Establishment of PMA Real-Time PCR Method to Detect Viable Cells of Listeria monocytogenes and Salmonella spp. in Milk and Dairy Products


  • Ho Chi Minh City Open University, Faculty of Biotechnology, Thanh Pho Ho Chi Minh, 700000, Viet Nam


The infection with Salmonella spp., Listeria monocytogenes are the most contamination events in the milk and dairy products. There are many disadvantages of conventional culture-based methods, which are still recognized as the “gold standard†for identifying pathogenic bacteria. Thus, the aim of current study was to develop reliable and rapid method for the detection of bacteria causing foodborne. For the establishment of PMA Real-time PCR method for co-detection of Salmonella spp., Listeria monocytogenes, bacteria strains, including Escherichia coli (ATCC 25922), Listeria monocytogenes (ATCC 19115; ATCC 19111), Salmonella enterica (ATCC 19115), Staphylococcus aureus (ATCC 25923), Vibrio parahaemolyticus (ATCC 17802), Shigella flexneri (ATCC 12022), Bacillus aureus (ATCC 11778) were enrolled into the establishment of current protocol. The concentration of PMA, primer concentration, probe concentration, and primer annealing temperature, specificity and sensitivity were evaluated. As the results, we successfully established the protocol of PMA Real-time PCR for co-detection of Salmonella spp. and Listeria monocytogenes on milk product. The concentration of PMA was determined as 50 μM. The sensitivity of established protocol were 101 CFU/ml for detection of Salmonella spp., and 102 CFU/ml for detection of Listeria monocytogenes. The current PMA Real-time PCR protocol was applied to detect the contamination of twenty local milk samples. No sample was co-contaminated with Salmonella spp. and Listeria monocytogenes. These results were similar to its performed by conventional culture-based methods. In summary, the current established PMA (50 μM) Real-time PCR could be applied for the co-detection of Salmonella spp. and Listeria monocytogenes on milk and dairy food.


Dairy Products, Listeria monocytogenes, PMA Real-time PCR, Salmonella spp.

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Aarestrup FM, Hendriksen RS, Lockett J, Gay K, Teates K, McDermott PF, White DG, Hasman H, Sørensen G, Bangtrakulnonth A, Pornreongwong S, Pulsrikarn C, Angulo FJ, Gerner-Smidt P. International spread of multidrug-resistant Salmonella Schwarzengrund in food products. Emerg Infect Dis. 2007; 13(5):726-731. PMid:17553251 PMCid:PMC2738437.

Cangelosi GA, Meschke JS. Dead or alive: Molecular assessment of microbial viability. Appl Environ Microbiol. 2014; 80(19):5884-5891. PMid:25038100 PMCid:PMC4178667.

Elizaquível P, Sánchez G, Aznar R. Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidiummonoazide and real-time PCR. Food Control. 2012; 25(2):704-708.

Law JW, Ab Mutalib NS, Chan KG, Lee LH. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Front Microbiol. 2015; 5:770. PMid:25628612 PMCid:PMC4290631.

Liang N, Dong J, Luo L, Li Y. Detection of viable Salmonella in lettuce by propidiummonoazide real-time PCR. J Food Sci. 2011; 76(4):M234-M237. PMid:22417362.

Liu Y, Cao Y, Wang T, Dong Q, Li J, Niu C. Detection of 12 common food-borne bacterial pathogens by TaqMan real-time PCR using a single set of reaction conditions. Front Microbiol. 2019; 10:222. PMid:30814987 PMCid:PMC6381072.

Ma Y, Deng Y, Xu Z, Liu J, Dong J, Yin H, Yu J, Chang Z, Wang D. Development of a propidiummonoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer. Braz J Microbiol. 2017; 48(4):740-746. PMid:28633981 PMCid:PMC5628306.

Nocker A, Camper AK. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol. 2006; 72(3):1997-2004. PMid:16517648 PMCid:PMC1393219.

Seinige D, Krischek C, Klein G, Kehrenberg C. Comparative analysis and limitations of ethidium monoazide and propidiummonoazide treatments for the differentiation of viable and nonviable campylobacter cells. Appl Environ Microbiol. 2014; 80(7):2186-2192. AEM.03962-13. PMid:24487529 PMCid:PMC3993131.

Shamloo E, Hosseini H, Abdi Moghadam Z, Halberg Larsen M, Haslberger A, Alebouyeh M. Importance of Listeria monocytogenes in food safety: A review of its prevalence, detection, and antibiotic resistance. Iran J Vet Res. 2019; 20(4):241-254.

Wiemer D, Loderstaedt U, von Wulffen H, Priesnitz S, Fischer M, Tannich E, Hagen RM. Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples. Int J Med Microbiol. 2011; 301(7):577-584. PMid:21855409.

Yang Y, Wan C, Xu H, Lai W, Xiong Y, Xu F, You X, Xu H, Aguilar ZP, Sun J, Wei H. Development of a multiplexed PCR assay combined with propidiummonoazide treatment for rapid and accurate detection and identification of three viable Salmonella entericaserovars. Food Control. 2012; 28(2):456-462.


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