Specific DNA probes in diagnostic microbiology

• US 4,358,535 A
• Filed: 12/08/1980
• Issued: 11/09/1982
• Est. Priority Date: 12/08/1980
• Status: Expired due to Term

• Current Assignee: Board of Regents of The University of Washington (University of Washington)
• Original Assignee: Board of Regents of The University of Washington (University of Washington)
• Inventors: Falkow, Stanley, Moseley, Stephen L.
• Primary Examiner(s): Warden, Robert J.
• Application Number:
  US06/213,803
• Time in Patent Office: 701 Days
• Field of Search: 435/5, 435/6, 435/7, 435/29, 435/34, 435/35, 435/36, 435/37, 435/38, 435/39, 435/40, 23/230 B, 424/2, 424/8, 424/12, 424/13
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

Method and compositions for infectious disease diagnosis and epidemiology involving labeled nucleotide probes complementary to nucleic acid coding for a characteristic pathogen product. Clinical isolates are cultivated, expanding the number of microorganisms, the resulting colonies lysed, the genome normally denatured and then fixed. Alternatively, clinical samples (stool, sputum, pus, etc.) are spotted onto an inert support. The sample is treated in such a way that the DNA is liberated from microbes present in the sample and complexed onto the support. The DNA is normally denatured and fixed in this process. Subsequently, a labelled polynucleotide probe specific for a DNA sequence characteristic of a pathogenic product suspected of being present in the clinical sample is contacted with the fixed genomic single stranded nucleic acid under hybridizing conditions. Hybridization of probes to the single stranded nucleic acid is diagnostic of the presence of the pathogen.

Claims

1. A method for detecting the presence of a pathogen in a clinical sample suspected of containing said pathogen, said method comprising:

depositing said sample on an inert support;

treating said sample to affix genetic material of any of said pathogen present in said sample to said support in substantially single stranded form at substantially the same site on said support where said sample was deposited;

contacting said fixed single stranded genetic material with a labeled probe having a nucleotide sequence of at least about 25 bases at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said pathogen, said contacting being under hybridizing conditions at a predetermined stringency; and

detecting duplex formation on said support by means of said label.

2. A method according to claim 1, including the additional step of cultivating said deposited sample to produce at least one individual colony.

3. A method according to claims 1 or 2, wherein said depositing is performed by streaking or spotting.

4. A method according to claim 2, wherein said support is an inert porous filter and said cultivating comprises:

maintaining said filter in contact with a nutrient gel; and

said contacting comprises

placing said filter on a bibulous material wetted with reagent solution capable of removing other than genetic material to leave single stranded genetic material.

5. A method according to claims 1, 2 or 4, wherein said pathogen is a unicellular organism.

6. A method according to claim 5, wherein said unicellular organism is a bacterium.

7. A method according to claims 1, 2 or 4, wherein said pathogen is a virus.

8. A method according to claims 1, 2 or 4, wherein said pathogen is a multicellular organism.

9. A method according to claims 1, 2 or 4, wherein said structural gene codes for an excreted product.

10. A method according to claims 1, 2 or 4, wherein said structural gene codes for a cytoplasmic product.

11. A method for detecting the presence of a unicellular pathogen in a sample suspected of containing said pathogen, said method comprising:

depositing said sample on an inert porous filter as a plurality of individual portions;

transferring said filter to a bibulous material wetted with a reagent solution capable of lysing said pathogen and denaturing the genetic material of said pathogen to provide single stranded DNA;

heating said filter to fix said single stranded DNA at substantially the same site as the individual portion from which said genetic material is derived;

contacting said fixed single stranded DNA with a labeled probe having a nucleotide sequence of at least about 25 bases at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said pathogen under hybridizing conditions of a predetermined stringency,; and

detecting duplex formation on said support by means of said label.

12. A method according to claim 11, including the step of:

cultivating said individual portions on said inert porous filter by contacting said filter with a nutrient gel to produce individual colonies of said pathogen, and wherein said reagent solution is a dilute aqueous alkaline solution.

13. A method according to claims 11 or 12, wherein said label is a radionuclide.

14. A method according to claims 11 or 12, wherein said label is a fluorescent molecule.

15. A method according to claims 11 or 12, wherein said unicellular pathogen is a bacterium.

16. A method according to claim 15, wherein said label is a radionuclide.

17. A method for detecting the presence of a gram negative bacillus in a clinical isolate suspected of containing said bacillus, said method comprising:

spotting said clinical isolate onto an inert porous filter;

contacting said spotted inert porous filter with a nutrient gel, whereby nutrients diffuse to said bacillus in said spot, whereby a colony forms;

transferring said filter supporting said colony onto a bibulous material containing a reagent solution for lysing said bacillus and denaturing the genome of said bacillus to provide single stranded DNA at substantially the same site as said colony;

heating said filter to fix said single stranded DNA to said filter;

contacting said filter with said fixed single stranded DNA, with a radioactively labeled probe having a nucleotide sequence of at least about 25 bases and at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said bacillus under hybridizing conditions of a predetermined stringency; and

detecting duplex formation on said support by means of said radioactive isotope.

18. A method according to claim 17, wherein said structural gene codes for a released product.

19. A method according to claim 18, wherein said released product is a toxin.

20. A method according to claims 17, 18 or 19, wherein said bacillus is enterotoxigenic Escherichia coli.