Food Analytical Methods

, Volume 10, Issue 6, pp 1681–1689 | Cite as

Development of a Real-Time Multiplex PCR Assay with Propidium Monoazide Treatment for Simultaneous Detection of Live Salmonella, and Salmonella Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum, in Rinse Water of Chicken Carcasses

  • So Youn Youn
  • Ok Mi Jeong
  • Byung Kook Choi
  • Suk Chan Jung
  • Min Su Kang


Salmonella is a major food-borne pathogen in humans and a cause of local or systemic disease in animals. Therefore, rapid and reliable methods to detect these poultry-associated Salmonella serotypes are necessary for efficient control of Salmonella in poultry. The present study aimed to develop a real-time multiplex PCR (MqPCR) method to simultaneously detect and/or differentiate Salmonella sp. and poultry-associated serotypes, including Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Pullorum, and Salmonella Gallinarum. A MqPCR method was designed using four specific primer pairs and probes for the detection of Salmonella sp., including S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. Additionally, a novel TaqMan-based MqPCR method combined with propidium monoazide (PMA) treatment was developed for the simultaneous quantification of viable cells of Salmonella sp. and these four Salmonella serotypes in rinse water of chicken carcasses. The MqPCR assay specifically detected Salmonella sp., S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum, showing 100% sensitivity and 100% specificity. This optimized PMA-MqPCR assay could detect live Salmonella (100–106 CFU/reaction) without enrichment in live/dead cell mixtures from spiked rinse water of chicken carcasses. The procedure for detecting live Salmonella required less than 2 h to complete. This PMA TaqMan-based MqPCR technique facilitates accurate and rapid monitoring of contamination with viable Salmonella. Also, the assay enables simultaneous identification of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum in rinse water of chicken carcasses. The assay developed in this study will be useful in diagnostic laboratories for improving Salmonella control in poultry and poultry products.


Salmonella Enteritidis Typhimurium Pullorum Gallinarum Real-time PCR 



This work was supported by a grant from the QIA, Ministry of Agriculture, Food and Rural Affaires, Korea.

Compliance with Ethical Standards


This study was funded by QIA, Ministry of Agriculture, Food and Rural Affaires, Korea.

Conflict of Interest

So Youn Youn declares that she has no conflict of interest. Ok Mi Jeong declares that she has no conflict of interest. Byung Kook Choi declares that he has no conflict of interest. Suk Chan Jung declares that he has no conflict of interest. Min Su Kang declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Publication has been approved by all individual participants.


  1. Aarestrup FM, Hendriksen RS, Lockett J, Gay K, Teates K, McDermott PF, White DG, Hasman H, Sorensen G, Bangtrakulnonth A, Pornreongwong S, Pulsrikarn C, Angulo FJ, Gerner-Smidt P (2007) International spread of multidrug-resistant Salmonella Schwarzengrund in food products. Emerg Infect Dis 13:726–731CrossRefGoogle Scholar
  2. Ahn YC, Cho MH, Yoon IK, Jung DH, Lee EY, Kim JH, Jang WC (2010) Detection of Salmonella using the loop mediated isothermal amplification and real-time PCR. J Kor Chemi Soci 54:215–221CrossRefGoogle Scholar
  3. Alizadeh AH, Behrouz N, Salmanzadeh S, Ranjbar M, Azimian MH, Habibi E, Jaafari F, Zolfagharian K, Zali MR (2007) Escherichia coli, Shigella and Salmonella species in acute diarrhoea in Hamedan, Islamic Republic of Iran. Eastern Mediterr Health J 13:243–249Google Scholar
  4. Cawthorn DM, Witthuhn RC (2008) Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide. J Appl Microbiol 105:1178–1185CrossRefGoogle Scholar
  5. Cenciarini-Borde C, Courtois S, La Scola B (2009) Nucleic acids as viability markers for bacteria detection using molecular tools. Future Microbiol 4:45–64CrossRefGoogle Scholar
  6. Centers for Disease Control and Prevention (CDC) (2010) Preliminary food net data on the incidence of infection with pathogens transmitted commonly through food-10 states, 2009. MMWR Morb Mortal Wkly Rep 59:418–422 Google Scholar
  7. Chen S, Wang F, Beaulieu JC, Stein RE, Ge BL (2011) Rapid detection of viable Salmonella in produce by coupling propidium monoazide with loop-mediated isothermal amplification. Appl Environ Microbiol 77:4008–4016CrossRefGoogle Scholar
  8. Chen Y, Song KY, Brown EW, Lampel KA (2010) Development of an improved protocol for the isolation and detection of Enterobacter sakazakii (Cronobacter) from powdered infant formula. J Food Prot 73:1016–1022CrossRefGoogle Scholar
  9. Cobb SP (2011) The spread of pathogens through trade in poultry meat: overview and recent developments. Rev Sci Tech 30:149–164CrossRefGoogle Scholar
  10. Contreras PJ, Urrutia H, Sossa K, Nocker A (2011) Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment. J Microbiol Methods 87:89–95CrossRefGoogle Scholar
  11. Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, Hein I (2007) Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR. Res Microbiol 158:405–412CrossRefGoogle Scholar
  12. Guy RA, Kapoor A, Holicka J, Shepherd D, Horgen PA (2006) A rapid molecular-based assay for direct quantification of viable bacteria in slaughterhouses. J Food Prot 69:1265–1272CrossRefGoogle Scholar
  13. Hoorfar J, Ahrens P, Radström P (2000) Automated 5′ nuclease PCR assay for identification of Salmonella enterica. J Clin Microbiol 38:3429–3435Google Scholar
  14. Josefsen MH, Lofstrom C, Hansen TB, Christensen LS, Olsen JE, Hoorfar J (2010) Rapid quantification of viable campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Appl Environ Microbiol 76:5097–5104CrossRefGoogle Scholar
  15. Josephson KL, Gerba CP, Pepper IL (1993) Polymerase chain reaction detection of nonviable bacterial pathogens. Appl Environ Microbiol 59:3513–3515Google Scholar
  16. Jothikumar N, Griffiths MW (2002) Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays. Appl Environ Microbiol 68:3169–3171CrossRefGoogle Scholar
  17. Kobayashi H, Oethinger M, Tuohy MJ, Hall GS, Bauer TW (2009) Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide. Lett Appl Microbiol 48:633–638CrossRefGoogle Scholar
  18. Kornschober C, Mikula C, Springer B (2009) Salmonellosis in Austria: situation and trends. Wien Klin Wochenschr 121:96–102CrossRefGoogle Scholar
  19. Kottwitz LBM, Back A, Leão JA, Alcocer I, Karan M, Oliveira TCRM (2008) Contaminação por Salmonella spp. em uma cadeia de produção de ovos de uma integração de postura commercial. Arq Bras Med Vet Zootec 60:496–498CrossRefGoogle Scholar
  20. Lampel KA, Chen Y (2009) Method for the isolation and detection of Enterobacter sakazakii (Cronobacter) from powdered infant formula. Int J Food Microbiol 136:179–184CrossRefGoogle Scholar
  21. Mainar-Jaime RC, Andres S, Vico JP, San RB, Garrido V, Grillo MJ (2013) Sensitivity of the ISO 6579:2002/Amd 1:2007 standard method for detection of Salmonella spp. on mesenteric lymph nodes from slaughter pigs. J Clin Microbiol 51:89–94CrossRefGoogle Scholar
  22. Malkawi HI, Gharaibeh R (2003) Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products. J Basic Microbiol 43:328–336CrossRefGoogle Scholar
  23. Malorny B, Bunge C, Helmuth R (2007) A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs. J Microbiol Methods 70:245–251CrossRefGoogle Scholar
  24. Malorny B, Paccassoni E, Fack P, Bunge C, Martin AP, Helmuth R (2004) Diagnostic real-time PCR for detection of Salmonella in food. Appl Environ Microbiol 70:7046–7052CrossRefGoogle Scholar
  25. Martin B, Raurich R, Garriga M, Aymerich T (2013) Effect of amplicon length in propidium monoazide quantitative PCR for the enumeration of viable cells of Salmonella in cooked ham. Food Anal Methods 6:683–690CrossRefGoogle Scholar
  26. Masters CI, Shallcross JA, Mackey BM (1994) Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain-reaction. J Appl Bacteriol 77:73–79CrossRefGoogle Scholar
  27. Mozola MA (2006) Genetics-based methods for detection of Salmonella spp. in foods. J AOAC Int 89:517–529Google Scholar
  28. Nocker A, Camper AK (2006) Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol 72:1997–2004CrossRefGoogle Scholar
  29. Nocker A, Cheung CY, Camper AK (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67:310–320CrossRefGoogle Scholar
  30. Nogva HK, Dromtrop SM, Nissen H, Rudi K (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. BioTechniques 34:804–813Google Scholar
  31. Rabsch W, Tschäpe H, Bäumler AJ (2001) Non-typhoidal salmonellosis: emerging problems. Microbes Infect 3:237–247CrossRefGoogle Scholar
  32. Rahn K, De Grandis SS, Clarke RC, McEwen SA, Galán JE, Ginocchio C, Curtiss R, Gyles CL (1992) Amplification of an invA gene sequence of Salmonella Typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 6:271–279CrossRefGoogle Scholar
  33. Rossen L, Norskov P, Holmstrom K, Rasmussen OF (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol 17:37–45CrossRefGoogle Scholar
  34. Rudi K, Moen B, Dromtorp SM, Holck AL (2005) Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environ Microbiol 71:1018–1024CrossRefGoogle Scholar
  35. Soejima T, Schlitt-Dittrich F, Yoshida S (2011) Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae. Anal Biochem 418:37–43CrossRefGoogle Scholar
  36. Tatusov RL, Koonin EV, Lipman DJ (1997) A genomic perspective on protein families. Science 278:631–637CrossRefGoogle Scholar
  37. Thorns CJ (2000) Bacterial food-borne zoonoses. Rev Sci Tech 19:226–239CrossRefGoogle Scholar
  38. Wang L, Mustapha A (2010) EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs. J Food Sci 75:M134–M139CrossRefGoogle Scholar
  39. Wang S, Levin RE (2006) Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. J Microbiol Methods 64:1–8CrossRefGoogle Scholar
  40. Wiemer D, Loderstaedt U, von Wulffen H, Priesnitz S, Fischer M, Tannich E, Hagen RM (2011) Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples. Int J Med Microbiol 301:577–584CrossRefGoogle Scholar
  41. Wilson DL, Abner SR, Newman TC, Mansfield LS, Linz JE (2000) Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay. J Clin Microbiol 38:3971–3978Google Scholar
  42. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63:3741–3751Google Scholar
  43. Yang YG, Song MK, Park SJ, Kim SW (2007) Direct detection of Shigella flexneri and Salmonella Typhimurium in human feces by real-time PCR. J Microbiol Biotechnol 17:1616–1621Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • So Youn Youn
    • 1
  • Ok Mi Jeong
    • 1
  • Byung Kook Choi
    • 1
  • Suk Chan Jung
    • 1
  • Min Su Kang
    • 1
  1. 1.Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural AffairsGimcheon-siRepublic of Korea

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