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

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.

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.

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.

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.