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J Clin Microbiol. Nov 1990; 28(11): 2485–2490.
PMCID: PMC268211

Practical and economical method for using biotinylated DNA probes with bacterial colony blots to identify diarrhea-causing Escherichia coli.


A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.

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Selected References

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  • Baudry B, Savarino SJ, Vial P, Kaper JB, Levine MM. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis. 1990 Jun;161(6):1249–1251. [PubMed]
  • Bhan MK, Bhandari N, Sazawal S, Clemens J, Raj P, Levine MM, Kaper JB. Descriptive epidemiology of persistent diarrhoea among young children in rural northern India. Bull World Health Organ. 1989;67(3):281–288. [PMC free article] [PubMed]
  • Bhan MK, Khoshoo V, Sommerfelt H, Raj P, Sazawal S, Srivastava R. Enteroaggregative Escherichia coli and Salmonella associated with nondysenteric persistent diarrhea. Pediatr Infect Dis J. 1989 Aug;8(8):499–502. [PubMed]
  • Bhan MK, Raj P, Levine MM, Kaper JB, Bhandari N, Srivastava R, Kumar R, Sazawal S. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J Infect Dis. 1989 Jun;159(6):1061–1064. [PubMed]
  • Bilge SS, Clausen CR, Lau W, Moseley SL. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. J Bacteriol. 1989 Aug;171(8):4281–4289. [PMC free article] [PubMed]
  • Black RE, Lopez de Romaña G, Brown KH, Bravo N, Bazalar OG, Kanashiro HC. Incidence and etiology of infantile diarrhea and major routes of transmission in Huascar, Peru. Am J Epidemiol. 1989 Apr;129(4):785–799. [PubMed]
  • Chatkaeomorakot A, Echeverria P, Taylor DN, Bettelheim KA, Blacklow NR, Sethabutr O, Seriwatana J, Kaper J. HeLa cell-adherent Escherichia coli in children with diarrhea in Thailand. J Infect Dis. 1987 Oct;156(4):669–672. [PubMed]
  • Grunstein M, Hogness DS. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. [PMC free article] [PubMed]
  • Haas MJ, Fleming DJ. Use of biotinylated DNA probes in colony hybridization. Nucleic Acids Res. 1986 May 12;14(9):3976–3976. [PMC free article] [PubMed]
  • Haas MJ, Fleming DJ. A simplified lysis method allowing the use of biotinylated probes in colony hybridization. Anal Biochem. 1988 Feb 1;168(2):239–246. [PubMed]
  • Huovinen S, Huovinen P, Jacoby GA. Reliability of biotinylated DNA probes in colony hybridization: evaluation of an improved colony lysis method for detection of TEM-1 beta-lactamase. Mol Cell Probes. 1988 Mar;2(1):83–85. [PubMed]
  • Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis. 1987 Mar;155(3):377–389. [PubMed]
  • Levine MM, Prado V, Robins-Browne R, Lior H, Kaper JB, Moseley SL, Gicquelais K, Nataro JP, Vial P, Tall B. Use of DNA probes and HEp-2 cell adherence assay to detect diarrheagenic Escherichia coli. J Infect Dis. 1988 Jul;158(1):224–228. [PubMed]
  • Levine MM, Xu JG, Kaper JB, Lior H, Prado V, Tall B, Nataro J, Karch H, Wachsmuth K. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. J Infect Dis. 1987 Jul;156(1):175–182. [PubMed]
  • Maas R. An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid. 1983 Nov;10(3):296–298. [PubMed]
  • MacDonald KL, O'Leary MJ, Cohen ML, Norris P, Wells JG, Noll E, Kobayashi JM, Blake PA. Escherichia coli O157:H7, an emerging gastrointestinal pathogen. Results of a one-year, prospective, population-based study. JAMA. 1988 Jun 24;259(24):3567–3570. [PubMed]
  • Moseley SL, Huq I, Alim AR, So M, Samadpour-Motalebi M, Falkow S. Detection of enterotoxigenic Escherichia coli by DNA colony hybridization. J Infect Dis. 1980 Dec;142(6):892–898. [PubMed]
  • Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine MM. Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe. J Infect Dis. 1985 Sep;152(3):560–565. [PubMed]
  • Sethabutr O, Hanchalay S, Echeverria P, Taylor DN, Leksomboon U. A non-radioactive DNA probe to identify Shigella and enteroinvasive Escherichia coli in stools of children with diarrhoea. Lancet. 1985 Nov 16;2(8464):1095–1097. [PubMed]
  • So M, Dallas WS, Falkow S. Characterization of an Escherichia coli plasmid encoding for synthesis of heat-labile toxin: molecular cloning of the toxin determinant. Infect Immun. 1978 Aug;21(2):405–411. [PMC free article] [PubMed]
  • Taub F, Thompson EB. An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: in situ purification and stable binding of DNA on paper filters. Anal Biochem. 1982 Oct;126(1):222–230. [PubMed]
  • Taylor DN, Echeverria P, Pál T, Sethabutr O, Saiborisuth S, Sricharmorn S, Rowe B, Cross J. The role of Shigella spp., enteroinvasive Escherichia coli, and other enteropathogens as causes of childhood dysentery in Thailand. J Infect Dis. 1986 Jun;153(6):1132–1138. [PubMed]
  • Taylor DN, Echeverria P, Pitarangsi C, Seriwatana J, Sethabutr O, Bodhidatta L, Brown C, Herrmann JE, Blacklow NR. Application of DNA hybridization techniques in the assessment of diarrheal disease among refugees in Thailand. Am J Epidemiol. 1988 Jan;127(1):179–187. [PubMed]
  • Wood PK, Morris JG, Jr, Small PL, Sethabutr O, Toledo MR, Trabulsi L, Kaper JB. Comparison of DNA probes and the Sereny test for identification of invasive Shigella and Escherichia coli strains. J Clin Microbiol. 1986 Sep;24(3):498–500. [PMC free article] [PubMed]

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