METHODS FOR IDENTIFICATION OF SEPSIS-CAUSING BACTERIA
First Claim
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1. An oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length, said primer pair configured to generate an amplification product between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number:
- 49175990, and said reverse primer configured to hybridize with at least 70% complementarity to said second portion of said region.
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Abstract
The present invention provides compositions, kits and methods for rapid identification and quantification of sepsis-causing bacteria by molecular mass and base composition analysis.
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Citations
55 Claims
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1. An oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length, said primer pair configured to generate an amplification product between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number:
- 49175990, and said reverse primer configured to hybridize with at least 70% complementarity to said second portion of said region.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. An oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length wherein said forward primer has at least 70% sequence identity with SEQ ID NO:
- 1448 and said reverse primer has at least 70% sequence identity with SEQ ID NO;
1461. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
- 1448 and said reverse primer has at least 70% sequence identity with SEQ ID NO;
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31. A kit for identifying a sepsis-causing bacterium, comprising:
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i) a first oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length, said primer pair configured to generate an amplification product that is between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number;
49175990, and said reverse primer configured to hybridize with at least 70% complementarity to a second portion of said region; andii) at least one additional primer pair, wherein the primers of each of said at least one additional primer pair are configured to hybridize to conserved sequence regions within a bacterial gene selected from the group consisting of;
16S rRNA, 23S rRNA, tufB, rpoB, valS, rplB, and gyrB. - View Dependent Claims (32, 33, 34)
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35. A method for identifying a sepsis-causing bacterium in a sample, comprising:
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a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length, said primer pair configured to generate an amplification product that is between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number;
49175990, and said reverse primer configured to hybridize with at least 70% complementarity to a second portion of said region;
wherein said amplifying step generates at least one amplification product that comprises between 45 and 200 linked nucleotides; andb) determining the molecular mass of said at least one amplification product by mass spectrometry. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44)
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45. A method for identification of a sepsis-causing bacterium in a sample comprising:
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obtaining a plurality of amplification products using one or more primer pairs that hybridize to ribosomal RNA and one or more primer pairs that hybridize to a housekeeping gene; measuring molecular masses of said plurality of amplification products; calculating base compositions of said amplification products from said molecular masses; and comparing said base compositions to known base compositions of amplification products of known sepsis-causing bacteria produced with said one or more primer pairs, thereby identifying said sepsis-causing bacterium in said sample. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53)
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54. A kit for identification of a sepsis-causing bacterium comprising one or more primer pairs that hybridize to ribosomal RNA wherein each member of said one or more primer pairs is between 13 to 35 nucleobases in length and has at least 70% sequence identity with the corresponding member of primer pair number 346 (SEQ ID NOs:
- 202;
1110), 347 (SEQ ID NOs;
560;
1278), 348 (SEQ ID NOs;
706;
895), 349 (SEQ ID NOs;
401;
1156), 360 (SEQ ID NOs;
409;
1434) or 361 (SEQ ID NOs;
697;
1398). - View Dependent Claims (55)
- 202;
Specification