheeses in Mexico.

1. (20204) Dairy market value worldwide. Statista 2020-2028.
2. (2023) Agri-Food and Fisheries Information Service (SIAP). Statistical yearbook of livestock production
3. Ibarra SLA, Van Tassell ML, Miller MJ (2017) Invited review: Hispanic-style cheeses and their association with listeria monocytogenes. Journal of Dairy Science 100(4): 2421-2432.
4. Ribeiro AC, Almeida Fad, Medeiros MM, Miranda BR, Pinto UM, et al. (2023) Listeria monocytogenes: An inconvenient hurdle for the dairy industry. Dairy 4(2): 316-344.
5. Rios Muñiz D, Cerna CJF, Lopez SC, Angeles-ME, Bobadilla del VM, et al. (2019) Longitudinal analysis of the microbiological quality of raw cow’s milk samples collected from three small family dairy farms in Mexico over a 2-year period. J Food Prot 82(12): 2194-2200.
6. (2010) Official Mexican Standard NOM-243-SSA1-2010, Products and services. Milk, milk formula, combined milk product and dairy products. Sanitary provisions and specifications. Test Methods.
7. Jin QC, Stephanie H, Patrick R, Pongpan L, Zonglin Hu (2017) PCR-based methodologies for detection and characterization of Listeria monocytogenes and Listeria ivanovii in foods and environmental sources. Food Science and Human Wellness 6(2): 39-59.
8. Garrido MA, Azinheiro S, Carvalho J, Prado M (2018) Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR. Food Microbiol 73: 254-263.
9. Law JW, 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.
10. Ryu J, Park SH, Yeom YS, Shrivastav A, Lee SH, et al. (2013) Simultaneous detection of listeria species isolated from meat processed foods using multiplex PCR. Food Cont 32(2): 659-664.
11. Fernández BA, Hernández PM, Moreno TY, García HJ (2023) Development of optical label-free biosensor method in detection of listeria monocytogenes from food. Sensors 23(12): 5570.
12. Gasanov U, Hughes D, Hansbro PM (2005) Methods for the isolation and identification of listeria spp. and Listeria monocytogenes: A review. FEMS Microbiology Reviews 29(5): 851-875.
13. Wernars K, Heuvelman CJ, Chakraborty T, Notermans SHW (1991) Use of the polymerase chain reaction for direct detection of listeria monocytogenes in soft cheese. Journal of Applied Microbiology 70(2): 121-126.
14. Gianfranceschi MV, Rodriguez LD, Hernandez M, González GP, Comin D, et al. (2014) European validation of a real-time PCR-based method for detection of listeria monocytogenes in soft cheese. International Journal of Food Microbiology 184: 128-33.
15. Heo EJ, Song BR, Park HJ, Kim YJ, Moon JS, et al. (2014) Rapid detection of Listeria monocytogenes by real-time PCR in processed meat and dairy products. J Food Prot 77(3): 453-458.
16. Zbrun MV, Moreno N, Camussone CM (2024) 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 55(2): 1783-1791.
17. Quero GM, Santovito E, Visconti A, Fusco V (2014) Quantitative detection of listeria monocytogenes in raw milk and soft cheeses: Culture-independent versus liquid-and solid-based culture-dependent real time PCR approaches. LWT-Food Science and Technology 58(1): 11-20.
18. Cremonesi P, Cortimiglia C, Picozzi C, Minozzi G, Malvisi M, et al. (2016) Development of a droplet digital polymerase chain reaction for rapid and simultaneous identification of common foodborne pathogens in soft cheese. Frontiers in Microbiol 7: 1725.
19. Garrido A, Chapela MJ, Román B, Fajardo P, Lago J, et al. (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.
20. Tirloni E, Bernardi C, Drago S, Stampone G, Pomilio F, et al. (2017) Evaluation of a loop-mediated isothermal amplification method for the detection of listeria monocytogenes in dairy food. Ital J Food Saf 6(4): 6890.
21. Chen JQ, Healey S, Regan P, Laksanalamai P, Hu Z, et al. (2017) PCR-based methodologies for detection and characterization of Listeria monocytogenes and Listeria ivanovii in foods and environmental sources. Food Science and Human Wellness 6(2): 39-59.
22. (2024) Catalogue of microbial analysis in food. Universidad Autónoma de Querétaro Mexico
23. (2024) Center for Research and Assistance in Technology and Design of the State of Jalisco (CIATEJ) Laboratory Services: Molecular Methods.
24. Hitchins AD, Jinneman K, Chen Y (2022) BAM chapter 10: Detection of Listeria monocytogenes in foods and environmental samples and enumeration of listeria monocytogenes in foods. Food & Drug.
25. (2024) Health Products and Food Branch (HPFB). Laboratory procedures for microbiological analysis of foods. Canada.