COMPOSITIONS FOR USE IN IDENTIFICATION OF STRAINS OF HEPATITIS C VIRUS
The present invention provides compositions, kits and methods for rapid identification and quantification of strains of hepatitis C viruses by molecular mass and base composition analysis.
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 9177 to 9337 of Genbank Accession Number: NC — 001433.1, and said reverse primer configured to hybridize with at least 70% complementarity to said second portion of said region.
2 . The oligonucleotide primer pair of claim 1 , wherein said forward primer has at least 70% sequence identity with SEQ ID NO: 2.
3 . The oligonucleotide primer pair of claim 2 , wherein said forward primer comprises at least 80% sequence identity with SEQ ID NO: 2.
4 . The oligonucleotide primer pair of claim 3 , wherein said forward primer comprises at least 90% sequence identity with SEQ ID NO: 2.
5 . The oligonucleotide primer pair of claim 1 , wherein said forward primer is SEQ ID NO: 29.
6 . The oligonucleotide primer pair of claim 1 , wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 29.
7 . The oligonucleotide primer pair of claim 6 , wherein said reverse primer comprises at least 80% sequence identity with SEQ ID NO: 29.
8 . The oligonucleotide primer pair of claim 7 , wherein said reverse primer comprises at least 90% sequence identity with SEQ ID NO: 29.
9 . The oligonucleotide primer pair of claim 1 , wherein said reverse primer is SEQ ID NO: 29.
10 . 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.
11 . The oligonucleotide primer pair of claim 10 , wherein at least one of said at least one modified nucleobases is a mass modified nucleobase.
12 . The oligonucleotide primer pair of claim 11 , wherein said mass modified nucleobase is 5-Iodo-C.
13 . The composition of claim 11 , wherein said mass modified nucleobase comprises a molecular mass modifying tag.
14 . The oligonucleotide primer pair of claim 10 , wherein at least one of said at least one modified nucleobases is a universal nucleobase.
15 . The oligonucleotide primer pair of claim 14 , wherein said universal nucleobase is inosine.
16 . 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.
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: 2 and said reverse primer has at least 70% sequence identity with SEQ ID NO: 29.
18 . The oligonucleotide primer pair of claim 17 , wherein said forward primer comprises at least 80% sequence identity with SEQ ID NO: 2.
19 . The oligonucleotide primer pair of claim 18 , wherein said forward primer comprises at least 90% sequence identity with SEQ ID NO: 2.
20 . The oligonucleotide primer pair of claim 17 , wherein said forward primer is SEQ ID NO: 2.
21 . The oligonucleotide primer pair of claim 17 , wherein said reverse primer comprises at least 80% sequence identity with SEQ ID NO: 29.
22 . The oligonucleotide primer pair of claim 21 , wherein said reverse primer comprises at least 90% sequence identity with SEQ ID NO: 29.
23 . The oligonucleotide primer pair of claim 17 wherein said reverse primer is SEQ ID NO: 29.
24 . The oligonucleotide primer pair of claim 17 , wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.
25 . The oligonucleotide primer pair of claim 24 , wherein at least one of said at least one modified nucleobases is a mass modified nucleobase.
26 . The oligonucleotide primer pair of claim 25 , wherein said mass modified nucleobase is 5-Iodo-C.
27 . The oligonucleotide primer of claim 25 , wherein said mass modified nucleobase comprises a molecular mass modifying tag.
28 . The oligonucleotide primer pair of claim 17 , wherein at least one of said at least one modified nucleobases is a universal nucleobase.
29 . The oligonucleotide primer pair of claim 28 , wherein said universal nucleobase is inosine.
30 . The oligonucleotide primer pair of claim 17 , wherein at least one of said forward primer and said reverse primer comprises a non-templated T residue at its 5′ end.
31 . A kit for identifying a strain of hepatitis C virus, comprising:
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 9177 to 9337 of Genbank Accession Number: NC — 001433.1, and said reverse primer configured to hybridize with at least 70% complementarity to a second portion of said region; and
ii) 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 genome segments of a hepatitis C genome, said genome segments selected from the group consisting of: NS2, NS3 and NS5.
32 . The kit of claim 31 , wherein each of said at least one additional primer pairs is a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each between 13 to 35 linked nucleotides in length and each having at least 70% sequence identity with the corresponding forward and reverse primers of primer pair numbers: 3683 (SEQ ID NOs: 4:21), 3684 (SEQ ID NOs: 13:17), 3685 (SEQ ID NOs: 7:18), 3686 (SEQ ID NOs: 7:30), 3687 (SEQ ID NOs: 5:24), 3688 (SEQ ID NOs: 14:24), or 3689 (SEQ ID NOs: 14:15),
33 . A method for identifying a strain of hepatitis C virus 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 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 9177 to 9337 of Genbank Accession Number: NC — 001433.1, 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; and
b) determining the molecular mass of said at least one amplification product by mass spectrometry.
34 . The method of claim 33 , further comprising comparing said 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 in said database identifies said strain of hepatitis C virus in said sample.
35 . The method of claim 33 , further comprising calculating a base composition of said at least one amplification product using said molecular mass.
36 . The method of claim 35 , 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 included in said database identifies said strain of hepatitis C virus in said sample.
37 . The method of claim 33 , wherein said forward primer has at least 70% sequence identity with SEQ ID NO: 2.
38 . The method of claim 33 , wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 29.
39 . The method of claim 33 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 genome segments of a hepatitis C genome, said genome segments selected from the group consisting of: NS2, NS3 and NS5.
40 . The method of claim 33 , wherein said molecular mass identifies the presence of said strain of hepatitis C virus in said sample.
41 . The method of claim 40 , further comprising determining either sensitivity or resistance of said strain of hepatitis C virus in said sample to one or more anti-viral drugs.
42 . The method of claim 33 , wherein said molecular mass identifies a sub-species characteristic, strain, or genotype of said strain of hepatitis C virus in said sample.
43 . The method of claim 42 , wherein said strain of hepatitis C virus is 1a-HCV-1, 1a-M67463, 1b-D90208, 1b-M58335, 1b-HCVT094, 1b-D89815, 1b-HCV-N, 1b-HCV-A, 1b-AB016785, 1b-AB016785, 1b-M96362, 1c-India, 2k-VAT96, 2a-HC-J6, 2b-MA, 2c-BEBE1, 3k-JK049, 3b-Tr.kj, 4a-ed43, 5a-EUH1480, 6a-6a33,6b-Th580, 6d-VN235, 6g-JK046, 6h-VN004, or 6k-VN 4 O 5 .
44 . The method of claim 43 , wherein said sample is a blood sample obtained from a human.
45 . The method of claim 44 , further comprising selecting an anti-viral drug known to decrease titer levels of said hepatitis C virus and treating said human with said anti-viral drug.
46 . The method of claim 43 further comprising analyzing said sample which contains mixed population of strains or quasispecies of hepatitis C virus and determining the relative ratio of a strain of hepatitis C virus which is resistant to a given anti-viral drug, relative to strains of hepatitis c virus which are sensitive to a given anti-viral drug.