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 three 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 and at least one detectably labeled detection probe oligomer, wherein
(a) at least one amplification oligomer is
an oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:28, including RNA equivalents and DNA/RNA chimerics thereof;
(b) at least one amplification oligomer 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 of the promoter primer; and
(c) at least one detectably labeled detection probe oligomer comprises 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 thererof.
2. The combination of at least three oligomers of claim 1 , 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.
3. The combination of at least three oligomers of claim 1 , wherein the at least one amplification oligomer of (a) comprises a target-hybridizing sequence consisting of SEQ ID NO:29, including RNA equivalents and DNA/RNA chimerics thereof.
4. The combination of at least three oligomers of claim 1 , wherein the at least one amplification oligomer of (a) comprises a target-hybridizing sequence consisting of SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:32, including RNA equivalents and DNA/RNA chimerics thereof.
5. The combination of at least three oligomers of claim 1 , 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.
6. The combination of at least three oligomers of claim 1 , 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.
7. The combination of at least three oligomers of claim 6 , wherein the nucleobase at position 1 of SEQ ID NO:56 is guanine (G).
8. The combination of at least three oligomers of claim 1 , wherein said combination comprises the amplification oligomer as in (a), a first amplification oligomer as in (b), a second amplification oligomer as in (b), and a fourth amplification oligomer,
wherein the fourth amplification oligomer comprises a target-hybridizing sequence consisting of 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.
9. The combination of at least three oligomers of claim 8 , wherein
the amplification oligomer as in (a) 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;
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; and
the fourth amplification oligomer comprises the target-hybridizing sequence consisting of SEQ ID NO:65, or an RNA equivalent or DNA/RNA chimeric thereof.
10. The combination of at least three oligomers of claim 1 , wherein said combination comprises a first amplification oligomer as in (a), a second amplification oligomer as in (a), a first amplification oligomer as in (b), and a second amplification oligomer as in (b).
11. The combination of at least three oligomers of claim 1 , 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 is
(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; or
(vi) SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof.
12. The combination of at least three oligomers of claim 1 , wherein the promoter sequence is a T7 promoter sequence.
13. The combination of at least three oligomers of claim 12 , wherein the T7 promoter sequence has the sequence shown in SEQ ID NO:73.
14. The combination of at least three oligomers of claim 1 , wherein the at least one detection probe oligomer contains a 2′-methoxy backbone at one or more linkages in the nucleic acid backbone.
15. A kit comprising the combination of at least three oligomers as in claim 1 .
16. A reaction mixture comprising the combination of at least three oligomers as in claim 1 .
17. A method for determining the presence or absence of hepatitis E virus (HEV) in a sample, said method comprising:
(1) contacting a sample, said sample suspected of containing HEV, with at least two oligomers for amplifying a target region of an HEV target nucleic acid, said oligomer combination comprising
(a) at least one amplification 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 of the promoter primer;
(2) performing an in vitro nucleic acid amplification reaction, wherein any HEV target nucleic acid present in the sample is used as a template for generating an amplification product; and
(3) contacting the amplification reaction with 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 thererof, wherein said contacting is performed under conditions whereby the presence or absence of the amplification product is determined, thereby determining the presence or absence of HEV in the sample.
18. The combination of at least three oligomers of claim 1 , 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.