Browse > Article

Rapid Detection of Enterobacter sakazakii Using TaqMan Real-Time PCR Assay  

Kang, Eun-Sil (Department of Food Science and Technology, Dongguk University)
Nam, Yong-Suk (KogeneBiotech Co.)
Hong, Kwang-Won (Department of Food Science and Technology, Dongguk University)
Publication Information
Journal of Microbiology and Biotechnology / v.17, no.3, 2007 , pp. 516-519 More about this Journal
Abstract
Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 168 rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/ assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.
Keywords
Enterobacter sakazakii; real-time PCR; 16S rRNA; rapid detection;
Citations & Related Records
Times Cited By KSCI : 4  (Citation Analysis)
Times Cited By Web Of Science : 0  (Related Records In Web of Science)
연도 인용수 순위
1 Bischoff, C., J. Luthy, M. Altwegg, and F. Baggi. 2005. Rapid detection of diarrheagenic E. coli by real-time PCR. J. Microbiol. Methods 61: 335-341   DOI   ScienceOn
2 Klappenbach, J. A., P. R. Saxman, J. R. Cole, and T. M. Schmidt. 2001. rrndb: The ribosomal RNA operon copy number database. Nucleic Acids Res. 29: 181-184   DOI
3 Koh, Y. H., M. D. Kim, N. S. Han, and J. H. Seo. 2002. Quantitative analysis of Leuconostoc mesenteroides and Lactobacillus plantarum populations by a competitive polymerase chain reaction. J. Microbiol. Biotechnol. 12: 801-806
4 Liu, Y., X. Cai, X. Zhang, Q. Gao, X. Yang, Z. Zheng, M. Luo, and X. Huang. 2006. Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula. J. Microbiol. Methods 65: 21-31   DOI   ScienceOn
5 Matsuki, T., K. Watanabe, J. Fujimoto, T. Takada, and R. Tanaka. 2004. Use of 16S rRNA gene-targeted groupspecific primers for real-time PCR analysis of predominant bacteria in human feces. Appl. Environ. Microbiol. 70: 7220-7228   DOI   ScienceOn
6 Poms R. E., E. Anklam, and M. Kuhn. 2004. Polymerase chain reaction techniques for food allergen detection. J. AOAC Int. 87: 1391-1397
7 Klein, D. 2002. Quantification using real-time PCR technology: Applications and limitations. Trends Mol. Med. 8: 257-260   DOI   ScienceOn
8 Nazarowec-White, M., R. C. Mckellar, and P. Piyasena. 1999. Predictive modelling of Enterobacter sakazakii inactivation in bovine milk during high-temperature short-time pasteurization. Food Res. Int. 32: 375-379   DOI   ScienceOn
9 Warsen, A. E., M. J. Krig, S. LaFrentz, D. R. Stanek, F. J. Long, and D. R. Call. 2004. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays. Appl. Environ. Microbiol. 70: 4216-4221   DOI   ScienceOn
10 Seo, K. H. and R. E. Brackett. 2005. Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J. Food Prot. 68: 59-63   DOI
11 Kandhai, M. C., M. W. Reij, L. Gorris, O. Guillaume-Gentil, and M. van Schothorst. 2004. Occurrence of Enterobacter sakazakii in food production environments and households. Lancet 363: 39-40   DOI   ScienceOn
12 Park, S. H., H. J. Kim, and H. Y. Kim. 2006. Simultaneous detection of Yersinia enterocolitica, Staphylococcus aureus, and Shigella spp. in lettuce using multiplex PCR method. J. Microbiol. Biotechnol. 16: 1301-1305   과학기술학회마을
13 Simmons, B. P., M. S. Gelfand, M. Hass, L. Metts, and J. Ferguson. 1989. Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect. Control Hosp. Epidemiol. 10: 398-401   DOI   ScienceOn
14 Biering, G., S. Karlsson, N. C. Clark, K. E. Jonsdottir, P. Ludvigsson, and O. Steingrimsson. 1989. Three cases of neonatal meningitis caused by Enterobacter sakazakii in powdered milk. J. Clin. Microbiol. 9: 2054-2056
15 Hawkins, R. E., C. R. Lissner, and J. P. Sanford. 1991. Enterobacter sakazakii bacteremia in an adult. South Med. J. 84: 793-795   DOI   ScienceOn
16 Van Acker, J., F. De Smet, G. Muyldermans, A. Bougatef, A. Naessens, and S. Lauwers. 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J. Clin. Microbiol. 39: 293-297   DOI   ScienceOn
17 Lai, K. K. 2001. Enterobacter sakazakii infections among neonates, infants, children, and adult. Medicine 80: 113-122   DOI   ScienceOn
18 Kandhai, M. C., M. W. Reij, K. van Puyvelde, O. Guillaume-Gentil, R. R. Beumer, and M. van Schothorst. 2004. A new protocol for the detection of Enterobacter sakazakii applied to environmental samples. J. Food Prot. 67: 1267-1270   DOI
19 Acinas, S. G., L. A. Marcelino, V. Klepac-Ceraj, and M. F. Polz. 2004. Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J. Bacteriol. 186: 2629-2635   DOI   ScienceOn
20 Iversen, C. and S. Forsythe. 2004. Isolation of Enterobacter sakazakii and other Enterobacteriaceae from powdered infant formula milk and related products. Food Microbiol. 21: 771-777   DOI   ScienceOn
21 Jung, S. J., H. J. Kim, and H. Y. Kim. 2005. Quantitative detection of Salmonella typhimurium contamination in milk, using real-time PCR. J. Microbiol. Biotechnol. 15: 1353-1358   과학기술학회마을
22 Lukinmaa, S., U. M. Nakari, M. Eklund, and A. Siitonen. 2004. Application of molecular genetic methods in diagnostics and epidemiology of food-borne bacterial pathogens. APMIS 112: 908-929   DOI   ScienceOn
23 Gurtler, J. B., J. L. Kornacki, and L. R. Beuchat. 2005. Enterobacter sakazakii: A coliform of increased concern to infant health. Int. J. Food Microbiol. 104: 1-34   DOI   ScienceOn
24 Iversen, C., M. Waddington, S. L. On, and S. Forsythe. 2004. Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter species. J. Clin. Microbiol. 42: 5368-5370   DOI   ScienceOn
25 Kim, S. B., H. J. Lim, W. K. Lee, I. G. Hwang, G. J. Woo, and S. R. Ryu. 2006. PCR-based detection and molecular genotyping of enterotoxigenic Clostridium perfringens isolates from swine diarrhea in Korea. J. Microbiol. Biotechnol. 16: 291-294   과학기술학회마을
26 Muytjens, H. L., H. Roelofs-Willemse, and G. H. Jaspar. 1988. Quality of powdered substitutes for breast milk with regard to members of the family Enterobacteriacea. J. Clin. Microbiol. 26: 743-746