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. Five strains of L. monocytogenes and single strains of each of the 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 the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. 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 confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.
If the inline PDF is not rendering correctly, you can download the PDF file here.
1. Barbau-Piednoir E., Botteldoorn N., Yde M., Mahillon J., Roosens N.H.: Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes. Appl Microbiol Biotechnol 2013, 97, 4021-4037.
2. Bassler H.A., Flood S.J.A., Livak K.J., Marmaro J., Knorr R., Batt C.A.: Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl Environ Microbiol 1995, 61, 3724-3728.
3. Dmowska K., Wieczorek K., Lynch O., Osek J.: Typing of Listeria monocytogenes isolated from slaughtered cattle and beef meat. Bull Vet Inst Pulawy 2013, 57, 179-183.
4. Drevets D.A., Bronze M.S.: Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion. FEMS Immunol Med Microbiol 2008, 53, 151-165.
5. 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.
6. Goulet V., Hedberg C., Le Monnier A., de Valk H.: Increasing incidence of listeriosis in France and other European countries. Emerg Infect Dis 2008, 14, 734-740.
7. Guilbaud M., de Coppet P., Bourion F., Rachman C., Prévost H., Dousset X.: Quantitative detection of Listeria monocytogenes in biofilms by real-time PCR. Appl Environ Microbiol 2005, 71, 2190-2194.
8. Heid C.A., Stevens J., Livak K.J., Williams P.M.: Real time quantitative PCR. Genome Res 1996, 6, 986-994.
9. Hein I., Klein D., Lehner A., Bubert A., Brandl E., Wagner M.: Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay. Res Microbiol 2001, 152, 37-46.
11. 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.
12. Le Monnier A., Abachin E., Beretti J.L, Berche P., Kayal S.: Diagnosis of Listeria monocytogenes meningoencephalitis by realtime PCR for the hly gene. J Clin Microbiol 2011, 49, 3917-3923.
13. Low J.C., Donachie W.: A review of Listeria monocytogenes and listeriosis. Vet J 1997, 153, 9-29.
14. Mędrala D., Dąbrowski W., Czekajło-Kołodziej U., Daczkowska-Kozon E., Koronkiewicz A., Augustynowicz E., Manzano M.: Persistence of Listeria monocytogenes strains isolated from products in a Polish fish-processing plant over a 1-year period. Food Microbiol 2003, 20, 715-724.
15. 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.
16. O’Grady J., Sedano-Balbás S., Maher M., Smith T., Barry T.: Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target. Food Microbiol 2008, 25, 75-84.
17. Oravcová K., Kuchta T., Kacliková E.: A novel real-time PCRbased method for the detection of Listeria monocytogenes in food. Lett Appl Microbiol 2007, 45, 568-573.
18. Ririe K. M., Rasmussen R.P., Wittwer C.T.: Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997, 245, 154-160.
19. 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.
20. Rossmanith P., Krassnig M., Wagner M., Hein I.: Detection of Listeria monocytogenes in food using a combined enrichment/realtime PCR method targeting the prfA gene. Res Microbiol 2006, 157, 763-771.
21. Rudi K., Naterstad K., Drømtorp S.M., Holo H.: Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR. Lett Appl Microbiol 2005, 40, 301-306.
22. Swaminathan B., Gerner-Smidt P.: The epidemiology of human listeriosis. Microbes Infect 2007, 9, 1236-1243.