1887

Abstract

A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing rapidly (within 60 min). The 24 strains of VT-producing were successfully amplified, but 6 strains of non-VT-producing and 46 bacterial species other than were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing ; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing in food because of the difficulty of detection in food samples. The recovery of VT-producing by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing .

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2007-03-01
2024-03-28
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References

  1. Beige J., Lokies J., Schaberg T., Finckh U., Fischer M., Mauch H., Lode H., Kohler B., Rolfs A. 1995; Clinical evaluation of a Mycobacterium tuberculosis PCR assay. J Clin Microbiol 33:90–95
    [Google Scholar]
  2. Bennett A. R., MacPhee S., Betts R. P. 1996; The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures. Lett Appl Microbiol 22:237–243 [CrossRef]
    [Google Scholar]
  3. Bettelheim K. A. 1995; Identification of enterohaemorrhagic Escherichia coli by means of their production of enterohaemolysin. J Appl Bacteriol 79:178–180 [CrossRef]
    [Google Scholar]
  4. Bettelheim K. A. 2000; Role of non-O157 VTEC. Symp Ser Soc Appl Microbiol 88:38S–50S [CrossRef]
    [Google Scholar]
  5. Chapman P. A., Ellin M., Ashton R. 2001; A comparison of immunomagnetic separation and culture, RevealTM and VIPTM for the detection of E. coli O157 in enrichment cultures of naturally-contaminated raw beef, lamb and mixed meat products. Lett Appl Microbiol 32:171–175 [CrossRef]
    [Google Scholar]
  6. Hara-Kudo Y., Onoue Y., Konuma H., Nakagawa H., Kumagai S. 1999; Comparison of enrichment procedures for isolation of Escherichia coli O157 : H7 from ground beef and radish sprouts. Int J Food Microbiol 50:211–214 [CrossRef]
    [Google Scholar]
  7. Hara-Kudo Y., Konuma H., Nakagawa H., Kumagai S. 2000; Escherichia coli O26 isolation from foods using enrichment procedure and immunomagnetic separation method. Lett Appl Microbiol 30:151–154 [CrossRef]
    [Google Scholar]
  8. Hara-Kudo Y., Ikedo M., Komatsu O., Yamamoto S., Kumagai S. 2002; Evaluation of a chromogenic agar medium for isolation of Escherichia coli O26. Food Control 13:377–379 [CrossRef]
    [Google Scholar]
  9. Hiramatsu R., Matsumoto M., Miwa Y., Suzuki Y., Saito M., Miyazaki Y. 2002; Characterization of Shiga toxin-producing Escherichia coli O26 strains and establishment of selective isolation media for these strains. J Clin Microbiol 40:922–925 [CrossRef]
    [Google Scholar]
  10. Ito H., Terai A., Kurazono H., Takeda Y., Nishibuchi M. 1990; Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91 : H21 isolated from a patient with the hemolytic uremic syndrome. Microb Pathog 8:47–60 [CrossRef]
    [Google Scholar]
  11. Ito H., Nishibuchi M., Takeda Y. 1991; Analysis of the antigenic difference between Vero toxin 2 (VT2) and VT2 variant (VT2vh) of Verotoxin-producing Escherichia coli by a site-directed mutagenesis. FEMS Microbiol Lett 15:27–30
    [Google Scholar]
  12. Jackson M. P., Neill R. J., O'Brien A. D., Holmes R. K., Newland J. W. 1987; Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933. FEMS Microbiol Lett 44:109–114 [CrossRef]
    [Google Scholar]
  13. Kawasaki S., Horikoshi N., Okada Y., Takeshita K., Sameshima T., Kawamoto S. 2005; Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes , and Escherichia coli O157 : H7 in meat samples. J Food Prot 68:551–556
    [Google Scholar]
  14. Lin Z., Yamasaki S., Kurazono H., Ohmura M., Karasawa T., Inoue T., Sakamoto S., Suganami T., Takeoka T. other authors 1993; Cloning and sequencing of two new Vero toxin 2 variant genes of Escherichia coli isolated from cases of human and bovine diarrhea. Microbiol Immunol 37:451–459 [CrossRef]
    [Google Scholar]
  15. Mori Y., Nagamine K., Tomita N., 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]
    [Google Scholar]
  16. Mullah B., Livak K., Andrus A., Kenney P. 1998; Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real-time PCR assay. Nucleic Acids Res 26:1026–1031 [CrossRef]
    [Google Scholar]
  17. Nagamine K., Watanabe K., Ohtsuka K., Hase T., Notomi T. 2001; Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem 47:1742–1743
    [Google Scholar]
  18. Nagamine K., Kuzuhara Y., Notomi T. 2002; Isolation of single-stranded DNA from loop-mediated isothermal amplification products. Biochem Biophys Res Commun 290:1195–1198 [CrossRef]
    [Google Scholar]
  19. National Institute of Infectious Diseases & Infectious Diseases Control Division, Ministry of Health and Welfare of Japan 2004; Enterohemorrhagic Escherichia coli infection, as of May 2004. Infect Agents Surveillance Rep 25:138–143 (in Japanese
    [Google Scholar]
  20. National Institute of Infectious Diseases& Infectious Diseases Control Division, Ministry of Health and Welfare of Japan 2005; Enterohemorrhagic Escherichia coli infection, as of May 2005. Infect Agents Surveillance Rep 26:137–138 (in Japanese
    [Google Scholar]
  21. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. 2000; Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63 [CrossRef]
    [Google Scholar]
  22. Okrend A. J. G., Rose B. E., Bennett B. 1990; A screening method for the isolation of Escherichia coli O157 : H7 from ground beef. J Food Prot 53:249–252
    [Google Scholar]
  23. Read S. C., Clarke R. C., Martin A., De Grandis S. A., Hii J., McEwen S., Gyles C. L. 1992; Polymerase chain reaction for detection of verotoxigenic Escherichia coli isolated from animal and food sources. Mol Cell Probes 6:153–161 [CrossRef]
    [Google Scholar]
  24. Weinstein D. L., Jackson M. P., Samuel J. E., Holmes R. K., O'Brien A. D. 1988; Cloning and sequencing of a Shiga-like toxin type II variant from an Escherichia coli strain responsible for edema disease of swine. J Bacteriol 170:4223–4230
    [Google Scholar]
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