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 primer and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, configured to generate an amplicon that is between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 80% complementarity to a first portion of a region of Genbank gi number: 57634611, and said reverse primer configured to hybridize with at least 80% complementarity to a second portion of said region of Genbank gi number: 57634611, wherein said region of Genbank gi number: 57634611 begins with the 5′ end of SEQ ID NO.: 180, and extends to the 5′ end of SEQ ID NO.: 1321.
2 . The oligonucleotide primer pair of claim 1 , wherein said forward primer comprises at least 90% complementarity to said first portion of said region of Genbank gi number 57634611, and wherein said first portion is within a region beginning with the 5′ end of SEQ ID NO: 180 and extending to the 3′ end of SEQ ID NO.: 607.
3 . The oligonucleotide primer pair of claim 2 , wherein said forward primer comprises at least 95% complementarity to said first portion.
4 . The oligonucleotide primer pair of claim 3 , wherein said forward primer comprises 100% complementarity to said first portion.
5 . The oligonucleotide primer pair of claim 1 , wherein said reverse primer comprises at least 90% complementarity to said second portion of said region of Genbank gi number 57634611, and wherein said second portion is within a region beginning with the 3′ end of SEQ ID NO: 982 and extending to the 5′ end of SEQ ID NO.: 1321.
6 . The oligonucleotide primer pair of claim 5 , wherein said reverse primer comprises at least 95% complementarity to said second portion.
7 . The oligonucleotide primer pair of claim 6 , wherein said reverse primer comprises 100% complementarity to said second portion.
8 . The oligonucleotide primer pair of claim 1 , wherein said forward primer comprises at least 70% sequence identity with SEQ ID NO: 430.
9 . The oligonucleotide primer pair of claim 1 , wherein said forward primer is SEQ ID NO: 430.
10 . The oligonucleotide primer pair of claim 1 , wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 1321.
11 . The oligonucleotide primer pair of claim 1 , wherein said reverse primer is SEQ ID NO: 1321.
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 a Staphylococcus aureus bioagent comprising:
i) a first oligonucleotide primer pair comprising a forward primer and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, configured to generate an amplicon that is between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 80% complementarity to a first portion of a region of Genbank gi number: 57634611, and said reverse primer configured to hybridize with at least 80% complementarity to a second portion of said region of Genbank gi number: 57634611, wherein said region of Genbank gi number: 57634611 begins with the 5′ end of SEQ ID NO.: 180, and extends to the 5′ end of SEQ ID NO.: 1321; and
ii) at least one additional primer pair, wherein the primers of each of said at least one additional primer pair are designed to hybridize to conserved sequence regions within a Staphylococcus aureus gene selected from the group consisting of mecA, mecR1, ermA, ermC, pvluk, mupR, and nuc.
20 . The kit of claim 19 wherein each of said at least one additional primer pair comprises SEQ ID NO: 217:SEQ ID NO: 1167, SEQ ID NO: 399:SEQ ID NO: 1041, SEQ ID NO: 456:SEQ ID NO: 1261, SEQ ID NO: 174:SEQ ID NO: 853, SEQ ID NO: 288:SEQ ID NO: 1269, SEQ ID NO: 698:SEQ ID NO: 1420, or SEQ ID NO: 205:SEQ ID NO: 876.
21 . The kit of claim 19 wherein said first oligonucleotide primer pair and said at least one additional primer pair consists of eight oligonucleotide primer pairs having at least 70% sequence identity with the primer pairs: SEQ ID NO: 217:SEQ ID NO: 1167, SEQ ID NO: 399:SEQ ID NO: 1041, SEQ ID NO: 456:SEQ ID NO: 1261, SEQ ID NO: 430:SEQ ID NO: 1321, SEQ ID NO: 288:SEQ ID NO: 1269, SEQ ID NO: 698:SEQ ID NO: 1420, SEQ ID NO: 205:SEQ ID NO: 876, and SEQ ID NO: 174:SEQ ID NO: 853.
22 . A method for identifying a Staphylococcus aureus bioagent in a sample comprising:
a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair comprising a forward primer and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, said forward primer configured to hybridize with at least 80% complementarity to a first portion of a region of Genbank gi number: 57634611, and said reverse primer configured to hybridize with at least 80% complementarity to a second portion of said region of Genbank gi number: 57634611, wherein said region of Genbank gi number: 57634611 begins with the 5′ end of SEQ ID NO.: 180, and extends to the 5′ end of SEQ ID NO.: 1321, wherein said amplifying generates at least one amplification product that comprises between 45 and 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 database comprising a plurality of molecular masses of bioagent identifying amplicons, wherein a match between said determined molecular mass and a molecular mass comprised in said database identifies 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, wherein a match between said calculated base composition and a base composition comprised in said database identifies 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: 430.
27 . The method of claim 22 , wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 1321.
28 . The method of claim 22 further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair wherein the primers of each of said at least one additional primer pair are designed to hybridize to conserved sequence regions within a Staphylococcus aureus gene selected from the group consisting of mecA, mecR1, ermA, ermC, pvluk, tufb, mupR, and nuc.
29 . The method of claim 22 , wherein said identifying comprises detecting the presence of said Staphylococcus aureus bioagent in said sample.
30 . The method of claim 22 , wherein said identifying comprises determining either the sensitivity or the resistance of said Staphylococcus aureus bioagent in said sample to one or more antibiotics.
31 . The method of claim 22 , wherein said identifying comprises identifying a sub-species characteristic, strain, or genotype of said Staphylococcus aureus bioagent in said sample.