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, each comprising between 13 and 35 linked nucleotides in length, designed to generate an amplicon that is between about 45 and about 200 linked nucleotides in length, wherein said forward primer comprises at least 80% complementarity to a first region within nucleotides 29-113 of a reference sequence, said reference sequence being a sequence extraction of coordinates 41798-41609 of Genbank gi number 21281729, and wherein said reverse primer comprises at least 80% complementarity to a second region within nucleotides 29-113 of said reference sequence.
2 . The oligonucleotide primer pair of claim 1 wherein said forward primer comprises at least 90% complementarity to said first region, and wherein said first region is within nucleotides 29-66 of said reference sequence.
3 . The oligonucleotide primer pair of claim 2 wherein said forward primer comprises at least 95% complementarity to said first region.
4 . The oligonucleotide primer pair of claim 3 wherein said forward primer comprises 100% complementarity to said first region.
5 . The oligonucleotide primer pair of claim 1 wherein said reverse primer comprises at least 90% complementarity to said second region, and wherein said second region is within nucleotides 81-113 of said reference sequence.
6 . The oligonucleotide primer pair of claim 5 wherein said reverse primer comprises at least 95% complementarity to said second region.
7 . The oligonucleotide primer pair of claim 6 wherein said reverse primer comprises 100% complementarity to said second region.
8 . The oligonucleotide primer pair of claim 1 wherein said forward primer comprises at least 70% sequence identity with SEQ ID NO: 698.
9 . The oligonucleotide primer pair of claim 8 wherein said forward primer is SEQ ID NO: 698.
10 . The oligonucleotide primer pair of claim 1 wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 1420.
11 . The oligonucleotide primer pair of claim 10 wherein said reverse primer is SEQ ID NO: 1420.
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 mecA, ermA, ermC, pvluk, tufB, nuc, mupR, or a combination thereof.
20 . The kit of claim 19 wherein said at least one additional primer pair comprises SEQ ID NOs: 217:1167, SEQ ID NOs: 399:1041, SEQ ID NOs: 456:1261, SEQ ID NOs: 174:853, SEQ ID NOs: 430:1321, SEQ ID NOs: 288:1269, SEQ ID NOs: 205:876 or a combination thereof.
21 . A kit for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent comprising eight oligonucleotide primer pairs having at least 70% sequence identity with the primer pairs represented by: SEQ ID NOs: 217:1167, SEQ ID NOs: 399:1041, SEQ ID NOs: 456:1261, SEQ ID NOs: 174:853, SEQ ID NOs: 430:1321, SEQ ID NOs: 288:1269, SEQ ID NOs: 698:1420, and SEQ ID NOs: 205:876.
22 . 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 mecR1 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 29-113 of a reference sequence, said reference sequence being a sequence extraction of coordinates 41798-41609 of Genbank gi number 21281729, and wherein said reverse primer comprises at least 70% complementarity to a second region within nucleotides 29-113 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.
23 . The method of claim 22 further comprising comparing said determined molecular mass to a plurality of molecular masses of bioagent identifying amplicons, each indexed to said oligonucleotide primer pair, 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.
24 . The method of claim 22 further comprising calculating a base composition of said at least one amplification product using said molecular mass.
25 . The method of claim 24 further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of bioagent identifying amplicons that are indexed to said oligonucleotide primer pair, 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.
26 . The method of claim 22 wherein said forward primer comprises at least 70% sequence identity with SEQ ID NO: 698.
27 . The method of claim 22 wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 1420.
28 . The method of claim 22 further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair designed to hybridize to a Staphylococcus aureus gene encoding mecA, ermA, ermC, pvluk, tufB, nuc, mupR, mecR1, or a combination thereof.
29 . The method of claim 22 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 tufB, mecA, or mupR.
30 . 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 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 linked nucleotides 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: 430:1321, SEQ ID NOs: 288:1269, SEQ ID NOs: 698:1420, and SEQ ID NOs: 205:876, 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 spectrometer.