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J Clin Microbiol. Mar 1997; 35(3): 656–662.
PMCID: PMC229645

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains.

Abstract

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.

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Selected References

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  • Barrett TJ, Kaper JB, Jerse AE, Wachsmuth IK. Virulence factors in Shiga-like toxin-producing Escherichia coli isolated from humans and cattle. J Infect Dis. 1992 May;165(5):979–980. [PubMed]
  • Barrett TJ, Lior H, Green JH, Khakhria R, Wells JG, Bell BP, Greene KD, Lewis J, Griffin PM. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing. J Clin Microbiol. 1994 Dec;32(12):3013–3017. [PMC free article] [PubMed]
  • Basta M, Karmali M, Lingwood C. Sensitive receptor-specified enzyme-linked immunosorbent assay for Escherichia coli verocytotoxin. J Clin Microbiol. 1989 Jul;27(7):1617–1622. [PMC free article] [PubMed]
  • Beebakhee G, Louie M, De Azavedo J, Brunton J. Cloning and nucleotide sequence of the eae gene homologue from enterohemorrhagic Escherichia coli serotype O157:H7. FEMS Microbiol Lett. 1992 Feb 1;70(1):63–68. [PubMed]
  • Bitzan M, Karch H, Maas MG, Meyer T, Rüssmann H, Aleksić S, Bockemühl J. Clinical and genetic aspects of Shiga-like toxin production in traditional enteropathogenic Escherichia coli. Zentralbl Bakteriol. 1991 Jan;274(4):496–506. [PubMed]
  • Cao C, Yamasaki S, Lin Z, Kurazono H, Takeda Y. Specific detection of a verotoxin 2 variant, VT2vp1, by a bead-enzyme-linked immunosorbent assay. Microbiol Immunol. 1994;38(6):435–440. [PubMed]
  • Cebula TA, Payne WL, 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 Jan;33(1):248–250. [PMC free article] [PubMed]
  • Chapman PA, Wright DJ, Siddons CA. A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. J Med Microbiol. 1994 Jun;40(6):424–427. [PubMed]
  • Clarke RC, McEwen SA, Gannon VP, Lior H, Gyles CL. Isolation of verocytotoxin-producing Escherichia coli from milk filters in south-western Ontario. Epidemiol Infect. 1989 Apr;102(2):253–260. [PMC free article] [PubMed]
  • Cordovéz A, Prado V, Maggi L, Cordero J, Martinez J, Misraji A, Rios R, Soza G, Ojeda A, Levine MM. Enterohemorrhagic Escherichia coli associated with hemolytic-uremic syndrome in Chilean children. J Clin Microbiol. 1992 Aug;30(8):2153–2157. [PMC free article] [PubMed]
  • Cubbon MD, Coia JE, Hanson MF, Thomson-Carter FM. A comparison of immunomagnetic separation, direct culture and polymerase chain reaction for the detection of verocytotoxin-producing Escherichia coli O157 in human faeces. J Med Microbiol. 1996 Mar;44(3):219–222. [PubMed]
  • Dorn CR, Francis DH, Angrick EJ, Willgohs JA, Wilson RA, Collins JE, Jenke BH, Shawd SJ. Characteristics of Vero cytotoxin producing Escherichia coli associated with intestinal colonization and diarrhea in calves. Vet Microbiol. 1993 Jul;36(1-2):149–159. [PubMed]
  • Downes FP, Green JH, Greene K, Strockbine N, Wells JG, Wachsmuth IK. Development and evaluation of enzyme-linked immunosorbent assays for detection of shiga-like toxin I and shiga-like toxin II. J Clin Microbiol. 1989 Jun;27(6):1292–1297. [PMC free article] [PubMed]
  • Fratamico PM, Bhaduri S, Buchanan RL. Studies on Escherichia coli serotype O157:H7 strains containing a 60-MDa plasmid and on 60-MDa plasmid-cured derivatives. J Med Microbiol. 1993 Nov;39(5):371–381. [PubMed]
  • Fratamico PM, Sackitey SK, Wiedmann M, Deng MY. Detection of Escherichia coli O157:H7 by multiplex PCR. J Clin Microbiol. 1995 Aug;33(8):2188–2191. [PMC free article] [PubMed]
  • Frost JA, Cheasty T, Thomas A, Rowe B. Phage typing of Vero cytotoxin-producing Escherichia coli O 157 isolated in the United Kingdom: 1989-91. Epidemiol Infect. 1993 Jun;110(3):469–475. [PMC free article] [PubMed]
  • Gannon VP, King RK, Kim JY, Thomas EJ. Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl Environ Microbiol. 1992 Dec;58(12):3809–3815. [PMC free article] [PubMed]
  • Gannon VP, Rashed M, King RK, Thomas EJ. Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J Clin Microbiol. 1993 May;31(5):1268–1274. [PMC free article] [PubMed]
  • Gunzburg ST, Tornieporth NG, Riley LW. Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene. J Clin Microbiol. 1995 May;33(5):1375–1377. [PMC free article] [PubMed]
  • Hii JH, Gyles C, Morooka T, Karmali MA, Clarke R, De Grandis S, Brunton JL. Development of verotoxin 2- and verotoxin 2 variant (VT2v)-specific oligonucleotide probes on the basis of the nucleotide sequence of the B cistron of VT2v from Escherichia coli E32511 and B2F1. J Clin Microbiol. 1991 Dec;29(12):2704–2709. [PMC free article] [PubMed]
  • Hofmann SL. Southwestern Internal Medicine Conference: Shiga-like toxins in hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. Am J Med Sci. 1993 Dec;306(6):398–406. [PubMed]
  • Jackson MP. Detection of Shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli by using polymerase chain reaction with incorporation of digoxigenin-11-dUTP. J Clin Microbiol. 1991 Sep;29(9):1910–1914. [PMC free article] [PubMed]
  • Jerse AE, Gicquelais KG, Kaper JB. Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli. Infect Immun. 1991 Nov;59(11):3869–3875. [PMC free article] [PubMed]
  • Jerse AE, Yu J, Tall BD, Kaper JB. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7839–7843. [PMC free article] [PubMed]
  • Johnson WM, Pollard DR, Lior H, Tyler SD, Rozee KR. Differentiation of genes coding for Escherichia coli verotoxin 2 and the verotoxin associated with porcine edema disease (VTe) by the polymerase chain reaction. J Clin Microbiol. 1990 Oct;28(10):2351–2353. [PMC free article] [PubMed]
  • Karch H, Heesemann J, Laufs R, O'Brien AD, Tacket CO, Levine MM. A plasmid of enterohemorrhagic Escherichia coli O157:H7 is required for expression of a new fimbrial antigen and for adhesion to epithelial cells. Infect Immun. 1987 Feb;55(2):455–461. [PMC free article] [PubMed]
  • Karch H, Meyer T. Evaluation of oligonucleotide probes for identification of shiga-like-toxin-producing Escherichia coli. J Clin Microbiol. 1989 Jun;27(6):1180–1186. [PMC free article] [PubMed]
  • Karch H, Meyer T. Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction. J Clin Microbiol. 1989 Dec;27(12):2751–2757. [PMC free article] [PubMed]
  • Karmali MA. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38. [PMC free article] [PubMed]
  • Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis. 1985 May;151(5):775–782. [PubMed]
  • Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun. 1977 Dec;18(3):775–779. [PMC free article] [PubMed]
  • Law D, Hamour AA, Acheson DW, Panigrahi H, Ganguli LA, Denning DW. Diagnosis of infections with Shiga-like toxin-producing Escherichia coli by use of enzyme-linked immunosorbent assays for Shiga-like toxins on cultured stool samples. J Med Microbiol. 1994 Apr;40(4):241–245. [PubMed]
  • Lawn AM, Orskov I, Orskov F. Morphological distinction between different H serotypes of Escherichia coli. J Gen Microbiol. 1977 Jul;101(1):111–119. [PubMed]
  • Levine MM, Nataro JP, Karch H, Baldini MM, Kaper JB, Black RE, Clements ML, O'Brien AD. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J Infect Dis. 1985 Sep;152(3):550–559. [PubMed]
  • Lin Z, Kurazono H, Yamasaki S, Takeda Y. Detection of various variant verotoxin genes in Escherichia coli by polymerase chain reaction. Microbiol Immunol. 1993;37(7):543–548. [PubMed]
  • Lindgren SW, Melton AR, O'Brien AD. Virulence of enterohemorrhagic Escherichia coli O91:H21 clinical isolates in an orally infected mouse model. Infect Immun. 1993 Sep;61(9):3832–3842. [PMC free article] [PubMed]
  • Louie M, de Azavedo J, Clarke R, Borczyk A, Lior H, Richter M, Brunton J. Sequence heterogeneity of the eae gene and detection of verotoxin-producing Escherichia coli using serotype-specific primers. Epidemiol Infect. 1994 Jun;112(3):449–461. [PMC free article] [PubMed]
  • McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1664–1668. [PMC free article] [PubMed]
  • Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun. 1983 Sep;41(3):1340–1351. [PMC free article] [PubMed]
  • Newland JW, Neill RJ. DNA probes for Shiga-like toxins I and II and for toxin-converting bacteriophages. J Clin Microbiol. 1988 Jul;26(7):1292–1297. [PMC free article] [PubMed]
  • O'Brien AD, Holmes RK. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. [PMC free article] [PubMed]
  • O'Brien AD, LaVeck GD, Thompson MR, Formal SB. Production of Shigella dysenteriae type 1-like cytotoxin by Escherichia coli. J Infect Dis. 1982 Dec;146(6):763–769. [PubMed]
  • Orskov I, Orskov F, Jann B, Jann K. Serology, chemistry, and genetics of O and K antigens of Escherichia coli. Bacteriol Rev. 1977 Sep;41(3):667–710. [PMC free article] [PubMed]
  • Pollard DR, Johnson WM, Lior H, Tyler SD, Rozee KR. Differentiation of Shiga toxin and Vero cytotoxin type 1 genes by polymerase chain reaction. J Infect Dis. 1990 Nov;162(5):1195–1198. [PubMed]
  • Read SC, Gyles CL, Clarke RC, Lior H, McEwen S. Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork, and chicken in southwestern Ontario. Epidemiol Infect. 1990 Aug;105(1):11–20. [PMC free article] [PubMed]
  • Samadpour M, Grimm LM, Desai B, Alfi D, Ongerth JE, Tarr PI. Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster. J Clin Microbiol. 1993 Dec;31(12):3179–3183. [PMC free article] [PubMed]
  • Samadpour M, Liston J, Ongerth JE, Tarr PI. Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples. Appl Environ Microbiol. 1990 May;56(5):1212–1215. [PMC free article] [PubMed]
  • Schmidt H, Beutin L, Karch H. Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect Immun. 1995 Mar;63(3):1055–1061. [PMC free article] [PubMed]
  • Schmidt H, Karch H, Beutin L. The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coli alpha-hemolysin family. FEMS Microbiol Lett. 1994 Apr 1;117(2):189–196. [PubMed]
  • Schmidt H, Plaschke B, Franke S, Rüssmann H, Schwarzkopf A, Heesemann J, Karch H. Differentiation in virulence patterns of Escherichia coli possessing eae genes. Med Microbiol Immunol. 1994 Feb;183(1):23–31. [PubMed]
  • Schoenhals G, Whitfield C. Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern. J Bacteriol. 1993 Sep;175(17):5395–5402. [PMC free article] [PubMed]
  • Siegler RL. Hemolytic uremic syndrome in children. Curr Opin Pediatr. 1995 Apr;7(2):159–163. [PubMed]
  • Smith HW, Green P, Parsell Z. Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs. J Gen Microbiol. 1983 Oct;129(10):3121–3137. [PubMed]
  • Tarr PI. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin Infect Dis. 1995 Jan;20(1):1–10. [PubMed]
  • Whittam TS, Wolfe ML, Wachsmuth IK, Orskov F, Orskov I, Wilson RA. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect Immun. 1993 May;61(5):1619–1629. [PMC free article] [PubMed]
  • Willshaw GA, Smith HR, Scotland SM, Field AM, Rowe B. Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol. 1987 May;133(5):1309–1317. [PubMed]
  • Yu J, Kaper JB. Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol. 1992 Feb;6(3):411–417. [PubMed]

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