Detection of Some Foodborne Pathogens In Some Food Samples Using Species - Specific Polymerase Chain Reaction Technique 

Sakar kamal Hamasaleh1 , Khulod Ibraheem Hassan1

 1 Food Sciences department, College of Agricultural sciences, Sulaimani University, Sulaimani, Kurdistan Region, Iraq

Original: 27/11/2017, Revised: 07/01/2018, Accepted: 06/02/2018, Published online:

DOI Link: https://doi.org/10.17656/jzs.10672


Polymerase Chain Reaction (PCR) techniques was  applied as a rapid method for detection of some food-borne pathogens directly without culturing, to detect three species of bacteria, including: Shigella flexneri, Escherichia coli 0157:H7 and Listeria monocytogenese by targeting specific genes for each pathogen from cultures of various types of artificially inoculated foods, which were spiked with reference bacteria at known concentrations. DNA was isolated from each food sample using phenol -chloroform based method.Positive results were obtained which produced specific amplicons of the expected sizes of each of them (691 bp in L. monocytogenese, 556 bp in E.coli o157:H7 and 600 bp in S. flexneri). The detection limit of the assay were 103 CFU/ ml For L. monocytogenese, and 104 CFU/ ml for both S. flexneri and E.coli o157:H7. overall results of this study indicate that PCR is a good tool for rapid screening of the three pathogens in food and this application can be followed for detection of any pathogenic bacteria transmitted through food.

  Key Words: PCR, food,  Shigella flexner,  Escherichia coli 0157, Listeria monocytogenese


[1] Jason,V.;  Jacxsens, L.; Luning , P.;  Rajkovic,  A., and Uyttendaele, M. " Alternative microbial methods: An overview and selection criteria", Food Microbiol.,  Vol. 27, pp. 710–730. (2010).       

[2] Zhao X.; Lin C.; Wang  W. and OhD, J. "Advances in rapid detection methods for foodborne pathogens", J. Microbiol. Biotechn., Vol. 24, pp.297–312.(2014). 

[3] Oliver, J. D. "The viable but nonculturable state and cellular resuscitation" , Atlantic Canada Society for Microbial Ecology, Halifax, Canada, pp. 723-730. (2000).

[4]  Shabir, B.;  Bell, D.; Bossuyt, P.;  Herring, A.;  Mabey, D.;  Poole6, F.; Smith, P. G.; Sriram, N.; Wongsrichanalai, C.;  Linke, R.;  O'Brien, R.;  Perkins, M.; Cunningham, J.; Matsoso, P.; Nathanson, C.M.; Olliaro, P.; Peeling R. W. and Ramsa,  A. "Detection of food born disease in food", J. Diarrheal Dis., Vol.35, pp.15-40. (2014).

[5] Germini, A., Masola, A.;  Carnevali,  P. and Marchelli, R. "Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR", Food quality Control, Vol. 20, pp.733-738. (2009).

[6]  Wong, W.C.; Pui, C.F.;  Chai,  L.C.; Lee, H.Y.; Ghazali, F.M.; Tang,  J.Y.H.; Ponniah, J.; Tuan, T.C.; Zainazor, Y.K. and Son, R. " Biosafety assessment of Listeria monocytogenes in vegetarian burger patties in Malaysia ", International J. Food Res.,  Vol.18, pp.459-463. (2011).

[7] AOAC International "Performance Tested Methods sm Validated Methods. http://www.aoac.org/testkits/testedmethods.html. Last updated: October 2011. Accessed 18 November 2011.

[8]  Fernandez-Prada C.M.; Hoover, D.L.; Tall D.;  Hartman, A. B.; Kopelowitz,  J. and Venkatesan, M.M. "Shigella flexneri IpaH(7.8) facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages" , Infect. Immun., Vol.6, pp.3608-19. (2000).

[9]  Jiménez, K.; McCoy, B.C.B. and Achí, R. "Detection of Shigella in lettuce by the use of a rapid molecular assay with increased sensitivity", Braz. J. Microbiol.,  Vol.41, No.4, pp.993-1000. (2010).

[10]  World Health Organization (WHO) "Guidelines for the control of Shigellosis, including epidemics due to Shigella dysenteriae type 1", (2005). Available at: http://www.who.int/vaccine_research/documents/Guidelines Shigellosis.pdf>. Accessed 24 May 2007.

[11] Kuo, CY., Su,  L.H.; Perera, J.;  Carlos, C.;  Tan, B.H.G.; Kumarasinghe,  T.;  Van, P.H.; Chongthaleong, A.; Song, J.H. and Chiu, C. H. "Antimicrobial susceptibility of Shigella isolates in eight Asian countries", J. Microbiol. Immunol. Infect., Vol. 41, pp.107-111. (2008).

[12]  Barbora, V.; Eva,  T.; Ľuboslav,  B.;  Viera,  H.  and  Andrej, G. "Multiplex PCR for detection of Escherichia coli O157:H7 in foods Find out how to access preview-only content", Close Biologia.,  Vol.66, pp.401-40. (2011).

[13]  Jeshveen, S.S.; Chai, L.C.; Pui, C.F. and Son, R. "Optimization of multiplex PCR conditions for rapid detection of Escherichia coli O157:H7 virulence genes", J. Int. Food Res., Vol.19, No.2, pp. 461-466. (2012).

[14] Iijima,Y.;  Matsumoto,  M.; Higuchi,  K.; Furuta, T. and Honda,  T. "Resistance to dryness of Escherichia coli O157:H7 strains from Outbreak in Saki City", Japan, Emerging Infectious Dis., Vol.4, pp. 340-341. (1998). 

[15] Meng, J., Doyle, M. P.;  Zhao, T. and  Zhao, S. "Enterohemorrhagic Escherichia coli" In Food microbiol: fundamentals and frontiers. American Society of Microbiology Press, Washington, DC. (2001)

[16]  Callaway, T. R., Edrington, T. S.; Brabban, A. D.; Keen, J. E.; Anderson, R. C.;  Rossman, M. L.; Engler, M. J.; Genovese, K. J.; Gwartney, B. L.;  Reagan, J. O.;  Poole, T. L.,  Harvey, R. B.;  Kutter, E. M. and Nisbet, D. J. "Fecalprevalence of  Escherichia coli O157, Salmonella, Listeria, and bacteriophage infecting E. coli O157:H7 in feed lot cattle in the southern plains region of the United States", Foodborne Pathog. Dis., Vol.3, pp.234-244. (2006).

[17]  Reinstein, S.; Fox, J. T.;  Shi, X. and Nagaraja, T.G. " Prevalence of Escherichia coli O157:H7 in gallbladders of beef cattle", Appl and Environ Microbiol., Vol.73, pp.1002-1004. (2007).

[18]  Almeida, P. F. and Almeida, R. C. C. "A PCR protocol using inl gene as a target for specific detection of Listeria monocytogenes" Food Control,  pp. 97-101. (2000).

[19] Gouws P.A. and Liedemanne, I. "Valuation of Diagnostic PCR for the Detection of Listeriamonocytogenes in Food Products", Food Biotechnol. Vol.43, pp.201–205. (2005).

[20]  Doganay, M. " Listeriosis: clinical presentation.FEMS Immunol", Med. Microbiol. Vol.35, pp.173–175. (2003).

[21] Jacquet, C.;  Doumith, M.;  Gordon, J.I.;  Martin, P.M.V.; Cossart, P. and Lecuit, M. " A molecular marker for evaluating the pathogenic potential of foodborne Listeria monocytogenese", J. Infect. Dis. Vol.189, pp.2094-2100. (2004)

[22] Doumith, M.; Buc hrieser, C.;  Glaser, P.; Jacquet, C. and Martin, P. "Differentiation of the major Listeria monocytogenes serovars by multiplex PCR", J. Clin. Microbiol., Vol.42, pp.3819–3822. (2004).

[23]  Akpolat,  N.O.;  Elci, S.; Atmaca, S. and Gül, K. "Listeria monocytogenes in Products of Animal Origin in Turkey", Vet.Res.Commun., Vol.7, pp.561-567. (2004).

[24] Wang, R.F.; Cao, W.W. and Cerniglia, C.E. "Auniversal protocol for PCR detection of 13 species of food borne pathogens in foods", J. Appl. Microbiol., Vol.84, pp.727-736. (1997).

[25] Huang, Q.; Hu, Q. and Li, Q. "Identification of 8 Foodborne Pathogens by Multicolor Combinational Probe Coding Technology in a Single Real-Time PCR", Clinical Chemistry, Vol.53, pp.1741–1748. (2007).

[26] Sambrook, J. and  Russel, D.W. " Molecular cloning: A laboratory manual" (3rd ed.). Cold Spring Harbor, NY, USA: Cold Spring Harbor Press. (2001).

[27] Lampel, K.; Orlandi P. A. and Kornegay, L. "Improved template preparations for PCR-based assays for detection of food- -borne bacterial pathogens", Appl. Environ. Microbio., Vol. 6, pp. 4539 – 4542. (2000).

[28] Anonymous. "In Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp." (ISO 6579:2002) Geneva, Switzerland", ISO (pp. 1–28). (2002).

[29] Kalia,  A.; Wdhesh, K.; Ashok R. and Prem, C. A. "Method for Extraction of High-Quality and High-Quantity Genomic DNA Generally Applicable to Pathogenic Bacteria" Anal. Biochem., Vol.275, pp.1–5. (1999).

[30] Atashpa, S.;  Khani, S.;  Barzegari, A.;  Barar, J.;  Vahed , SZ.;  Azarbaijani,  R. and Omidi, Y. " A robust universal method for extraction of genomic DNA from bacterial species", Mikrobiologiia,  Vol.4, pp. 62-566. (2010).

[31]. Yoshimura, K.; Toibana,  A.  and  Nakahama, k. "Human lysozyme: sequencing of a DNA, and expression and secretion by Saccharomyces cerevisiae", Biochem. Biophys. Res. Commun., Vol.150, No.2, pp. 794–801. (1988).

[32] Oliveira, S.D.;  Santos, L.R.; Schuch, D.M.; Silva, A.B.;  Salle, C.T. and  Canal, C.W. "Detection and identification of salmonella from poultry-related samples by PCR", Vet. Microbiol.,  Vol.87, pp. 25-35. (2002).

[33] Nucera, D.M.; Maddox , C.W.; Hoien-Dalen, P. and  Weigel, R.M. "Comparison of API 20E and invA PCR for Identification of Salmonella enterica Isolates from Swine Production Units",  J. Clin. Microbiol Vol.44, pp. 3388-3390. (2006).

[34] Thong, K.L.; Hoe, S. L. L.; Puthucheary,  S. D. and Yasin, R. M. "Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay", BMC Infec. Dis.,  Vol. 5, pp.1–7. (2005).

[35] Silva, T.; Nogueira, P.A.; Magalhães, G.F.; Grava, A.F.; Silva,  L. H. P. and Orlandi, P.P. " Characterization of Shigella spp. byantimicrobial resistance and PCR detection of ipa genes in an infantile population from Porto Velho", (Western Amazon region), Braz Mem. Inst. Oswaldo Cruz., Vol.103, No.7, pp. 731-733. (2008).

[36] Li, X.;  Zhang , S.H.; Zhang, L.; Tao, H.; Yu, J.; Zheng, W.; Liu, C.; Lü, D.;  Xiang, R.; Liu, Y.   "A loop-mediated isothermal amplification method targets the phoP gene for the detection of Salmonella in food samples", Int. J. Food Microbiol.,  Vol.13, pp. 252-258. (2009).

[37] Haraga, A. and Miller, S.I. "A Salmonella enterica serovar typhimurium translocated leucine-rich repeat effector protein inhibits NF-kappa B-dependant gene expression", Infect. Immun., Vol.71, pp.4052-4058. (2003).

[38] Toyotome, T.;  Suzuki, T.; Kuwae, A.; Nonaka, T.; Fukuda, H. and Majoh, O." Shigella protein IpaH 9.8 is secreted from bacteria within mammalian cells and transported to the nucleus" J. Biol. Chem., Vol. 276, pp. 32071–32079. (2001).

[39] Heijnen, L. and Medema, G. "Quantitative detection Of  E. coli, E. coli O157 and other Shiga toxin producing E. coli in water samples using a culture method combined with real-time PCR", J. Water  Health, Vol.4, pp. 487-498. (2006).

[40] Theron, J.; Morar, D.; du Preez, M.;  Broézel, V.S. and Venter, S.N. "A sensitive seminested PCR method for the detection of Shigella in spiked environmental water samples", Water Res., Vol.35, pp. 869-874. (2001).

[41] Cui, SH. "Detection and characterization of Escherichia coli O157: H7 and Salmonella in food". Dissertation directed by Professor Jianghong Meng Department of Nutrition and Food Science. (2004)"

[42] Wang, G. and Doyle, M. P. "Survival of enterohemorrhagic Escherichia coli O157:H7 in water", Journal of Food Protection, Vol. 61, pp. 662 - 667. (1998)

[43] Barkocy-Gallagher, G. A.; Arthur, T. M.; Siragusa, G. R.; Keen, J. E.; Elder, R. O.; Laegreid, W. W.; and Koohmaraie, M. "Genotypicanalyses of Escherichia coli O157:H7 and O157 nonmotile isolatesrecovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States", Appl. Environ. Microbiol., Vol.67, pp.3810–3818. (2001).

[44] Gilot, P. and Content, J. "Specific identification of Listeria welshimeri and Listeria monocytogenes by PCR assays targeting a gene encoding a fibronectin-binding protein", J. Clin. Microbiol., Vol. 40,pp.698–703, (2002).

[45] Malorny, B.; Tassios, P.T.; Radstrom,  P.;  Cook,  N.;  Wagner,  M. and Hoorfar, J. "Standardization of diagnostic PCR for the detection of foodborne pathogens", Int. J. Food Microbiol., Vol. 83, pp. 39-48. ,(2003).