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Comparison of Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, and Loop-Mediated Isothermal Amplification for the Detection of Cronobacter sakazakii in Milk Powder
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 Title & Authors
Comparison of Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, and Loop-Mediated Isothermal Amplification for the Detection of Cronobacter sakazakii in Milk Powder
Kim, Young-Joo; Seo, Sheungwoo; Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa; Son, Na Ry; Lee, Bog-Hieu; Choi, Changsun;
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Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk.
loop-mediated isothermal amplification (LAMP);Cronobacter sakazakii;ompA;specificity;sensitivity;
 Cited by
Alex, P. and Georg, E. S. (1998) Structure of the outer membrane protein A transmembrane domain. Nat. Struct. Biol. 5, 1013-1017. crossref(new window)

Arad, I., Baras, M., Gofin, R., Bar-Oz, B., and Peleg, O. (2001) Does parity affect the neonatal outcome of very low birth weight infants. Eur. J. Obstet. Gynecol. Reprod. Biol. 94, 283-288. crossref(new window)

Derzelle, S., Dilasser, F., Maladen, V., Soudrie, N., Leclercq, A., Lombard, B., and Lafarge, V. (2007) Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories. J. Food Prot. 70, 1678-1684.

Enosawa, M., Kageyama, S., Sawai, K., Watanabe, K., Notomi, T., Onoe, S., Mori, Y., and Yokomizo, Y. (2003) Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis. J. Clin. Microbiol. 41, 4359-4365. crossref(new window)

Farmer, J. J., Asbury, M. A., Hickman, F. W., and Brenner, D. J. (1980) Enterobacter sakazakii: A new species of "Enterobacteriaceae" isolated from clinical specimens. Int. J. Syst. Bacteriol. 30, 569-584. crossref(new window)

Friedemann, M. (2007) Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). Int. J. Food Microbiol. 116, 1-10. crossref(new window)

Guillaume-Gentil, O., Sonnard, V., Kandhai, M. C., Marugg, J. D., and Joosten, H. (2005) A Simple and rapid cultural method for detection of Enterobacter sakazakii in environmental samples. J. Food Prot. 68, 64-69.

Hara-Kudo, Y., Nemoto, J., Ohtsuka, K., Segawa, Y., Takatori, K., Kojima, T., and Ikedo, M. (2007) Sensitive and rapid detection of vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J. Med. Microbiol. 56, 398-406. crossref(new window)

Hara-Kudo, Y., Yoshino, M., Kojima, T., and Ikedo, M. (2005) Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol. Lett. 253, 155-161. crossref(new window)

Hu, L., Zhang, W., Zhang, X., Yuan, Y., Zhang, Y., Zhang, H., Ma, X., and Su, X. (2009) Development of a loop-mediated isothermal amplification assay for rapid detection of Enterobacter sakazakii in powdered infant formula. Wei Sheng Wu Xue Bao. 49, 378-382.

Iversen, C. and Forsythe, S. J. (2003) Risk profile of Enterobacter sakazakii, an emergent pathogen associated with infant milk formula. Trends Food Sci. Tech. 14, 443-454. crossref(new window)

Iversen, C., Mullane, N., McCardell, B., Tall, B. D., and Lehner, A. (2008) Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov. Int. J. Syst. Evol. Microbiol. 58, 1442-1447. crossref(new window)

Liu, C., Zheng, W., Zhang, H., Hou, Y., and Liu, Y. (2009) Sensitive and rapid detection of Enterobacter sakazakii in infant formula by loop-mediated isothermal amplification method. J. Food Safety 29, 83-94. crossref(new window)

Liu, Y., Cai, X., Zhang, X., Gao, Q., Yang, X., Zheng, Z., Luo, M., and Huang, X. (2006) Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula. J. Microbiol. Meth. 65, 21-31. crossref(new window)

Mohan Nair, M. K. and Venkitanarayanan, K. (2007) Role of bacterial OmpA and host cytoskeleton in the invasion of human intestinal epithelial cells by Enterobacter sakazakii. Pediatr. Res. 62, 664-669. crossref(new window)

Mori, Y., Nagamine, K., Tomita, N., and Notomi, T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150-154. crossref(new window)

Nagamine, K., Hase, T., and Notomi, T. (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probe. 16, 223-229. crossref(new window)

Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63. crossref(new window)

Orlandi, P. A., and Lampel, K. L. (2000) Extraction-free, filter-based template preparation for rapid and sensitive PCR detection of pathogenic parasitic protozoa. J. Clin. Microbiol. 38, 2271-2277.

Saito, R., Misawa, Y., Moriya, K., Koike, K., Ubukata, K., and Okamura, N. (2005) Development and evaluation of a loopmediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae. J. Med. Microbiol. 54, 1037-1041. crossref(new window)

Singh, S. P., Williams, Y. U., Miller, S., and Nikaido, H. (2003) The C-terminal domain of Salmonella enterica serovar typhimurium ompA is an immunodominant antigen in mice but appears to be only partially exposed on the bacterial cell surface. Infect. Immun. 71, 3937-3946. crossref(new window)

Yamazaki, W., Taguchi, M., Ishibashi, M., Kitazato, M., Nukina, M., Misawa, N., and Inoue, K. (2008) Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. J. Med. Microbiol. 57, 444-451. crossref(new window)

Ye, Y., Wu, Q., Yao, L., Dong, X., Wu, K., and Zhang, J. (2009) A comparison of polymerase chain reaction and international organization for standardization methods for determination of Enterobacter sakazakii contamination of infant formulas from Chinese mainland markets. Foodborne Pathog. Dis. 6, 1229-1234. crossref(new window)

Yoshida, A., Nagashima, S., Ansai, T., Tachibana, M., Kato, H., Watari, H., Notomi, T., and Takehara, T. (2005) Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. J. Clin. Microbiol. 43, 2418-2424. crossref(new window)