Compositions and methods for detecting HEV nucleic acid
View Patent ↗Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of Hepatitis E Virus (HEV) nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.
1. A combination of at least two oligomers for determining the presence or absence of hepatitis E virus (HEV) in a sample, said oligomer combination comprising:
at least two amplification oligomers for amplifying a target region of an HEV target nucleic acid, wherein
(a) at least one amplification oligomer is selected from the group consisting of
(i) an oligomer comprising a target-hybridizing sequence consisting of a sequence that is from about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO:63 and that includes at least the sequence of SEQ ID NO:26, including RNA equivalents and DNA/RNA chimerics thereof, and
(ii) an oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:28, including RNA equivalents and DNA/RNA chimerics thereof; and
(b) at least one amplification oligomer that is a promoter primer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 or SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof and further comprising a promoter sequence joined to the 5′ end of the target hybridizing sequence.
2. A kit comprising a combination of at least two oligomers for determining the presence or absence of hepatitis E virus (HEV) in a sample, said oligomer combination comprising:
at least two amplification oligomers for amplifying a target region of an HEV target nucleic acid, wherein
(a) at least one amplification oligomer is selected from the group consisting of
(i) an oligomer comprising a target-hybridizing sequence consisting of a sequence that is from about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO:63 and that includes at least the sequence of SEQ ID NO:26, including RNA equivalents and DNA/RNA chimerics thereof, and
(ii) an oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:28, including RNA equivalents and DNA/RNA chimerics thereof; and
(b) at least one amplification oligomer that is a promoter primer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 or SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof and further comprising a promoter sequence joined to the 5′ end of the target hybridizing sequence.
3. A reaction mixture comprising a combination of at least two oligomers for determining the presence or absence of hepatitis E virus (HEV) in a sample, said oligomer combination comprising:
at least two amplification oligomers for amplifying a target region of an HEV target nucleic acid, wherein
(a) at least one amplification oligomer is selected from the group consisting of
(i) an oligomer comprising a target-hybridizing sequence consisting of a sequence that is from about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO:63 and that includes at least the sequence of SEQ ID NO:26, including RNA equivalents and DNA/RNA chimerics thereof, and
(ii) an oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:28, including RNA equivalents and DNA/RNA chimerics thereof; and
(b) at least one amplification oligomer that is a promoter primer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 or SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof and further comprising a promoter sequence joined to the 5′ end of the target hybridizing sequence.
4. The reaction mixture of claim 3 , wherein the at least one amplification oligomer of (a) comprises a target-hybridizing sequence consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66, including RNA equivalents and DNA/RNA chimerics thereof.
5. The reaction mixture of claim 3 , wherein the at least one amplification oligomer of (a) comprises a target-hybridizing sequence consisting of SEQ ID NO:29 or SEQ ID NO:64, including RNA equivalents and DNA/RNA chimerics thereof.
6. The reaction mixture of claim 3 , wherein the at least one amplification oligomer of (a) comprises the target-hybridizing sequence consisting of SEQ ID NO:28, including RNA equivalents and DNA/RNA chimerics thereof.
7. The reaction mixture of claim 6 , wherein the at least one amplification oligomer of (a) comprises a target-hybridizing sequence consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, including RNA equivalents and DNA/RNA chimerics thereof.
8. The reaction mixture of claim 3 , wherein the at least one amplification oligomer of (b) comprises a target-hybridizing sequence consisting of SEQ ID NO:24 or SEQ ID NO:46, including RNA equivalents and DNA/RNA chimerics thereof.
9. The reaction mixture of claim 3 , wherein the at least one amplification oligomer of (b) comprises a target-hybridizing sequence consisting of SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof.
10. The reaction mixture of claim 9 , wherein the nucleobase at position 1 of SEQ ID NO:56 is guanine (G).
11. The reaction mixture of claim 3 , wherein said combination comprises an amplification oligomer as in (a)(i), an amplification oligomer as in (a)(ii), a first amplification oligomer as in (b), and a second amplification oligomer as in (b).
12. The reaction mixture of claim 11 , wherein
the amplification oligomer as in (a)(i) comprises the target-hybridizing sequence consisting of SEQ ID NO: 65, or an RNA equivalent or DNA/RNA chimeric thereof;
the amplification oligomer as in (a)(ii) comprises the target-hybridizing sequence consisting of SEQ ID NO: 29, or an RNA equivalent or DNA/RNA chimeric thereof;
the first amplification oligomer as in (b) comprises the target-hybridizing sequence consisting of SEQ ID NO:24, or an RNA equivalent or DNA/RNA chimeric thereof; and
the second amplification oligomer as in (b) comprises the target-hybridizing sequence consisting of SEQ ID NO:56, or an RNA equivalent or DNA/RNA chimeric thereof.
13. The reaction mixture of claim 3 , wherein said combination comprises a first amplification oligomer as in (a)(ii), a second amplification oligomer as in (a)(ii), a first amplification oligomer as in (b), and a second amplification oligomer as in (b).
14. The reaction mixture of claim 3 , wherein the combination comprises a set of first, second, and third amplification oligomers comprising a set of first, second, and third target-hybridizing sequences, respectively, wherein the set of target-hybridizing sequences consists of:
(i) SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:24, including RNA equivalents and DNA/RNA chimerics thereof;
(ii) SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof;
(iii) SEQ ID NO:29, SEQ ID NO:24, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof;
(iv) SEQ ID NO:66, SEQ ID NO:24, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof;
(v) SEQ ID NO:65, SEQ ID NO:24, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof; [randl] or
(vi) SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof.
15. The reaction mixture of claim 3 , wherein the at least one amplification oligomer of (a) comprises a target-hybridizing sequence consisting of SEQ ID NO:31, SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:66, SEQ ID NO:65, SEQ ID NO:64, SEQ ID NO:62, SEQ ID NO:35, SEQ ID NO:34, SEQ ID NO:33, or SEQ ID NO:61, including RNA equivalents and DNA/RNA chimerics thereof.
16. The reaction mixture of claim 3 , wherein the promoter sequence is a T7 promoter sequence.
17. The reaction mixture of claim 16 , wherein the T7 promoter sequence has the sequence shown in SEQ ID NO:73.
18. The reaction mixture of claim 3 , further comprising at least one detectably labeled detection probe oligomer comprising a target-hybridizing sequence that is from about 14 to about 28 nucleotides in length and is configured to specifically hybridize to a target sequence contained within SEQ ID NO:39 or the complement thereof, and wherein the at least one detection probe oligomer contains a 2′-methoxy backbone at one or more linkages in the nucleic acid backbone.
19. The reaction mixture of claim 3 , wherein at least one of the amplification oligomers of (a) and (b) contains a 2′-methoxy backbone at one or more linkages in the nucleic acid backbone.