Quantitative detection of specific nucleic acid sequences using lambdoid bacteriophages linked by oligonucleotides to solid support

• US 5,679,510 A
• Filed: 01/05/1995
• Issued: 10/21/1997
• Est. Priority Date: 04/27/1993
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Hybridon, Inc. (Aceragen Incorporated)
• Inventors: Lin, Edmund C. C., Ray, Bryan L.
• Primary Examiner(s): Elliott, George G.
• Assistant Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/368,870
• Time in Patent Office: 1,020 Days
• Field of Search: 435/5, 435/6, 435/91.2, 435/29, 435/39, 435/181
• US Class Current: 435/5
• CPC Class Codes: A61K 47/6901 Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2795/10321 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2795/10322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2795/10345 Special targeting system for viral vectors
• CPC Class Codes: C12N 2795/10362 by genetic engineering
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The present invention provided compositions, methods and kits for detection and quantitation of pathogenic organisms. The composition of the invention is an oligonucleotide probe comprising a bacteriophage covalently linked to one site on an oligonucleotide probe complementary to a conserved region of a pathogenic organism. At a second site, the oligonucleotide probe is linked to a matrix. The oligonucleotide probe contains a region complementary to one strand of a restriciton endonuclease recognition site or an oligoribonucleotide moiety. The number of pathogenic organisms present in a biological fluid sample may be quantitated in accordance with the method of the invention by combining the composition of the invention with the sample, allowing hybridization to occur. Hybridization generates a DNA-RNA hybrid, and by adding the appropriate nucleolytic enzyme capable of cleaving DNA-RNA hybrids; bacteriophage will be released for measurement. The kit of the invention provides components which allow the method of the invention to be performed.

Claims

1. A method for quantitating a pathogenic organism in a biological fluid, the pathogenic organism being characterized by a double stranded deoxyribonucleic acid genome, which method comprises:

a) processing the biological fluid to release the genome of the pathogen;

b) combining the biological fluid with an oligoribonucleotide probe comprising a lambdoid bacteriophage having a surface polypeptide covalently linked to one site on a single stranded oligoribonucleotide, said oligoribonucleotide being linked via a second site to a matrix and being characterized by a nucleic acid sequence complementary to a conserved region of the genome of the pathogenic organism;

c) incubating the biological fluid and the oligoribonucleotide probe at a temperature and for a time sufficient to allow nucleic acid hybridization to occur without irreversibly destroying the infectivity of the lambdoid bacteriophage, thereby forming a hybridization mixture;

d) incubating the hybridization mixture with a ribonuclease at a temperature and for a time sufficient to allow cleavage of all deoxyribonucleic acid-ribonucleic acid hybrids, thereby releasing infective lambdoid bacteriophage;

e) measuring the number of infective lambdoid bacteriophage released in step d, wherein the number of infective lambdoid bacteriophage released corresponds to the number of pathogen genomes present in the biological fluid.

2. A method for quantitating a pathogenic organism in a biological fluid, the pathogenic organism being characterized by a double stranded deoxyribonucleic acid genome, which method comprises:

a) processing the biological fluid to release the genome of the pathogen;

b) combining the biological fluid with an oligoribonucleotide probe comprising a bacteriophage capable of infecting Bacillus subtilis, said bacteriophage having a surface polypeptide covalently linked to one site on a single stranded oligoribonucleotide, said oligoribonucleotide being linked via a second site to a matrix and being characterized by a nucleic acid sequence complementary to the conserved region of a genome of the pathogenic organism;

c) incubating the biological fluid and the oligoribonucleotide probe at a temperature and for a time sufficient to allow nucleic acid hybridization to occur without irreversibly destroying the infectivity of the bacteriophage, thereby forming a hybridization mixture;

d) incubating the hybridization mixture with a ribonuclease at a temperature and for a time sufficient to allow cleavage of all deoxyribonucleic acid-ribonucleic acid hybrids, thereby releasing infective bacteriophage;

e) measuring the number of infective bacteriophage released in step d, wherein the number of infective bacteriophage released corresponds to the number of pathogen genomes present in the biological fluid.

3. A method for quantitating a pathogenic organism in a biological fluid, the pathogenic organism being characterized by a double stranded deoxyribonucleic acid genome, which method comprises:

a) processing the biological fluid to release the genome of the pathogen;

b) combining the biological fluid with an oligoribonucleotide probe comprising a bacteriophage capable of infecting Lactobacillus lactis, said bacteriophage having a surface polypeptide covalently linked to one site on a single stranded oligoribonucleotide, said oligoribonucleotide being linked via a second site to a matrix and being characterized by a nucleic acid sequence complementary to a conserved region of the genome of the pathogenic organism;

c) incubating the biological fluid and the oligoribonucleotide probe at a temperature and for a time sufficient to allow nucleic acid hybridization to occur without irreversibly destroying the infectivity of the bacteriophage, thereby forming a hybridization mixture;

d) incubating the hybridization mixture with a ribonuclease at a temperature and for a time sufficient to allow cleavage of all deoxyribonucleic acid-ribonucleic acid hybrids, thereby releasing infective bacteriophage;

e) measuring the number of infective bacteriophage released in step d, wherein the number of infective bacteriophage released corresponds to the number of pathogen genomes present in the biological fluid.

4. A method for quantitating a pathogenic organism in a biological fluid, the pathogenic organism being characterized by a double stranded deoxyribonucleic acid genome, which method comprises:

a) processing the biological fluid to release the genome of the pathogen;

b) combining the biological fluid with an oligoribonucleotide probe comprising a bacteriophage capable of infecting attenuated strains of Pseudomonas, said bacteriophage having a surface polypeptide covalently linked to one site on a single stranded oligoribonucleotide, said oligoribonucleotide being linked via a second site to a matrix and being characterized by a nucleic acid sequence complementary to a conserved region of the genome of the pathogenic organism;

c) incubating the biological fluid and the oligoribonucleotide probe at a temperature and for a time sufficient to allow nucleic acid hybridization to occur without irreversibly destroying the infectivity of the bacteriophage, thereby forming a hybridization mixture;

d) incubating the hybridization mixture with a ribonuclease at a temperature and for a time sufficient to allow cleavage of all deoxyribonucleic acid-ribonucleic acid hybrids, thereby releasing infective bacteriophage;

e) measuring the number of infective bacteriophage released in step d, wherein the number of infective bacteriophage released corresponds to the number of pathogen genomes present in the biological fluid.

5. A method for quantitating a pathogenic organism in a biological fluid, the pathogenic organism being characterized by a double stranded deoxyribonucleic acid genome, which method comprises:

a) processing the biological fluid to release the genome of the pathogen;

b) combining the biological fluid with an oligoribonucleotide probe comprising a bacteriophage capable of infecting attenuated strains of Salmonella, said bacteriophage having a surface polypeptide covalently linked to one site on a single stranded oligoribonucleotide, said oligoribonucleotide being linked via a second site to a matrix and being characterized by a nucleic acid sequence complementary to a conserved region of the genome of the pathogenic organism;

c) incubating the biological fluid and the oligoribonucleotide probe at a temperature and for a time sufficient to allow nucleic acid hybridization to occur without irreversibly destroying the infectivity of the bacteriophage, thereby forming a hybridization mixture;

d) incubating the hybridization mixture with a ribonuclease at a temperature and for a time sufficient to allow cleavage of all deoxyribonucleic acid-ribonucleic acid hybrids, thereby releasing infective bacteriophage;

e) measuring the number of infective bacteriophage released in step d, wherein the number of infective bacteriophage released corresponds to the number of pathogen genomes present in the biological fluid.