Compositions and methods 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 combination of oligomers for determining the presence or absence of hepatitis E virus (HEV) in a sample, said oligomer combination comprising at least three amplification oligomers and at least one detection probe oligomer, wherein:
 the oligomer combinations are selected from the group consisting of 
 (a) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:46 and a further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:37; 
 (b) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:37; 
 (c) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:67; 
 (d) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:71; 
 (e) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:67; 
 (f) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:71; 
 (g) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:37; 
 (h) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:67; 
 (i) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:71; 
 (j) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a third amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:37; 
 (k) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a third amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:67; and 
 (1) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a third amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:71. 
   
     
     
         2 . The combination of oligomers of  claim 1 , wherein the third amplification oligomer of (a), (e), (f), (g), (h), (i), (j), (k) and/or (1) comprises a nucleotide sequence consisting of SEQ ID NO:15. 
     
     
         3 . The combination of oligomers of  claim 1 , wherein the third amplification oligomer of (b), (c), and/or (d) comprises a nucleotide sequence consisting of SEQ ID NO: 12. 
     
     
         4 . The combination of oligomers of  claim 1 , wherein the second amplification oligomer of (g), (h), (i), (j), (k) and/or (1) comprises a nucleotide sequence of SEQ ID NO: 12. 
     
     
         5 . The combination of oligomers of  claim 1 , wherein the combination comprises three detectably labeled detection probe oligomers, wherein (a), (b), (g), and (j) each further comprises a second detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:67 and a third detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:71. 
     
     
         6 . The combination of  claim 1 , wherein the detectable label is an acridinium ester compound. 
     
     
         7 . A combination of oligomers, wherein the oligomer combination comprises at least four amplification oligomers and at least one detection probe oligomer, wherein:
 the oligomer combination is selected from the group consisting of 
 (a) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a fourth amplification oligomer comprises a target-hybridizing sequence consisting of SEQ ID NO:29 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:37; 
 (b) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a fourth amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:67; 
 (c) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, a fourth amplification oligomer comprises a target-hybridizing sequence consisting of SEQ ID NO:29 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a detectably labeled detection probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:71. 
   
     
     
         8 . The combination of oligomers of  claim 7 , wherein the combination comprises at least two detectably labeled detection probe oligomers, wherein the at least two detection probe oligomers comprise 
 (i) a first detectably labeled detection probe oligomer comprising the target-hybridizing sequence consisting of SEQ ID NO:37 and a second detectably labeled detection probe oligomer consisting of the target-hybridizing sequence consisting of SEQ ID NO:67, or   (ii) a first detectably labeled detection probe oligomer comprising the target-hybridizing sequence consisting of SEQ ID NO:37 and a second detectably labeled detection probe oligomer consisting of the target-hybridizing sequence consisting of SEQ ID NO:71.   
     
     
         9 . The combination of oligomers of  claim 7 , wherein the third amplification oligomer promoter sequence and/or the fourth amplification oligomer promoter sequence consists of the sequence of SEQ ID NO:73. 
     
     
         10 . A kit comprising the combination of oligomers of  claim 1  and at least one reagent suitable for performing an in vitro nucleic acid amplification reaction. 
     
     
         11 . The kit according to  claim 10 , wherein the kit comprises an RNA polymerase. 
     
     
         12 . A kit comprising the combination of oligomers of  claim 7  and at least one reagent suitable for performing an in vitro nucleic acid amplification reaction. 
     
     
         13 . The kit according to  claim 12 , wherein the kit comprises an RNA polymerase. 
     
     
         14 . An amplification reaction mixture comprising the combination of oligomers of  claim 1 . 
     
     
         15 . An amplification reaction mixture comprising the combination of oligomers of  claim 7 . 
     
     
         16 . 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 three amplification oligomers for amplifying a target region of an HEV target nucleic acid, said amplification oligomer combination selected from the group consisting of 
 (a) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
 (b) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
 (c) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
 (d) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
 (e) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
 (f) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:29, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
 (g) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; and 
 (h) a first amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:32, a second amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:24 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence, and a third amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:46 and further comprising a promoter sequence joined to the 5′ end of the target-hybridizing sequence; 
   (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) detecting the presence or absence of the amplification product by using at least one detectably labeled detection probe oligomer selected from the group consisting of SEQ ID NO:37, SEQ ID NO:67, and SEQ ID NO:71, thereby determining the presence or absence of HEV in the sample.   
     
     
         17 . The method of  claim 16 , wherein the third amplification oligomer of (a), (d), (e), (f), (g), and/or (h) comprises a nucleotide sequence consisting of SEQ ID NO: 15, and/or wherein the third amplification oligomer of (b) and/or (c) comprises a nucleotide sequence consisting of SEQ ID NO: 12, and/or wherein the second amplification oligomer of (e), (f), (g), and/or (h) comprises a nucleotide sequence consisting of SEQ ID NO: 12. 
     
     
         18 . The method of  claim 16 , wherein the detectable label is an acridinium ester compound. 
     
     
         19 . The method of  claim 16 , wherein the amplification reaction at step (2) is an isothermal amplification reaction. 
     
     
         20 . The method of  claim 19 , wherein the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction.