KITS FOR DETECTING HEV NUCLEIC ACID
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 kit for determining the presence or absence of hepatitis E virus (HEV) in a sample, the kit comprising:
(1) an amplification reagent 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 is a promoter primer comprising a target-hybridizing sequence consisting of SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:51, 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; and
(2) one or more reagents selected from the group consisting of
(a) a capture probe oligomer comprising a target-hybridizing sequence that is configured to specifically hybridize to an HEV target sequence, wherein the capture probe target-hybridizing sequence is covalently attached to a sequence or moiety that binds to an immobilized probe;
(b) a 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
(c) an enzyme reagent comprising a reverse transcriptase and an RNA polymerase.
2 . The kit 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, 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.
3 . The kit of claim 1 , 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.
4 . The kit of claim 1 , 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.
5 . The kit of claim 4 , 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.
6 . The kit 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.
7 . The kit 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.
8 . The kit of claim 7 , wherein the nucleobase at position 1 of SEQ ID NO:56 is guanine (G).
9 . The kit of claim 1 , wherein the amplification reagent 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).
10 . The kit of claim 9 , 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.
11 . The kit of claim 1 , wherein the amplification reagent 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).
12 . The kit of claim 1 , wherein the amplification reagent 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; or
(vi) SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA/RNA chimerics thereof.
13 . The kit of claim 1 , 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.
14 . The kit of claim 1 , wherein the capture probe target-hybridizing sequence consists of SEQ ID NO:4 or SEQ ID NO:42, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
15 . The kit of claim 1 , wherein the detection probe target-hybridizing sequence consists of SEQ I NO:55 or SEQ ID NO:67, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
16 . The kit of claim 1 , wherein the detection probe oligomer comprises a label selected from the group consisting of
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher; and
(d) a combination of two or more of (a), (b), and (c).
17 . The kit of claim 16 , wherein the label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the at least one detection probe oligomer.
18 . The kit of claim 1 , wherein the promoter sequence is a T7 promoter sequence and the RNA polymerase is a T7 RNA polymerase.
19 . The kit of claim 18 , wherein the T7 promoter sequence has the sequence shown in SEQ ID NO:73.
20 . The kit of claim 1 , wherein the reverse transcriptase is an MMLV reverse transcriptase.