Quantitative detection of specific nucleic acid sequences...

C - Chemistry – Metallurgy – 12 – Q

Patent

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Details

C12Q 1/70 (2006.01) C12N 11/06 (2006.01) C12Q 1/06 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2209523

The present invention provides 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 restriction 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 the intact restriction endonuclease site or a DNA-RNA hybrid, and by adding the appropriate restriction endonuclease or a 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.

La présente invention concerne des compositions, des procédés et des matériels pour détecter et quantifier des organismes pathogènes. La composition de l'invention est une sonde oligonucléotidique comprenant un bactériophage lié de manière covalente à un site sur une sonde oligonucléotidique complémentaire d'une région conservée d'un organisme pathogène. Sur un second site, la sonde oligonucléotidique est liée à une matrice. La sonde oligonucléotidique comprend une région complémentaire d'un brin d'un site de reconnaissance d'une endonucléase de restriction ou une fraction oligoribonucléotidique. Le nombre d'organismes pathogènes présents dans un échantillon de liquide biologique peut être quantifié selon le procédé décrit par l'invention en combinant la composition de l'invention avec l'échantillon et en permettant à l'hybridation de se faire. L'hybridation génère le site intact de l'endonucléase de restriction ou un hybride ADN-ARN, et par l'adjonction de l'endonucléase de restriction appropriée ou d'un enzyme nucléolytique capable de cliver les hybrides ADN-ARN, le bactériophage sera libéré et pourra être mesuré. Le matériel de l'invention comporte des constituants permettant de mettre en application le procédé décrit par l'invention.

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