Oligonucleotides For the Detection of Hepatitis B Virus
The invention relates to specific oligonucleotide primers and probes for rapid and sensitive detection of hepatitis B virus by amplification of the HBV nucleic acid, and to methods using the same. It is also concerned with kits useful to perform these detection tests.
1 : An oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and their complementary sequences.
2 : The oligonucleotide according to claim 1 , which consists of SEQ ID NO: 2 or its complementary sequence.
3 : The oligonucleotide according to claim 1 , which consists of SEQ ID NO: 3, or its complementary sequence.
4 : The oligonucleotide according to claim 1 , which consists of SEQ ID NO: 4, or its complementary sequence.
5 : The oligonucleotide according to claim 1 , which consists of SEQ ID NO: 5, or its complementary sequence.
6 : The oligonucleotide according to claim 1 , which consists of SEQ ID NO: 6, or its complementary sequence.
7 : The oligonucleotide according to claim 1 , which consists of SEQ ID NO: 7, or its complementary sequence.
8 : Use of an oligonucleotide as defined in claim 1 , as a probe or primer, for hybridizing with and optionally amplifying a nucleic acid from a hepatitis B virus (HBV).
9 : Use of an oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence, as a probe for hybridizing with a nucleic acid from HBV.
10 : The use according to claim 9 , wherein said oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence.
11 : The use according to claim 9 , wherein said oligonucleotide includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence and carries a fluorophore moiety at one terminus, and a quencher moiety at the other terminus.
12 : The use according to claim 11 , wherein said oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, and carries a fluorophore moiety at one terminus, and a quencher moiety at the other terminus.
13 : A set of oligonucleotides consisting of an oligonucleotide that includes SEQ ID NO:2, and at least an oligonucleotide selected from the group consisting of an oligonucleotide that includes SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
14 : A set of oligonucleotides according to claim 13 , which consists of:
(i) an oligonucleotide that includes SEQ ID NO:2, and an oligonucleotide that includes SEQ ID NO:3;
(ii) an oligonucleotide that includes SEQ ID NO:2, and an oligonucleotide that includes SEQ ID NO:4;
(iii) an oligonucleotide that includes SEQ ID NO:2, and an oligonucleotide that includes SEQ ID NO:5;
(iv) an oligonucleotide that includes SEQ ID NO:2, and an oligonucleotide that includes SEQ ID NO:6;
(v) an oligonucleotide that includes SEQ ID NO:2, and an oligonucleotide that includes SEQ ID NO:7;
(vi) an oligonucleotide that includes SEQ ID NO:2, an oligonucleotide that includes SEQ ID NO:4 and an oligonucleotide that includes SEQ ID NO:5; and
(vii) an oligonucleotide that includes SEQ ID NO:2, an oligonucleotide that includes SEQ ID NO:6 and an oligonucleotide that includes SEQ ID NO:7.
15 : A set of oligonucleotides comprising:
a) a set of oligonucleotides according to claim 13 ; and
b) an oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence.
16 : A set of oligonucleotides that comprises:
a) a set of oligonucleotides according to claim 13 ; and
b) an oligonucleotide that consists of a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, and carries a fluorophore moiety at one terminus, and a quencher moiety at the other terminus.
17 : A method for specifically detecting a HBV by amplification in a biological sample, which method comprises the steps consisting of:
a) contacting a set of oligonucleotides according to claim 13 with a biological sample or nucleic acid preparation obtained from a biological sample, under conditions suitable for the oligonucleotides to hybridize to a HBV nucleic acid present in the sample;
b) amplifying said HBV nucleic acid using said oligonucleotides as primers;
c) detecting the amplification product, indicative of the presence of a HBV in the biological sample.
18 : The method according to claim 17 , wherein HBV nucleic acid is amplified by polymerase chain reaction.
19 : The method according to claim 17 , wherein the detection of said amplification product is performed by using an oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence, and that is detectably labelled, as a probe.
20 : The method according to claim 19 , wherein said oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence, and carries a fluorophore moiety at one terminus, and a quencher moiety at the other terminus.
21 : The method according to claim 19 , wherein said oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence, is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.
22 : A kit for amplifying HBV in a biological sample, which kit comprises:
at least a set of oligonucleotides according to claim 13 , useful as primers;
means for amplifying a HBV nucleic acid.
23 : The kit according to claim 22 , which further comprises means for the detection of the amplified product.
24 : The kit according to claims 22 , wherein the means for amplifying HBV nucleic acid are means for amplification by Polymerase Chain Reaction.
25 : The kit according to claim 22 , which comprises an oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence, detectably labelled and useful as a probe.
26 : The kit according to claim 25 , wherein said oligonucleotide that includes a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and their complementary sequence, is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.