US00534.8854A United States Patent (19) 11 Patent Number: 5,348,854 Webster, Jr. 45) Date of Patent: Sep. 20, 1994 54 METHOD FORDETECTINGPROKARYOTIC vol. 10, No. 2, "Overview of Automation and Identifi ORGANISMS cation,” pp. 18-20, William J. Martin (1979). 76 Inventor: John A. Webster, Jr., 5 Kenmar Dr., American Society for Microbiology News, vol. 49, No. 2, Bldg. 5, Apt. 21, Billerica, Mass. "Impact of Modern Taxonomy on Microbiology,” Don 01821 J. Brenner. International Code of Nomenclature of Bacteria and 21) Appl. No.: 21,551 Selected Statutes... Bacteriological Code, 1976 Revi 22 Filed: Mar. 2, 1987 sions; ASM, Washington, D.C. (1975). Arnot et al., Mol. Biochem. Parasitol. 3:47-56 (1981). Related U.S. Application Data Dunn et al., Cell 12:23-36 (1977). Mattei et al., Chem. Absts, vol. 86, No. 19, p. 267, Ab 63) Continuation of Ser. No. 695,223, Jan. 25, 1985, aban doned, Continuation-in-part of Ser. No. 305,498, Sep. stract No. 1362(e) (1977). 25, 1981, Pat. No. 4,717,653. Moseley, S. L. et al., J. Infect. Dis. 142:892-898 (1980). Acore, R. U., Current Topics in Microbiology and Im 51 Int. Cl. ............................................... C12Q 1/68 munobiology 64:105-128 (1974), edited by Springer, 52 U.S. C. .......................................... 435/6; 435/34; New York. 435/172.1; 435/810; 436/504; 436/545; 436/501; 436/804 Boros et al., Nucl. Acids Res, 6:1817-1830 (1979). 58) Field of Search .................. 435/6, 34, 172.1, 810; Saillard, Colette, J. N. Bove, "Methods in Mycro 436/504, 543, 545, 801, 501; 535/695, 223, 78, plasma,’ vol. 1, New York, pp. 313–318. 27, 28 Degorce-Dumas, S. R., Ricard Berenico, Bove, J. N. "Methods in Mycroplasma,’ vol. 1, New York, pp. (56) References Cited 319-325 (1983). U.S. PATENT DOCUMENTS Brenner (1973) Int. J. Syst Bacteriol 23(4): 298–307. 4,234,683 11/1980 McMillan . Fox et al. (1980) Science 209:457-463. 4,252,897 2/1981 Oxford et al. Mordarski (1980) J. Gen Microbiol 118:313-319. 4,358,535 11/1982 Falkow et al. .......................... 435/5 Doi (1966) J. Bacteriol 92(1):88-96. 4,396,713 8/1983 Simpson et al. ....... Bohnert et al. (1980) Molec. gen. Genet. 179:539-549. 4,717,653 1/1988 Webster .................................. 435/6 (List continued on next page.) OTHER PUBLICATIONS Primary Examiner-Robert J. Hill, Jr. Sawada et al., Mol. Gen. Genet. 182:502-504 (1981). Assistant Examiner-Laurie Scheiner Amikam et al., Nucl. Acids Res. 10:4215-4222 (1982). Attorney, Agent, or Firm-Jorge A. Goldstein Tu et al., Nucl. Acids Res. 10:7231-7235 (1982). Amikam et al., J. Bacteriol. 158:376-378 (Apr. 1984). 57 ABSTRACT Schmickel et al., Am. J. Human Gen. 32:890-897 (1980). A method for detecting a prokaryotic organism while in Tompkins et al., J. Infect. Dis. 141:625-636 (1980). the presence of or associated with a eukaryotic organ Taylor et al., Biochim. Biophys. Acta. 442:324-330 ism which comprises selectively hybridizing ribosomal (1978). RNA (rRNA) sequences of the prokaryotic organism Ostapchuket al., Molec. Gen. Genet. 180:475-477 (1980). with a detectably labelled prokaryotic rRNA informa Lamppa et al., ibid. 182:310-320 (1981). tion-containing hybridization probe; and detecting the Arnheim et al., Proc. Natl. Acad. Sci. USA 77:7323-7327 label on the probe. (1980). Arnheim et al., Cell 11:363-370 (1977). South Central Association for Clinical Microbiology News 20 Claims, 16 Drawing Sheets 5,348,854 Page 2 OTHER PUBLICATIONS tection of Mycoplasma Contamination in Tissue. Cul Moore et al. (1967) J. Bacteriol 94(4):1066-1074. tures. Sinclair et al. (1971) Biochemistry 10:2761-2769. Amikam et al. Journal of Bacteriology 158:376-378 (Apr. Woese (1981) Sci American 244(6):98-122. 1984), “Mycoplasmas (Mollicutes) Have a Law Num Fox et al (1977) Int. J. Syst Bacteriol. 27(1):44-57. ber of rRNA Genes'. Amikam et al. (1982) Nucleic Acidso Research ofRazin Mycoplasmas etal, In Vitro Infecting 20:404-408 Cell (MayCultures 1984), by "DetectionDNA Hy 10(14):4215-4222. bridization'ridization'. Gobel et al (1984), Science 226:1211-1213. Kohne, D. E., PCT/US84/0016, published Jul. 19, Goebel et al. Abstracts of the Annual Meeting, 1984, G23, 1984, based on U.S. Application Ser. No. 456,729 filed "Use of Cloned Mycoplasma Ribosomal Genes for De- Jan. 10, 1983. U.S. Patent Sep. 20, 1994 Sheet 1 of 16 5,348,854 U.S. Patent Sep. 20, 1994 Sheet 2 of 16 5,348,854 RH809 RH811 RH815 Risi Ripo& Rh2 Rh U.S. Patent Sep. 20, 1994 Sheet 3 of 16 5,348,854 RSTUTZER RFLUORESCENS PPUTIDA A.ANITRATUS WOFF U.S. Patent Sep. 20, 1994 Sheet 4 of 16 5,348,854 PAERUGINOSA PSTUZER PFLUORESCENS PPUTDA A. ANTRATUS A.IWOFFII 94 - BAFSKLOBASE , ,-- U.S. Patent Sep. 20, 1994 Sheet 5 of 16 5,348,854 RH5063Ripelips RH3066 RH3068 | His RH3069 RH3OTO RH307 | grRH302 Rio RH306 ::::::::::::: & 3:33 KLOBASE PARS 2.6- 2.5- 2.5- 2.1- U.S. Patent Sep. 20, 1994 Sheet 8 of 16 5,348,854 RH3063 RH3O66 RH3068 RH3O69 RH3Of RH3O2 RH3O6 Ripsailipsirisposipo Ripte Rissol -- KLOBASE PARS 23 18 6. 3 1 9 O TO FIG. 8 U.S. Patent Sep. 20, 1994 Sheet 9 of 16 5,348,854 RH302 RH3033 RH3073 Rajor Rise? KILOBASE PAIRS U.S. Patent Sep. 20, 1994 Sheet 10 of 16 5,348,854 RH3021|Rior RH3033 Ripse RH3073 25 - KILOBASE PARS 3.4 - 3.2- FIG.O U.S. Patent Sep. 20, 1994 Sheet 11 of 16 5,348,854 Bg II SocI Rh3021 RH3021 8.5- TO - KILOBASE PARS 2.9- 2.5- FIG.IIA FIGifB U.S. Patent Sep. 20, 1994 Sheet 12 of 16 5,348,854 Mouse DNA Lg RH3077DNA with reOSSociated with Organs from RH3077 mouse DNA to total 18S (nd 28S infected mouse 10 pig per well rRNA probe RH3O77 lungs "Iheart liver. 19'- 10-in-2 toin-3 KLOBASE FIG2A FIG.12B FIG12C U.S. Patent Sep. 20, 1994 Sheet 13 of 16 5,348,854 MuS musculus meloSsinus MuS musculus domesticus Canis fomiliaris Cavia porcellus Cricetulus griseus Homo sapiens Felis Cotus Ratus norvegicus & MuS muSCulus MuS Cervicolor. Cervicolor Mus cervicolor papeuS MLS poly. 10 - 12 13 COOK --- KLOBASE PARS 26 - FIG.13 U.S. Patent Sep. 20, 1994 Sheet 14 of 16 5,348,854 Mus musculus domesticus Felis Cotus 1 | U.S. Patent Sep. 20, 1994 Sheet 15 of 16 5,348,854 Erythrocebus patas Ratus norvegicus Mus musculus domesticus Felis Catus Homo sapiens MOCOCOmulato -1- U.S. Patent Sep. 20, 1994 Sheet 16 of 16 5,348,854 22.0- i. 7.6- KILOBASE PAIRS 5,348,854 1. 2 (Bonen, L. and Gray, M. W., ibid, 8:319-335 (1980)) METHOD FOR DETECTING PROKARYOTC also further supports the general concept. ORGANISMS In this model the eukaryotic cell is a phylogenetic "chimera' with organelle components that are clearly This application is a continuation of application Ser. 5 prokaryotic in nature. The “prokaryotic-eukcaryotic' No. 695,223, filed Jan. 25, 1985, now abandoned, which dichotomy then, also has drawbacks, even as a broad is a continuation-in-part of application Ser. No. 305,498, classification method. filed Sep. 25, 1981, now U.S. Pat. No. 4,717,653. Where classification of organisms becomes more than BACKGROUND OF THE INVENTION a scientific exercise is in the identification of plants and animals for hybridization and breeding purposes, and in 1. Field of the Invention the accurate and reliable identification of microorgan The present invention relates to a method for the isms which may infect so-called "higher' organisms or rapid and accurate characterization and identification of other media. For example, the plant-breeder, cattle organisms, including parokaryotic and eukaryotic or breeder, or fish breeder may wish to have a quick and ganisms, such as bacteria, plants, animals. 15 2. Description of the Prior Art reliable means of identifying different species and The classification of living organisms has tradition strains of their subjects. The veterinarian, physician, or ally been done along more or less arbitrary and some horticulturist may wish to have an accurate identifica what artificial lines. For example, the living world has tion of any infectious organisms (parasites, fungi, bac been divided into two kingdoms: Plantae (plants) and 20 teria, etc. ) and viruses present in examined plant or Animalia (animals). This classification works well for animal tissues. The correct identification of species of generally familiar organisms, but becomes difficult for these organisms and viruses is of particular importance. such organisms as unicellular ones (e.g., green flagel The problem can best be illustrated by referring to lates, bacteria, blue-green algae), since these differ in the identification of bacteria. Names of bacterial species fundamental ways from the “plants' and "animals'. 25 usually represent many strains, and a strain is consid It has been suggested to simply divide organisms with ered to be a population derived from a single cell. respect to the internal architecture of the cell. In this Strains of a species have similar sets of attributes which scheme, all cellular organisms are either prokaryotic or serve to define the species. Precise definitions of bacte eukaryotic. Prokaryotes are less complex than euka rial species are difficult to express because subjective ryotes, they lack internal compartmentalization by unit 30 limits to strain diversity within species are required to membrane systems, and lack a defined nucleus. Prokar define species boundaries. (Buchanan, R. E., Interna yotic genetic information is carried in the cytoplasm on tional Bulletin of Bacteriological Nomenclature and double-stranded, circular DNA; no other DNA is pres Taxonomy, 15:25-32 (1965)).