Simultaneous Detection and Identification of Bacillus cereus Group Bacteria Using Multiplex PCR

  • Park, Si-Hong (Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University) ;
  • Kim, Hyun-Joong (Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University) ;
  • Kim, Jae-Hwan (Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University) ;
  • Kim, Tae-Woon (Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University) ;
  • Kim, Hae-Yeong (Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University)
  • 발행 : 2007.07.31

초록

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.

키워드

참고문헌

  1. Asano, S. I., Y. Nukumizu, H. Bando, T. Iizuka, and T. Yamamoto. 1997. Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 63: 1054-1057
  2. Ash, C., J. A. Farrow, M. Dorsch, E. Stackebrandt, and M. D. Collins. 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41: 343-346 https://doi.org/10.1099/00207713-41-3-343
  3. Carlson, C. R., D. Caugant, and A. Kolsto. 1994. Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains. Appl. Environ. Microbiol. 60: 1719-1725
  4. Chang, Y. H., Y. H. Shangkuan, H. C. Lin, and H. W. Liu. 2003. PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells. Appl. Environ. Microbiol. 69: 4502-4510 https://doi.org/10.1128/AEM.69.8.4502-4510.2003
  5. Chen, M. L. and H. Y. Tsen. 2002. Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites. J. Appl. Microbiol. 92: 912-919 https://doi.org/10.1046/j.1365-2672.2002.01606.x
  6. Daffonchio, D., N. Raddadi, M. Merabishvili, A. Cherif, L. Carmagnola, L. Brusetti, A. Rizzi, N. Chanishvili, R. Sharp, and S. Borin. 2006. Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis. Appl. Environ. Microbiol. 72: 1295-1301 https://doi.org/10.1128/AEM.72.2.1295-1301.2006
  7. Harrell, L. J., G. L. Andersen, and K. H. Wilson. 1995. Genetic variability of Bacillus anthracis and related species. J. Clin. Microbiol. 33: 1847-1850
  8. Henderson, I., C. J. Duggleby, and P. C. Turnbull. 1994. Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR. Int. J. Syst. Bacteriol. 44: 99-105 https://doi.org/10.1099/00207713-44-1-99
  9. Jang, S. S., E. Choo, K. Han, T. Miyamoto, S. Heu, and S. Ryu. 2006. Antibiotic resistance and genetic diversity of Listeria monocytogenes isolated from chicken carcasses in Korea. J. Microbiol. Biotechnol. 16: 1276-1284
  10. Juergensmeyer, M. A., B. A. Gingras, L. Restaino, and E. W. Frampton. 2006. A selective chromogenic agar that distinguishes Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. J. Food Prot. 69: 2002-2006 https://doi.org/10.4315/0362-028X-69.8.2002
  11. Keirn, P., A. Kalif, J. Schupp, K. Hill, S. E. Travis, K. Richmond, D. M. Adair, M. Hugh-Jones, C. R. Kuske, and P. Jackson. 1997. Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J. Bacteriol. 179: 818-824 https://doi.org/10.1128/jb.179.3.818-824.1997
  12. Kim, J. S., J. Y. Choi, J. H. Chang, H. J. Shim, J. Y. Roh, B. R. Jin, and Y. H. Je. 2005. Characterization of an improved recombinant baculovirus producing polyhedra that contains Bacillus thuringiensis Cry1Ac crystal protein. J. Microbiol. Biotechnol. 15: 710-715
  13. Manzano, M., L. Cocolin, C. Cantoni, and G. Comi. 2003. Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides differentiation using a PCR-RE technique. Int. J. Food Microbiol. 81: 249-254 https://doi.org/10.1016/S0168-1605(02)00222-2
  14. Okinawa, R. T., K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keirn, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson. 1999. Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J. Bacteriol. 181: 6509-6515
  15. 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
  16. Park, T. J., J. P. Park, G M. Seo, Y. G. Chai, and S. Y. Lee. 2006. Rapid and accurate detection of Bacillus anthracis spores using peptide-quantum dot conjugates. J. Microbiol. Biotechnol. 16: 1713-1719
  17. Rasko, D. A., M. R. Altherr, C. S. Han, and J. Ravel. 2005. Genomics of the Bacillus cereus group of organisms. FEMS Microbiol. Rev. 29: 303-329
  18. Sergeev, N., M. Distler, M. Vargas, V. Chizhikov, K. E. Herold, and A. Rasooly. 2005. Microarray analysis of Bacillus cereus group virulence factors. J. Microbiol. Methods 65: 488-502 https://doi.org/10.1016/j.mimet.2005.09.013
  19. Turnbull, P. C. B., R. A. Hutson, M. J. Ward, M. N. Jones, C. P. Quinn, N. J. Finnie, C. J. Duggleby, J. M. Krammer, and J. Melling. 1992. Bacillus anthracis but not always anthrax. J. Appl. Microbiol. 72: 21-28 https://doi.org/10.1111/j.1365-2672.1992.tb05181.x
  20. Zani, J. L., F. W. da Cruz, A. F. dos Santos, and C. GilTumes. 1998. Effect of probiotic CenBiot on the control of diarrhea and feed efficiency in pigs. J. Appl. Microbiol. 84: 68-71 https://doi.org/10.1046/j.1365-2672.1997.00309.x