COMPOSITIONS FOR USE IN IDENTIFICATION OF BACTERIA
The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.
1 . An oligonucleotide primer pair comprising a forward and a reverse primer, wherein said forward primer comprises the sequence of SEQ ID NO: 437, and said reverse primer comprises a sequence 90% identical to SEQ ID NO: 1232.
2 .- 8 . (canceled)
9 . The oligonucleotide primer pair of claim 1 wherein said forward primer is SEQ ID NO: 437.
10 . (canceled)
11 . The oligonucleotide primer pair of claim wherein said reverse primer is SEQ ID NO: 1232.
12 . The oligonucleotide primer pair of claim 1 wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.
13 . The oligonucleotide primer pair of claim 12 wherein at least one of said at least one modified nucleobase is a mass modified nucleobase.
14 . The oligonucleotide primer pair of claim 13 wherein said mass modified nucleobase is 5-Iodo-C.
15 . The composition of claim 13 wherein said mass modified nucleobase comprises a molecular mass modifying tag.
16 . The oligonucleotide primer pair of claim 12 wherein at least one of said at least one modified nucleobase is a universal nucleobase.
17 . The oligonucleotide primer pair of claim 16 wherein said universal nucleobase is inosine.
18 . The oligonucleotide primer pair of claim 1 wherein at least one of said forward primer and said reverse primer comprises a non-templated T residue at its 5′ end.
19 . A kit for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent comprising the oligonucleotide primer pair of claim 1 and at least one additional primer pair designed to hybridize to a Staphylococcus aureus gene encoding aroE, gmk, pta, tpi, yqi, or a combination thereof.
20 . The kit of claim 19 further comprising at least one other additional primer pair designed to hybridize to a Staphylococcus aureus gene encoding mecA, mecR1, pvluk, or a combination thereof.
21 . The kit of claim 19 wherein said at least one additional primer pair comprises SEQ ID NO: 590:891, SEQ ID NO: 474:869, SEQ ID NO: 268:1284, SEQ ID NO: 418:1301, SEQ ID NO: 318:1300, SEQ ID NO: 440:1076, SEQ ID NO: 219:1013, or a combination thereof.
22 . The kit of claim 19 wherein said oligonucleotide primer pair of claim 1 and said at least one additional primer pair consists of eight oligonucleotide primer pairs having at least 70% sequence identity with the primer pairs represented by: SEQ ID NOs:, SEQ ID NOs: 590:891, SEQ ID NOs: 474:869, SEQ ID NOs: 268:1284, SEQ ID NOs: 418:1301, SEQ ID NOs: 318:1300, SEQ ID NOs: 440:1076, and SEQ ID NOs: 219:1013.
23 . A method for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent in a sample comprising:
a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair targeted to a Staphylococcus aureus arcC gene comprising a forward and a reverse primer, each being between 13 and 35 linked nucleotides in length, wherein said forward primer comprises at least 70% complementarity to a first region within nucleotides 37-353 of a reference sequence, said reference sequence being a sequence extraction of coordinates 2725050-2724595 of Genbank gi number 21281729, and wherein said reverse primer comprises at least 70% complementarity to a second region within nucleotides 37-353 of said reference sequence, wherein said amplifying generates at least one amplification product that comprises between about 45 and about 200 linked nucleotides; and
b) determining the molecular mass of said at least one amplification product by mass spectrometry.
24 . The method of claim 23 further comprising comparing said determined molecular mass to a plurality of molecular masses of bioagent identifying amplicons, each indexed to said oligonucleotide primer pair and a Staphylococcus aureus bioagent, wherein a match between said determined molecular mass and one of said plurality of molecular masses identifies, determines one or more characteristic of, or detects said Staphylococcus aureus bioagent in said sample.
25 . The method of claim 23 further comprising calculating a base composition of said at least one amplification product using said molecular mass.
26 . The method of claim 25 further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of bioagent identifying amplicons, each indexed to said oligonucleotide primer pair and to a Staphylococcus aureus bioagent, wherein a match between said calculated base composition and a base composition in said database identifies, determines one or more characteristics of, or detects said Staphylococcus aureus bioagent in said sample.
27 . The method of claim 23 wherein said forward primer comprises at least 70% sequence identity with SEQ ID NO: 437.
28 . The method of claim 23 wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 1232.
29 . The method of claim 23 further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair designed to hybridize a Staphylococcus aureus gene encoding aroE, gmk, pta, tpi, or Yqi, arcC, or a combination thereof.
30 . The method of claim 23 further comprising repeating said amplifying and determining steps using at least three additional primer pairs, each designed to hybridize to a Staphylococcus aureus gene encoding aroE or gmk.
31 . A method for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent in a sample comprising:
c) amplifying a nucleic acid from said sample using at least four oligonucleotide primer pairs, each designed to hybridize to a Staphylococcus aureus gene and comprising a forward and a reverse primer, each being between 13 and 35 nucleobases in length, four of said at least four primer pairs comprising at least 70% sequence identity with the four primer pairs represented by SEQ ID NOs: 437:1232, SEQ ID NOs: 590:891, SEQ ID NOs: 474:869, and SEQ ID NOs: 268:1284, wherein said amplifying generates at least one amplification product that comprises between about 45 and about 200 linked nucleotides; and
d) determining the molecular mass of said at least one amplification product by mass spectrometry.