Compositions for use in identification of bacteria
The present invention provides oligonucleotide primers and compositions and kits containing the same for rapid identification of bacteria by amplification of a segment of bacterial nucleic acid followed by molecular mass analysis.
1 .- 30 . (canceled)
31 . A purified oligonucleotide primer pair comprising a forward primer and a reverse primer, said primer pair configured to generate an amplicon of between 54 consecutive nucleobases in length and 75 consecutive nucleobases in length from the sequence shown in GenBank accession number Y14051, said forward primer consisting of 15 to 24 consecutive nucleobases from SEQ ID NO: 183, and said reverse primer consisting of 15 to 27 consecutive nucleobases from SEQ ID NO: 538.
32 - 34 . (canceled)
35 . The purified oligonucleotide primer pair of claim 31 wherein the forward primer is SEQ ID NO: 183.
36 . The purified oligonucleotide primer pair of claim 31 wherein the reverse primer is SEQ ID NO: 538.
37 . The purified oligonucleotide primer pair of claim 31 wherein at least one of said forward primer or said reverse primer comprises at least one modified nucleobase.
38 . The purified oligonucleotide primer pair of claim 37 wherein said modified nucleobase is a mass modified nucleobase.
39 . The purified oligonucleotide primer pair of claim 37 wherein said mass modified nucleobase is 5-Iodo-C.
40 . The purified oligonucleotide primer pair of claim 37 wherein said modified nucleobase is a universal nucleobase.
41 . The purified oligonucleotide primer pair of claim 40 wherein said universal nucleobase is inosine.
42 . The purified oligonucleotide primer pair of claim 31 wherein at least one of said forward primer or said reverse primer comprises a non-templated T residue at its 5′-end.
43 . The purified oligonucleotide primer pair of claim 37 wherein said modified nucleobase comprises a molecular mass modifying tag.
44 - 53 . (canceled)
54 . A purified oligonucleotide pair, comprising a forward primer and a reverse primer, wherein said forward primer consists of 15 to 24 consecutive nucleobases selected from the sequence of SEQ ID NO: 183 and said reverse primer consists of 15 to 27 consecutive nucleobases selected from the sequence of SEQ ID NO: 538, which primer pair is configured to generate an amplicon between 54 and 100 consecutive nucleobases in length from the sequence shown in GenBank accession number Y14051.
55 . The purified oligonucleotide primer pair of claim 54 wherein at least one of said forward primer or said reverse primer comprises at least one modified nucleobase.
56 . The purified oligonucleotide primer pair of claim 55 wherein said modified nucleobase is a mass modified nucleobase.
57 . The purified oligonucleotide primer pair of claim 55 wherein said mass modified nucleobase is 5-Iodo-C.
58 . The purified oligonucleotide primer pair of claim 55 wherein said modified nucleobase is a universal nucleobase.
59 . The purified oligonucleotide primer pair of claim 58 wherein said universal nucleobase is inosine.
60 . The purified oligonucleotide primer pair of claim 54 wherein at least one of said forward primer or said reverse primer lacks a non-templated T residue at its 5′-end.
61 . The purified oligonucleotide primer pair of claim 55 wherein said modified nucleobase comprises a molecular mass modifying tag.
62 - 65 . (canceled)
66 . A kit comprising a purified oligonucleotide primer pair and at least one additional purified oligonucleotide primer pair selected from Table 1.
67 . A kit comprising a first primer pair as defined in claim 31 , a second primer pair configured to identify a respiratory pathogen by generating an amplicon from a gene encoding TUFB, and a third primer pair configured to identify a respiratory pathogen by generating an amplicon from at least one of a gene encoding 16S rRNA, a gene encoding 23S rRNA, a gene encoding INFB, a gene encoding RPLB, a gene encoding RPOC, or a combination thereof.
68 . The kit of claim 67 wherein said primer pair configured to generate an amplicon from a respiratory pathogen comprises primer pair no. 346, primer pair no. 361, primer pair no. 347, primer pair no. 348, primer pair no. 349, primer pair no. 360, primer pair no. 352, primer pair no. 356, primer pair no. 449, primer pair no. 354, primer pair no. 367 or a combination thereof.
69 . The kit of claim 67 wherein said first primer pair comprises a forward primer and reverse primer that hybridize between residues 4507 and 4610 of accession number Y14051.
70 . The kit of claim 69 wherein said first primer pair comprises a forward primer and reverse primer hybridize between residues 4507 and 4581 of accession number Y14051.
71 . The kit of claim 70 wherein said first primer pair is SEQ ID NOS: 183:539.
72 . The kit of claim 60 wherein said second primer pair is primer pair no. 367.