Detection of Listeria spp. and Listeria monocytogenes in biological samples by SYBR Green I and TaqMan probe-based real-time PCRs

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Introduction: The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood.

Material and Methods: Five strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted.

Results: The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.

Conclusion: Both real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.

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  • 1. Amagliani G. Omiccioli E. Brandi G. Bruce I.J. Magnani M.: A multiplex magnetic capture hybridisation and multiplex real-time PCR protocol for pathogen detection in sea food. Food Microbiol 2010 27 580–585.

  • 2. Barkallah M. Gharbi Y. Hmani M. Mallek Z. Gautier M. Gdoura R. Fendri I.: Locked nucleic acid probe-based real-time PCR for the diagnosis of Listeria monocytogenes in ruminants. Mol Cell Probe 2016 30 138–145.

  • 3. Budniak S. Kędrak-Jabłońska A. Szczawińska A. Reksa M. Krupa M. Szulowski K.: Comparison of two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples. J Vet Res 2016 60 411–416.

  • 4. Dadkhah H. Bassami M.R. Hashemi S. Shahraz F. Hosseini H. Karatzas K.A.G. Khaksar R.: Evaluation and comparison of SYBR Green I real-time PCR and TaqMan real-time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk. Afr J Microbiol Res 2012 6 1908–1917.

  • 5. Denis E. Bielińska K. Wieczorek K. Osek J.: Multiplex real-time PCRs for detection of SalmonellaListeria monocytogenes and verotoxigenic Escherichia coli in carcasses of slaughtered animals. J Vet Res 2016 60 287–292.

  • 6. Drevets D.A. Bronze M.S.: Listeria monocytogenes: epidemiology human disease and mechanisms of brain invasion. FEMS Immunol Med Microbiol 2008 53 151–165.

  • 7. European Food Safety Authority and European Centre for Disease Prevention and Control: The European Union summary report on trends and sources of zoonoses zoonotic agents and food-borne outbreaks in 2015. EFSA J 2016 14 4634 1–231. doi10.2903/j.efsa.2016.4634.

  • 8. Gasanov U. Hughes D. Hansbro P.M.: Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review. FEMS Microbiol Rev 2005 29 851–875.

  • 9. Gray M.L. Killinger A.H.: Listeria monocytogenes and Listeric infections. Bacteriol Rev 1966 30 309–382.

  • 10. Heid C.A. Stevens J. Livak K.J. Williams P.M.: Real time quantitative PCR. Genome Res 1996 6 986–994.

  • 11. Hough A.J. Harbison S.A. Savill M.G. Melton L.D. Fletcher G.: Rapid enumeration of Listeria monocytogenes in artificially contaminated cabbage using real–time polymerase chain reaction. J Food Prot 2002 65 1329–1332.

  • 12. Kubista M. Andrade J.M. Bengtsson M. Forootan A. Jonák J. Lind K. Sindelka R. Sjöback R. Sjögreen B. Strömbom L. Ståhlberg A. Zoric N.: The real-time polymerase chain reaction. Mol Aspects Med 2006 27 95–125.

  • 13. Low J.C. Donachie W.: A review of Listeria monocytogenes and listeriosis. Vet J 1997 153 9–29.

  • 14. Murphy N.M. McLauchlin J. Ohai C. Grant K.A.: Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica Int J Food Microbiol 2007 120 110–119.

  • 15. Murray E.G.D. Webb R.A. Swann M.R.B.: A disease of rabbits characterized by a large mononuclear leukocytosis caused by a hitherto undescribed bacillus Bacterium monocytogenes (n.sp.). J Pathol 1926 29 407–439.

  • 16. National Institute of Public Health and Chief Sanitary Inspectorate: Infectious diseases and poisonings in Poland 2015. Warsaw 2016 pp. 1–166.

  • 17. Nogva H.K. Rudi K. Naterstad K. Holck A. Lillehaug D.: Application of 5’-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures water skim milk and unpasteurized whole milk. Appl Environ Microbiol 2000 66 4266–4271.

  • 18. Poltronieri P. de Blasi M.D. D’Urso O. F.: Detection of Listeria monocytogenes through real-time PCR and biosensor methods. Plant Soil Environ 2009 55 363–369.

  • 19. Rocourt J.: Risk factors for listeriosis. Food Control 1996 7 195–202.

  • 20. Rodríguez-Lázaro D. Hernández M. Pla M.: Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay. FEMS Microbiol Letters 2004 233 257–267.

  • 21. Schlech W.F. Lavigne P.M. Bortolussi R.A. Allen A.C. Haldane E.V. Wort A.J. Hightower A.W. Johnson S.E. King S.H. Nicholls E.S. Broome M. D.: Epidemic listeriosis-evidence for transmission by food. N Engl J Med 1983 308 203–206.

  • 22. Schuchat A. Swaminathan B. Broome C.V.: Epidemiology of human listeriosis. Clin Microbiol Rev 1991 4 169–183.

  • 23. Skerniškyté J. Armalyté J. Kvietkauskaité R. Šeputiené V. Povilonis J. Sužiedéliené E.: Detection of Salmonella spp. Yersinia enterocoliticaListeria monocytogenes and Campylobacter spp. by real-time multiplex PCR using amplicon DNA melting analysis and probe-based assay. Int J Food Sci 2016 51 519–529.

  • 24. Swaminathan B. Gerner-Smidt P.: The epidemiology of human listeriosis. Microbes Infect 2007 9 1236–1243.

  • 25. Todd E.C.D. Notermans S.: Surveillance of listeriosis and its causative pathogen Listeria monocytogenes. Food Control 2011 22 1484–1490.

  • 26. Traunšek U. Toplak N. Jeršek B. Lapanje A. Majstorović T. Kovač M.: Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes. J Microbiol Methods 2011 85 40–46.

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