Kits for detecting hev nucleic acid

Abstract

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.

What is claimed is: 
     
         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.