Opracowanie testow multiplex PCR do identyfikacji i charakterystyki shigatoksycznych Escherichia coli

• Autorzy: Weiner M

Warianty tytułu

EN

Development of multiplex PCR assays for identifying and defining the characteristics of Shiga toxin-producing Escherichia coli

• Języki publikacji: PL

Abstrakty

EN

The aim of the study was to develop multiplex PCR (mPCR) assays which allow identification of shigatoxigenic Escherichia coli (STEC) strains and a characterization of their virulence properties. As an internal control, a fragment of 16S rRNA gene - two amplicons of 230 bp (stx gene) and 401 bp (16S rRNA) were obtained in the test, designated as mPCR-1 for the amplification of the stx gene, characteristic for all known Shiga toxin variants. The mPCR-2 assay was developed to characterize the stx-positive bacterial colonies, which allowed the detection of Shiga toxin 1 and/or 2, the affiliation of STEC O157:H7 serotype (rfbO157 and fliCн₇ genes), and internal control, resulting in amplicons of 348 bp (stx1), 584 (stx2), 420 bp (rfbO157), 247 bp (fliCн₇) and 798 bp (16S rRNA). The detection of markers O26wzx, rfbO111 and rfbO113, typical for E. coli O26, O111 and O113, respectively, was performed with mPCR-3. The products of molecular masses 153, 406 and 593 bp were observed. All STEC’s were also tested using mPCR-4, for the presence of enterohemolysin (ehlyA) and intimin (eaeA) markers generating specific bands of 837 bp (eaeA gene), 534 bp (ehlyA) and 401 (16S rRNA). The mPCR assays developed allow STEC to be identified and characterized and may be used in routine diagnosis of these bacteria.

Słowa kluczowe

PL

szczepy shigatoksyczne; Escherichia coli; lancuchowa reakcja polimerazy; metoda multiplex PCR; identyfikacja

• Wydawca: -
• Czasopismo: Medycyna Weterynaryjna
• Rocznik: 2008
• Tom: 64
• Numer: 03
• Strony: 310-313
• Opis fizyczny: s.310-313,tab.,bibliogr.

Twórcy

autor

Weiner M

• Panstwowy Instytut Weterynaryjny - Panstwowy Instytut Badawczy, Al.Partyzantow 57, 24-100 Pulawy

Bibliografia

• 1. Batchelor M., Knutton S., Caprioli A., Huter V., Zanial M., Dougan G., Frankel G.: Development of a universal intimin antiserum and PCR primers. J. Clin. Microbiol. 1999, 37, 3822-3827.
• 2. Blanco J., Blanco M., Blanco J. E., Mora A., Gonzalez E. A., Bernardez M. I.: Verotoxin-producing Escherichia coli in Spain: Prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans. Exp. Biol. Med. 2003, 228, 345-351.
• 3. Caprioli A., Morabito S., Brugere H., Oswald E.: Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Vet. Res. 2005, 36, 289-311.
• 4. Cebula T. A., Payne W. I., Feng P.: Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol. 1995, 33, 248-250.
• 5. DebRoy C., Roberts E., Kundrat J., Davis M. A., Briggs C. E., Fratamico P. M.: Detection of Escherichia coli serogroups O26 and O113 by PCR amplification of wzx and wzy genes. Appl. Environ. Microbiol. 2004, 70, 3379-3383.
• 6. Ehresmann C., Stiegler P., Fellner P., Ebel P.: The determination of the primary structure of the 16S ribosomal RNA of Escherichia coli. 2. Nucleotide sequence of products from partial enzymatic hydrolysis. Biochemie 1972, 54, 901-967.
• 7. Karch H., Meyer T.: Single primer pair for amplifying segments of distinct Shiga-like toxin genes by polymerase chain reaction. J. Clin. Microbiol. 1989, 27, 2751-2757.
• 8. Karch H., Tarr P. I., Bielaszewska M.: Enterohaemorrhagic Escherichia coli in human medicine. Int. J. Med. Microbiol. 2005, 295, 405-418.
• 9. Maurer J. J., Schmidt D., Petrosco P., Sanchez S., Bolton L., Lee M. D.: Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR. Appl. Environ. Microbiol. 1999, 65, 2954-2960.
• 10. Nataro J. P., Kaper J. B.: Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 1998, 11, 142-201.
• 11. O'Brien A. D., Tesh V. L., Donohue-Rolfe A., Jackson M. P., Olsnes S., Sandvig K., Lindberg A. A., Keusch G. T.: Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Curr. Top. Microbiol. Immunol. 1992, 180, 65-94.
• 12. Osek J.: Molekularne podstawy chorobotwórczości Escherichia coli O157. Medycyna Wet. 2002, 58, 750-755.
• 13. Osek J., Weiner M.: Metoda real-time PCR (r-PCR) w diagnostyce molekularnej shigatoksycznych Escherichia coli. Medycyna Wet. 2004, 60, 687-691.
• 14. Osek J., Weiner M.: Opracowanie testów multiplex PCR do identyfikacji odmian toksyny Shiga wytwarzanych przez Escherichia coli. Medycyna Wet. 2004, 60, 207-210.
• 15. Paton A. W., Paton J. C.: Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbо₁₁₁, and rfbо₁₅₇. J. Clin. Microbiol. 1998, 36, 598-602.
• 16. Paton A. W., Paton J. C.: Molecular characterization of the locus encoding biosynthesis of the lipopolysaccharide O antigen of Escherichia coli serotype O113. Infect Immun. 1999, 67, 5930-5937.
• 17. Paton J. C., Paton A. W.: Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infection. Clin. Microbiol. Rev. 1998, 11, 450-479.
• 18. Schmidt H., Beutin L., Karch H.: Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect. Immun. 1995, 63, 1055-1061.
• 19. Schmidt H., Karch H.: Enterohemolytic phenotypes and genotypes of Shiga toxin-producing Escherichia coli O111 strains from patients with diarrhea and hemolytic uremic syndrome. J. Clin. Microbiol. 1996, 34, 2364-2367.
• 20. Wang G., Clark C., Rodgers G.: Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J. Clin. Microbiol. 2002, 40, 3613-3619.