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United States Patent (19) 11 Patent Number: 5,087,558 Webster, Jr

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United States Patent (19) 11 Patent Number: 5,087,558 Webster, Jr HHHHHHHHH USOO5087558A United States Patent (19) 11 Patent Number: 5,087,558 Webster, Jr. 45 Date of Patent: Feb. 11, 1992 54 METHOD FOR IDENTIFYING AND 1979, vol. 10, No. 2, "Overview of Automation and CHARACTERIZING ORGANISMS Identification," pp. 18-20, William J. Martin. 76) Inventor: John A. Webster, Jr., 8343 Carrleigh American Society for Microbiology News, vol. 49, No. 2, Pkwy., Springfield, Va. 22152 "Impact of Modern Taxonomy on Clinical Microbiol ogy," Don J. Brenner. - (*) Notice: The portion of the term of this patent Journal of Bacteriology, 158:376-378 (Apr. 1984), p. 378, subsequent to Jan. 5, 2005 has been Amikam et al. disclaimed. International Code of Nomenclature of Bacteria and Se (21) Appl. No.: 294,903 lected Statutes... Bacteriological Code, 1976 Revision; 22 Filed: Jan. 4, 1989 ASM, Washington, D.C., 1975. Nucleic Acids Research, vol. 10, No. 14, Jul. 24, 1982, Related U.S. Application Data Amikam et al, "Ribosomal RNA Genes in Myco 63) Continuation of Ser. No. 892,064, Aug. 1, 1986, aban plasma,' pp. 4215-4222. doned, which is a continuation of Ser. No. 477,431, Sawada et al., Mol. Gen. Genet. 182:502-504 (1981). Mar. 21, 1983, abandoned, which is a continuation-in Tu et al., Nucl. Acids Res, 10:7231-7235 (1982). part of Ser. No. 305,498, Sep. 25, 1981, Pat. No. Dunn et al., Cell 12:23-36 (1977). 4,717,653. Mattei et al., Chem. Absts, vol. 86, No. 19, p. 267, Ab 51 Int. Cl. .......................... C12Q 1/70; C12O 1/68 stract No. 1362(e) (1977). 52) U.S. C. ........................................... 435/5; 435/4; Moseley, S. L. et al., J. Infect. Dis. 142:892-898 (1980). 435/6; 435/34; 435/810; 436/804; 536/27; Acore, R. U., Current Topics in Microbiology and Im 935/78 munobiology 64:105-128 (1974), edited by Springer, New York. 58) Field of Search ........................................ 435/4-6, Boros et al., Nucl. Acids Res. 61817-1830 (1979). 435/29, 34, 35, 39, 41, 172.3, 803, 810; 436/64, Saillard, Colette, J. N. Bove, "Methods in Mycro 73, 81, 94,501, 504,543, 545, 804; 536/27, 28; plasma," vol. 1, New York, pp. 313-318. 935/78 Degorce-Dumas, S. R., Ricard Berenico, Bove, J. N., 56 References Cited "Methods in Mycroplasma," vol. 1, New York, pp. U.S. PATENT DOCUMENTS 319-325 (1983). International Search Report mailed Jul. 12, 1984 in 4.252,8974,234,683 11/19802/1981 Alfordeal.".McMillan ... a - - - - - - - 435/34 International Application No. PCT/US84/00382. 4,358,535 ll/1982 Falkow et al., .......................... 435/5 Primary Examiner-Robert A. Wax 4,396,713 8/1983 Simpson et al. ........................ 435/6 Assistant Examiner-Laurie Scheiner 4,717,653 1/1988 Webster .................................. 435/6 Attorney, Agent, or Firm-Sterne, Kessler, Goldstein & Fox OTHER PUBLICATIONS (57 ABSTRACT Arnot et al., Mol. Biochem. Parasitol., 3: 47-56 (1981). A method of characterizing an unknown organism spe Schmickel et al, Am. J. Ham. Genet; 32: 890-897 (1980). cies which comprises, determining the position of part Fox et al, Inter. J. System. Bacter, 27(1): 44-57 (1977). or whole of evolutionarily conserved DNA sequences Tompkins et al, J. Infect. Dis., 141 (5): 625-636 (1980). in DNA of the organism, relative to the position of Taylor et al, Biochim. Biophys. Acta, 442: 324-330 restriction endonuclease cleavage sites in the DNA, (1978). thereby to obtain an identifying genetic characteriza Ostapchuk et al, Molec. gen. Genet., 180: 475-477 tion of the unknown organism, and comparing the char. (1980). acterization with information from at least two sets of Lamppa et al, Mol. Gen. Genet., 182: 310-320 (1981). identifying genetic characterizations derived from the Arnheim et al, Natl. Acad. Sci. U.S.A., 77(12): same conserved sequences, each of the sets defining a 7323-7327 (1980). known organism species. Arnheim et al, Cell, 11:363-370 (1977). South Central Association for Clinical Microbiology News, 43 Claims, 16 Drawing Sheets U.S. Patent Feb. 11, 1992 Sheet 1 of 16 5,087,558 FIG. I. !9!!Hae O. - 94 - 7.6 - 59 - an a to a U.S. Patent Feb. 11, 1992 Sheet 3 of 16 5,087,558 FG, 3 s G s 2 g an9 O 88 s S.s is he O. C. B ni of n n < p his O. - p o O 9. 4 - a 5.9 - b his U.S. Patent Feb. 11, 1992 Sheet 4 of 16 5,087,558 FIG. 4 e isN s ass is g t E o al a nil all d -l M s P- 9.4- c oG 7. - 9 6.4 - Na U.S. Patent Feb. 11, 1992 Sheet 5 of 16 5,087,558 co N. co to O. d T S 9 cd co co co co to N N N S 9 5 c o o o o O O O 9 9 SR 9 o o co co co co co co Q co t t t t t E E E F.G. 5 in or n or or n or or or or or or Y V Ol Od M s O C2 N1 2.6 - 2.5 - 2. 15 - U.S. Patent Feb. 11, 1992 Sheet 7 of 16 5,087,558 9 O. E. H. ob6 a H i O 8 - a A. O. E. H. O C H OAO E. H. 69 O.S HS 99 OC H 99 O 9 A 9 O. E. H. 99 oe Hu 79 O C H S E 9 O E - U.S. Patent Feb. 11, 1992 Sheet 9 of 16 5,087,558 tas vs. c. C. c. N N Co go N O O o O O FIG. 9 c c. c. c. () & . s C Cld M t O 9 S2 2.6 are 2.3 - 2.2 - 2. 15 - 2. 13 - 2. Wimpos U.S. Patent Feb. 11, 1992 Sheet 10 of 16 5,087,558 ves R e N c N s c gs al F.G. 10 c o o O 9 co cy c 2 - 5 - C d M s O C Sa 44 - 3.4 - 3.2 - U.S. Patent Feb. 11, 1992 Sheet 11 of 16 5,087,558 F.G. ll B g Sac I v v N N C O c i i s as 7.0 - F. 2.9 - U.S. Patent Feb. 11, 1992 Sheet 12 of 16 5,087,558 ug RH 3077 DNA with Organs from RH 3O 77 no use DNA to total infected mouse 10 ug per well / Mouse DNA reas so cated S 18 S and 28 S Kilob as e 5 g 2 - 1 -2 -3 rRNA probe ad pairs u 1 O 10 104 10 O - 7.0 - O N- T 4.0 - p - 8.0 - C - 6.8- W () 8.0 - N- 3.3 - F.G. 12 U.S. Patent Feb. 11, 1992 Sheet 13 of 16 5,087,558 ?T?T55-ojeusn|no8nuu 5ño?yseuopsn?no8nuuºn s C FIG. 13 U.S. Patent Feb. 11, 1992 Sheet 14 of 16 5,087,558 StElg s KilobaseO SS pairs 8.3 - e. 6.8 - O F.G. 14 U.S. Patent Feb. 11, 1992 Sheet 15 of 16 5,087,558 coi t di s C g S.o: g g a a G o to 2 s E c co s O P s s E O ce E 9 s o s ei till c 8.5 - M . Cl M C 9 S2 3 - 7 - U.S. Patent Feb. 11, 1992 Sheet 16 of 16 5,087,558 5???t?š??r?5õT?T?T? CN GN. e | 1.5 - - 7. 6 - CN - 6 - FIG. 16 5,087,558 1. 2 wards, K. and Kossel, H., Nucleic Acids Research, METHOD FOR DENTIFYING AND 9:2853-2869 (1981); other related work (Bonen, L. and CHARACTERIZING ORGANISMS Gray, M. W., ibid, 8:319-335 (1980)) also further sup ports the general concept. This is a continuation of application Ser. No. 5 In this model the eukaryotic cell is a phylogenetic 06/892,064 filed Aug. 1, 1986, abandoned, which is a "chimera' with organelle components that are clearly continuation of application Ser. No. 06/477,431, filed prokaryotic in nature. The "prokaryotic-eukaryotic" Mar. 21, 1983, abandoned, which is a continuation-in dichotomy then, also has drawbacks, even as a broad part of application Ser. No. 305,498, filed Sept. 25, classification method. 1981, now U.S. Pat. No. 4,717,653, issued Jan. 5, 1988. 10 Where classification of organisms becomes more than a scientific exercise is in the identification of plants and FIELD OF THE INVENTION animals for hybridization and breeding purposes, and in The present invention relates to a method for the the accurate and reliable identification of microorgan rapid and accurate characterization and identification of isms which may infect so-called "higher' organisms or organisms, including prokaryotic and eukaryotic organ 15 other media. For example, the plant-breeder cattle breeder, or fish breeder may wish to have a quick and isms, such as bacteria, plants, and animals. reliable means of identifying different species and BRIEF DESCRIPTION OF THE PRIOR ART strains of their subjects. The veterinarian, physician, or The classification of living organisms has tradition horticulturist may wish to have an accurate identifica ally been done along more or less arbitrary and some 20 tion of any infectious organisms (parasites, fungi, bac what artificial lines. For example, the living world has teria, etc.) and viruses present in examined plant or been divided into two kingdoms: Plantae (plants) and animal tissues.
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