Compositions and methods for detecting HEV nucleic acids

Abstract

Compositions, kits and methods are provided for detecting the presence or absence of HEV in a specimen with high specificity and sensitivity. In one embodiment, the present invention provides a combination of at least two oligomers for determining the presence or absence of Hepatitis E virus (HEV) in a sample, the combination of oligomers comprising at least two amplification oligomers for amplifying complementary nucleic acid strands of a target region of an HEV target nucleic acid.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of Provisional Application No. 61 / 865,848, filed August 14, 2013, and Provisional Application No. 61 / 941,303, filed February 18, 2014, the entire contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Background of the Invention Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus classified in the family Hepeviridae and is the only member of the genus Hepevirus, of which mammalian HEV and avian HEV are the two major known species. Dalton et al., Lancet Infect. Dis. 8:698-709, 2008; Baylis et al., J. Clin. Microbiol. 49:1234-1239, 2011. Mammalian HEV, with swine and potentially other mammalian reservoirs, is the leading cause of acute hepatitis in humans. See Dalton et al., supra. The virus is primarily transmitted via the fecal-oral route and is associated with sporadic infections and epidemics in developing countries, particularly in areas with poor sanitation and weak public health infrastructure. In developed countries, HEV infection is considered rare and occurs primarily in individuals infected during travel to areas where the virus is endemic. However, endemic infections have recently been reported more frequently in developed regions, including North America, Europe, Japan, New Zealand, and Australia. Thus, endemic hepatitis E in developed countries may be more common than previously recognized and may be more common than hepatitis A. Dalton et al., Lancet Infect. Dis. 8:698-709, 2008.

[0003] Four major genotypes of HEV are known to cause infection in humans. Baylis et al., supra. Clinical features of HEV infection can include mild to severe hepatitis and subacute liver failure. See, e.g., Pina et al., J. Hepatol. 33:826-833, 2000; Sainokami et al., J. Gastroenterol. 39:640-648, 2004; Tsang et al., Clin. Infect. Dis. 30:618-619, 2000; Widdowson et al., Clin. Infect. Dis. 36:29-33, 2003; Dalton et al., Eur. J. Gastroenterol. Hepatol. 20:784-790, 2008. HEV infection has a poor prognosis in pregnant women, as well as in individuals with pre-existing chronic liver disease. See Borkakoti et al., J. Med. Virol. 85:620-626, 2013; Baylis et al., supra. Diagnostic testing for HEV in patients with symptoms of hepatitis is important, especially in patients in whom other causes of acute hepatitis have been ruled out. Baylis et al., supra; Waar et al., J. Clin. See Virol. 33:145-149, 2005. Thus, there is a need for compositions, kits, and methods for detecting the presence or absence of HEV in a specimen with high specificity and sensitivity. Such compositions, kits, and methods are particularly useful for diagnosing HEV, screening for and / or monitoring the presence of HEV in blood or plasma donations, or monitoring a patient's response to treatment. The present invention fulfills these and other needs. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Dalton et al., Lancet Infect. Dis. 8:698-709, 2008 [Non-patent document 2] Baylis et al., J. Clin. Microbiol. 49:1234-1239, 2011 [Non-patent document 3] Pina et al., J. Hepatol. 33:826-833, 2000 [Non-patent document 4] Sainokami et al., J. Gastroenterol. 39:640-648, 2004 [Non-patent document 5] Tsang et al., Clin. Infect. Dis. 30:618-619, 2000 [Non-patent document 6] Widdowson et al., Clin. Infect. Dis. 36:29-33, 2003 [Non-Patent Document 7] Dalton et al., Eur. J. Gastroenterol. Hepatol. 20:784-790, 2008 [Non-patent document 8] Borkakoti et al., J. Med. Virol. 85:620-626, 2013 [Non-Patent Document 9] Waar et al., J. Clin. Virol. 33:145-149, 2005 Summary of the Invention [Means for solving the problem]

[0005] In one embodiment, the present invention provides a combination of at least two oligomers for determining the presence or absence of Hepatitis E virus (HEV) in a sample, the combination of oligomers comprising at least two amplification oligomers for amplifying complementary nucleic acid strands of a target region of an HEV target nucleic acid, wherein: (a) at least one amplification oligomer (i) an oligomer comprising a target hybridizing sequence comprising at least the sequence of SEQ ID NO: 26, which is about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 63, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 26; and (ii) an oligomer comprising a target hybridizing sequence of about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 16, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 16; (b) At least one amplification oligomer comprises a target hybridizing sequence comprising at least the sequence of SEQ ID NO: 25, which is about 17 to about 28 contiguous nucleotides contained in the sequence of SEQ ID NO: 47, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 25.

[0006] Suitable amplification oligomers as identified above in (a) include oligomers comprising a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66. In some preferred variations, the (a) amplification oligomer comprises a target-hybridizing sequence selected from SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In some embodiments, the (a) amplification oligomer comprises a target-hybridizing sequence that is about 14 to about 20 nucleotides contained in the sequence of SEQ ID NO:13 (e.g., SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66).

[0007] In particular variations, the amplification oligomer identified in (a) comprises a sequence that hybridizes to a target that is 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16 and comprises at least the sequence of SEQ ID NO: 27, including the RNA equivalent and DNA / RNA chimeras of SEQ ID NO: 27 (e.g., a sequence that hybridizes to a target selected from SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, including the RNA equivalent and DNA / RNA chimeras of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32). In some embodiments, the amplification oligomer in (a) comprises a sequence that hybridizes to a target of SEQ ID NO: 28, including the RNA equivalent and DNA / RNA chimeras of SEQ ID NO: 28 (e.g., a sequence that hybridizes to a target selected from SEQ ID NO: 29 and SEQ ID NO: 32, including the RNA equivalent and DNA / RNA chimeras of SEQ ID NO: 29 and SEQ ID NO: 32).

[0008] Suitable amplification oligomers identified above in (b) include oligomers comprising a sequence that hybridizes to a target selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:56. In some preferred variations, the amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:24 and SEQ ID NO:56. In other preferred variations, the amplification oligomer of (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51. In particular variations including an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G) (i.e., SEQ ID NO:46, or RNA equivalents and DNA / RNA chimeras of SEQ ID NO:46).

[0009] In some embodiments, the combination of at least two oligomers as described above comprises an amplification oligomer identified in (a)(i) and an amplification oligomer identified in (a)(ii). In some such embodiments, the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In certain preferred variations, the amplification oligonucleotide of (a)(i) comprises a sequence that hybridizes to a target selected from SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0010] In certain embodiments comprising an amplification oligomer identified in (a)(i) and an amplification oligomer identified in (a)(ii), the amplification oligomer described in (a)(ii) comprises a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54. In certain preferred variations, the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32. In particular variations, (I) the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64; the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29; or (II) the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO: 65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 65; and the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO: 29 or SEQ ID NO: 31, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29 or SEQ ID NO: 31.

[0011] In some embodiments, the combination of at least two oligomers as described above includes a first amplification oligomer as identified in (a)(ii) and a second amplification oligomer as identified in (a)(ii). In some such embodiments, each of the first and second amplification oligomers described in (a)(ii) above comprises a target-hybridizing sequence comprising at least the sequence of SEQ ID NO:27, which is 15-17 nucleotides contained in the sequence of SEQ ID NO:16, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO:27 (e.g., a target-hybridizing sequence selected from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32). In particular variations, each of the first and second amplification oligomers described in (a)(ii) above comprises a target-hybridizing sequence of SEQ ID NO:28, which includes the RNA equivalent and DNA / RNA chimera of SEQ ID NO:28. In a particular variation, the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29, and the second amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:32.

[0012] In some embodiments of the oligomer combinations described above, the combination comprises a first and a second amplification oligomer as identified in (b). In some such embodiments, the first amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:24 and SEQ ID NO:56. In other embodiments, the first amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51. In particular variations, (I) the first amplification oligomer in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24, and the second amplification oligomer in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:56; or (II) the first amplification oligomer in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:23 or SEQ ID NO:51, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:23 or SEQ ID NO:51, and the second amplification oligomer in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:45, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:45. In certain variations involving amplification oligomers having a sequence that hybridizes to a target of SEQ ID NO: 56, the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G) (i.e., SEQ ID NO: 46 or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46).

[0013] The oligomer combination as described above may comprise an amplification oligomer as set forth in (a)(i), an amplification oligomer as set forth in (a)(ii), a first amplification oligomer as set forth in (b), and a second amplification oligomer as set forth in (b). In particular variations, the amplification oligomer as set forth in (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO:64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:64; the amplification oligomer as set forth in (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; the first amplification oligomer as set forth in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; and the second amplification oligomer as set forth in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:56. In another variation, the amplification oligomer of (a)(i) comprises a sequence that hybridizes to the target SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; and the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO:65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:65. A chimera, comprising a sequence that hybridizes to a target; the first amplification oligomer of (b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; The second amplification oligomer described in (b) above comprises a sequence that hybridizes to a target of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. In certain variations that include an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO: 56, the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G) (i.e., SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46).

[0014] The combination of oligomers as described above may include a first amplification oligomer described in (a)(ii), a second amplification oligomer described in (a)(ii), a first amplification oligomer described in (b), and a second amplification oligomer described in (b). In particular variations, the first amplification oligomer in (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; the second amplification oligomer in (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:32; the first amplification oligomer in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; and the second amplification oligomer in (b) comprises a sequence that hybridizes to a target of SEQ ID NO:46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:46.

[0015] In still other embodiments of the combination of amplification oligomers as described above, the (a) amplification oligomer comprises a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, and the (b) amplification oligomer comprises a sequence that hybridizes to a target selected from SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:24 and SEQ ID NO:56. In some such embodiments, the combination comprises first, second, and third sets of amplification oligomers comprising first, second, and third sets of target-hybridizing sequences, respectively, selected from sets (i)-(vi) as follows: (i) SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:24, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:24; (ii) SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:56; (iii) SEQ ID NO:2 (iv) SEQ ID NO:66, SEQ ID NO:24, and SEQ ID NO:56, including the RNA equivalents and DNA / RNA chimeras of SEQ ID NO:66, SEQ ID NO:24, and SEQ ID NO:56; (v) SEQ ID NO:65, SEQ ID NO:24, and SEQ ID NO:56, including the RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:24, and SEQ ID NO:56; and (vi) SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56, including the RNA equivalents and DNA / RNA chimeras of SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56. In certain variations involving amplification oligomers having a sequence that hybridizes to a target of SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G) (i.e., SEQ ID NO:46, or the RNA equivalent or DNA / RNA chimera of SEQ ID NO:46).

[0016] In some embodiments, the oligomer combination as described above comprises first and second sets of amplification oligomers comprising a first (A) and a second (B) set of target-hybridizing sequences, respectively, wherein the set of target-hybridizing sequences is selected from sets (i) through (xiv) as follows: (i) (A) SEQ ID NO: 54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 54, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 53, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 53, and (B) SEQ ID NO: 23, 24, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 52, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (iv) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xiii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 41, 52, 44, 45, 46, or 47, or its RNA equivalent or DNA / RNA chimera; and (xiv) (A) SEQ ID NO: 61, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50 or 51, or its RNA equivalent or DNA / RNA chimera. In certain embodiments comprising an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO: 56, the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G) (i.e., SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46). In certain variations, the set of sequences A and B that hybridize to the target is selected from sets (i) through (xiii) as follows: (i) (A) SEQ ID NO: 54, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (ii) (A) SEQ ID NO: 53, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera; (iii) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 56, 48, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 24, 45, 56 or 50, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 24, 45 or 56, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; and (xiii) (A) SEQ ID NO: 61, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45 or 56, or their RNA equivalents or DNA / RNA chimeras.

[0017] In yet other embodiments, the combination of oligomers as described above comprises first and second sets of amplification oligomers comprising a first (A) and second (B) set of target-hybridizing sequences, respectively, wherein the set of target-hybridizing sequences is selected from the following sets (i) through (x): (i) (A) SEQ ID NO: 48, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 52, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (x) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65 or 66, or an RNA equivalent or DNA / RNA chimera thereof. In certain embodiments comprising an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO: 56, the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G) (i.e., SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46). In certain variations, the set of sequences A and B that hybridize to the target is selected from sets (i) through (ix) as follows: (i) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 49, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 31, 61, 62, 64 or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 33, 34, 35, 62, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 54, 62, or 64, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (ix) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 31, 33, 34, 35, 53, 61, 62, 64, 65 or 66, or an RNA equivalent or DNA / RNA chimera thereof.

[0018] In some embodiments of the oligomer combination described above, the amplification oligomer described in (b) is a promoter primer that further comprises a promoter sequence (for example, a T7 promoter sequence) located 5' of the sequence that hybridizes to the target.A particularly suitable promoter sequence is the T7 promoter sequence having the sequence shown in SEQ ID NO: 73.

[0019] In certain embodiments, the oligomer combination further comprises at least one detection probe oligomer. In certain embodiments, the detection probe oligomer comprises a target-hybridizing sequence about 14 to about 28 nucleotides in length configured to specifically hybridize to the target sequence contained in SEQ ID NO: 39 or its complement. In certain variations, the detection probe target-hybridizing sequence is selected from SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71. The detection probe oligomer may comprise a 2'-methoxy backbone at one or more junctions of its nucleic acid backbone.

[0020] In some variations, the oligomer combination includes at least two detection probe oligomers. For example, the oligomer combination may further include at least two detection probe oligomers comprising target-hybridizing sequences about 14 to about 28 nucleotides in length and configured to specifically hybridize to the target sequence contained within SEQ ID NO: 39 or its complement. In some such embodiments, the target-hybridizing sequence of each detection probe is individually selected from SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71. In certain variations, the oligomer combination includes a first detection probe oligomer containing a target-hybridizing sequence of SEQ ID NO:55 or its complement, or its RNA equivalent or DNA / RNA chimera; and a second detection probe oligomer containing a target-hybridizing sequence of SEQ ID NO:67 or its complement, or its RNA equivalent or DNA / RNA chimera. In other variations, the oligomer combination includes at least three detection probe oligomers. For example, the at least three detection probe oligomers can include a first detection probe oligomer containing a target-hybridizing sequence of SEQ ID NO:37 or its complement, or its RNA equivalent or DNA / RNA chimera; a second detection probe oligomer containing a target-hybridizing sequence of SEQ ID NO:67 or its complement, or its RNA equivalent or DNA / RNA chimera; and a third detection probe oligomer containing a target-hybridizing sequence of SEQ ID NO:71 or its complement, or its RNA equivalent or DNA / RNA chimera. One or more (eg, each) of the at least two detection probe oligomers may each contain a 2'-methoxy backbone at one or more junctions of its nucleic acid backbone.

[0021] In some embodiments, the oligomer combination further comprises a capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe. Particularly suitable target-hybridizing sequences include the sequences set forth in SEQ ID NO:4 and SEQ ID NO:42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO:4 and SEQ ID NO:42. In some variations comprising a target-hybridizing sequence of SEQ ID NO:4, the nucleobase at position 20 of SEQ ID NO:4 is adenine (A). In some such variations, the nucleobase at position 19 of SEQ ID NO:4 is cytosine (C) or uracil (U). In more particular variations, the capture probe oligomer has a sequence selected from SEQ ID NO:3, SEQ ID NO:7, and SEQ ID NO:43. In certain embodiments, the oligomer combination further comprises at least two or at least three capture probe oligomers as described above. For example, an oligomer combination may include a first capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:2 or its complement, or its DNA equivalent or DNA / RNA chimera; a second capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:6 or its complement, or its DNA equivalent or DNA / RNA chimera; and a third capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:42 or its complement, or its DNA equivalent or DNA / RNA chimera. In a more particular variation, the first, second, and third capture probe oligomers have the sequences of SEQ ID NO:3, SEQ ID NO:7, and SEQ ID NO:43, respectively.

[0022] In another aspect, the present invention provides a kit or reaction mixture comprising a combination of oligomers as described above.

[0023] In yet another aspect, the present invention provides a method for determining the presence or absence of hepatitis E virus (HEV) in a sample. The method generally comprises the steps of: (1) contacting a sample suspected of containing HEV with at least two oligomers for amplifying a target region of an HEV target nucleic acid; (2) performing an in vitro nucleic acid amplification reaction in which any HEV target nucleic acid present in the sample is used as a template to generate an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of HEV in the sample. The at least two amplification oligomers are (a) at least one amplification oligomer selected from: (i) an oligomer comprising a target-hybridizing sequence comprising at least the sequence of SEQ ID NO: 26, which is about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 63, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 26; and (ii) an oligomer comprising a target-hybridizing sequence comprising about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 16, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 16; (b) at least one amplification oligomer comprising a sequence that hybridizes to a target, the sequence comprising at least the sequence of SEQ ID NO: 25, which is about 17 to about 28 contiguous nucleotides contained in the sequence of SEQ ID NO: 47, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 25;

[0024] Suitable amplification oligomers as identified in (a) include oligomers comprising a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66. In some preferred variations, the (a) amplification oligomer comprises a target-hybridizing sequence selected from SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In some embodiments, the (a) amplification oligomer comprises a target-hybridizing sequence that is about 14 to about 20 nucleotides contained in the sequence of SEQ ID NO:13 (e.g., SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66).

[0025] In certain variations of the methods for determining the presence or absence of HEV, the amplification oligomer specified in (a) comprises a sequence that hybridizes to a target that is 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16 and includes at least the sequence of SEQ ID NO: 27, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO: 27 (e.g., a sequence that hybridizes to a target selected from SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32). In some embodiments, the amplification oligomer in (a) comprises a sequence that hybridizes to a target of SEQ ID NO: 28, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO: 28 (e.g., a sequence that hybridizes to a target selected from SEQ ID NO: 29 and SEQ ID NO: 32, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO: 29 and SEQ ID NO: 32).

[0026] Suitable amplification oligomers as identified above in (b) include oligomers comprising a sequence that hybridizes to a target selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:56. In some preferred variations, the amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:24 and SEQ ID NO:56. In other preferred variations, the amplification oligomer of (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51. In particular variations including an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G) (i.e., SEQ ID NO:46, or the RNA equivalent or DNA / RNA chimera of SEQ ID NO:46).

[0027] In some embodiments of the methods for determining the presence or absence of HEV as described above, the at least two amplification oligomers comprise the amplification oligomer identified in (a)(i) and the amplification oligomer identified in (a)(ii). In some such embodiments, the amplification oligomer in (a)(i) comprises a sequence that hybridizes to a target selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In certain preferred variations, the amplification oligomer in (a)(i) comprises a sequence that hybridizes to a target selected from SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0028] In certain embodiments of the method in which the amplification oligomers identified in (a)(i) and (a)(ii) are used for amplification of an HEV target region, the amplification oligomer described in (a)(ii) comprises a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54. In certain preferred variations, the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32. In particular variations, (I) the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64, and the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29; or (II) the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO: 65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 65, and the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO: 29 or SEQ ID NO: 31, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29 or SEQ ID NO: 31.

[0029] In some embodiments of the methods for determining the presence or absence of HEV, the combination of at least two oligomers described above comprises a first amplification oligomer identified in (a)(ii) and a second amplification oligomer identified in (a)(ii). In some such embodiments, each of the first and second amplification oligomers described in (a)(ii) comprises a target-hybridizing sequence comprising at least the sequence of SEQ ID NO:27, which is 15-17 nucleotides contained in the sequence of SEQ ID NO:16, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO:27 (e.g., a target-hybridizing sequence selected from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32). In certain variations, each of the first and second amplification oligomers described in (a)(ii) comprises the RNA equivalent and DNA / RNA chimera of SEQ ID NO:28. In a particular variation, the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29, and the second amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:32.

[0030] In some embodiments of the method as described above, the amplification step utilizes the first and second amplification oligomers identified in (b). In some such embodiments, the first amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:24 and SEQ ID NO:56. In other embodiments, the first amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51. In particular variations, (I) the first amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24, and the second amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:56; or (II) the first amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:23 or SEQ ID NO:51, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:23 or SEQ ID NO:51, and the second amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:45, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:45. In particular variations involving amplification oligomers having a sequence that hybridizes to a target of SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G) (i.e., , SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46).

[0031] To amplify the HEV target region in the above-described method, an amplification oligomer described in (a)(i), an amplification oligomer described in (a)(ii), a first amplification oligomer described in (b), and a second amplification oligomer described in (1)(b) may be used. In particular variations, the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64; the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29; the first amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO: 24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 24; and the second amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. In another variation, the amplification oligomer of (a)(i) comprises a sequence that hybridizes to a target of SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; the amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:65; the first amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; and the second amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:56. In a particular variation comprising an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G) (i.e., SEQ ID NO:46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:46).

[0032] In other embodiments for amplifying the HEV target region in the method as described above, a first amplification oligomer described in (a)(ii), a second amplification oligomer described in (a)(ii), a first amplification oligomer described in (b), and a second amplification oligomer described in (b) may be used. In particular variations, the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; the second amplification oligomer of (a)(ii) comprises a sequence that hybridizes to a target of SEQ ID NO:32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:32; the first amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; and the second amplification oligomer of (b) comprises a sequence that hybridizes to a target of SEQ ID NO:46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:46.

[0033] In still other embodiments of the method for determining the presence or absence of HEV as described above, the amplification oligomer (a) comprises a sequence that hybridizes to a target selected from SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, and the amplification oligomer (b) comprises a sequence that hybridizes to a target selected from SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:24 and SEQ ID NO:56. In some such embodiments, the amplifying step uses a combination of oligomers comprising first, second, and third sets of amplification oligomers, each comprising a first, second, and third set of target-hybridizing sequences, wherein the sets of target-hybridizing sequences are selected from sets (i)-(vi) as follows: (i) SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:24, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:24; (ii) SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29, and SEQ ID NO:56. (iii) SEQ ID NO:29, SEQ ID NO:24, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:24, and SEQ ID NO:56; (iv) SEQ ID NO:66, SEQ ID NO:24, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:66, SEQ ID NO:24, and SEQ ID NO:56; (v) SEQ ID NO:65, SEQ ID NO:24, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:24, and SEQ ID NO:56; and (vi) SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:62, SEQ ID NO:29, and SEQ ID NO:56. In certain variations involving amplification oligomers having a sequence that hybridizes to a target of SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G) (i.e., SEQ ID NO:46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:46).

[0034] In some embodiments, the amplifying step uses a combination of oligomers comprising first and second sets of amplification oligomers comprising a first (A) and second (B) set of target-hybridizing sequences, respectively, wherein the sets of target-hybridizing sequences are selected from sets (i) through (xiv) as follows: (i) (A) SEQ ID NO: 54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 54, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 53, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 53, and (B) SEQ ID NO: 23, 24, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 52, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (iv) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xiii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or its RNA equivalent or DNA / RNA chimera; and (xiv) (A) SEQ ID NO: 61, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50 or 51, or its RNA equivalent or DNA / RNA chimera. In certain embodiments comprising an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO: 56, the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G) (i.e., SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46). In certain variations, the set of sequences A and B that hybridize to the target is selected from sets (i) through (xiii) as follows: (i) (A) SEQ ID NO: 54, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (ii) (A) SEQ ID NO: 53, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera; (iii) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 56, 48, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 24, 45, 56 or 50, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 24, 45 or 56, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; and (xiii) (A) SEQ ID NO: 61, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45 or 56, or their RNA equivalents or DNA / RNA chimeras.

[0035] In yet other embodiments, the amplifying step uses a combination of oligomers comprising first and second sets of amplification oligomers comprising a first (A) and second (B) set of sequences that hybridize to the target, respectively, wherein the set of sequences that hybridize to the target is selected from sets (i) through (x) as follows: (i) (A) SEQ ID NO: 48, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 52, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (x) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65 or 66, or an RNA equivalent or DNA / RNA chimera thereof. In certain embodiments comprising an amplification oligomer having a sequence that hybridizes to a target of SEQ ID NO: 56, the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G) (i.e., SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46). In certain variations, the set of sequences A and B that hybridize to the target is selected from sets (i) through (ix) as follows: (i) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 49, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 31, 61, 62, 64 or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 33, 34, 35, 62, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 54, 62, or 64, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (ix) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 31, 33, 34, 35, 53, 61, 62, 64, 65 or 66, or an RNA equivalent or DNA / RNA chimera thereof.

[0036] In some embodiments of the method for determining the presence or absence of HEV as described above, the amplification oligomer described in (b) is a promoter primer that further comprises a promoter sequence (for example, a T7 promoter sequence) located 5' of the sequence that hybridizes with the target.A particularly suitable promoter sequence is the T7 promoter sequence having the sequence shown in SEQ ID NO: 73.

[0037] Typically, the method for determining the presence or absence of HEV further comprises purifying the HEV target nucleic acid from other components in the sample prior to the amplification step (1). In certain embodiments, the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe. Particularly suitable target-hybridizing sequences include those set forth in SEQ ID NO:4 and SEQ ID NO:42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO:4 and SEQ ID NO:42. In some variations of methods involving the use of a target-hybridizing sequence of SEQ ID NO:4, the nucleobase at position 20 of SEQ ID NO:4 is adenine (A). In some such variations, the nucleobase at position 19 of SEQ ID NO:4 is cytosine (C) or uracil (U). In more particular variations, the capture probe oligomer has a sequence selected from SEQ ID NO:3, SEQ ID NO:7, and SEQ ID NO:43. In certain embodiments, the purifying step comprises the use of at least two or at least three capture probe oligomers as described above. For example, the purifying step may comprise the use of a first capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:2, or its complement, or its DNA equivalent or DNA / RNA chimera; a second capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:6, or its complement, or its DNA equivalent or DNA / RNA chimera; and a third capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:42, or its complement, or its DNA equivalent or DNA / RNA chimera. In more particular variations, the first, second, and third capture probe oligomers have the sequences of SEQ ID NO:3, SEQ ID NO:7, and SEQ ID NO:43, respectively.

[0038] In some embodiments, the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer configured to specifically hybridize to the amplification product under conditions that determine the presence or absence of the amplification product, thereby determining the presence or absence of HEV in the sample. In certain embodiments, the detection probe oligomer comprises a target-hybridizing sequence that is about 14 to about 28 nucleotides in length and configured to specifically hybridize to the target sequence contained in SEQ ID NO: 39 or its complement. In certain variations, the detection probe target-hybridizing sequence is selected from SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71. The detection probe oligomer may contain a 2'-methoxy backbone at one or more junctions of its nucleic acid backbone.

[0039] In some variations, the detecting step comprises contacting the in vitro nucleic acid amplification reaction with at least two detection probe oligomers. For example, the in vitro nucleic acid amplification reaction may be contacted with at least two detection probe oligomers comprising target-hybridizing sequences about 14 to about 28 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 39 or its complement. In some such embodiments, the target-hybridizing sequence of each detection probe is individually selected from SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71. In certain variations, the detecting step comprises contacting the in vitro nucleic acid amplification reaction with a first detection probe oligomer comprising a sequence that hybridizes to a target of SEQ ID NO: 55 or its complement, or its RNA equivalent, or a DNA / RNA chimera; and a second detection probe oligomer comprising a sequence that hybridizes to a target of SEQ ID NO: 67 or its complement, or its RNA equivalent, or a DNA / RNA chimera. In other variations, the detecting step comprises contacting the in vitro nucleic acid amplification reaction with at least three detection probe oligomers. The at least three detection probe oligomers may include a first detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 37 or its complement, or its DNA equivalent or DNA / RNA chimera; a second detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 67 or its complement, or its DNA equivalent or DNA / RNA chimera; and a third detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 71 or its complement, or its DNA equivalent or DNA / RNA equivalent. One or more (e.g., each) of the at least two detection probe oligomers may contain a 2'-methoxy backbone at one or more junctions of its nucleic acid backbone.

[0040] In some embodiments of methods utilizing detection probe oligomers, the detection probe comprises at least one label. In certain variations, the one or more labels are selected from a chemiluminescent label, a fluorescent label, a quencher, or any combination thereof. In certain embodiments, the detecting step (3) detects hybridization of the labeled at least one detection probe oligomer to the amplification product in a homogeneous detection system. A particularly suitable label for use in a homogeneous detection system is a chemiluminescent acridinium ester (AE) compound, wherein the label is linked between two nucleobases of the at least one detection probe oligomer.

[0041] In certain variations of the method for determining the presence or absence of HEV as described above, said amplification reaction of step (2) is an isothermal amplification reaction, such as, for example, a transcription-mediated amplification (TMA) reaction.

[0042] These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention and the accompanying drawings. In certain embodiments, for example, the following are provided: (Item 1) A combination of at least two oligomers for determining the presence or absence of Hepatitis E virus (HEV) in a sample, the combination of oligomers comprising: at least two amplification oligomers for amplifying a target region of an HEV target nucleic acid, wherein (a) at least one amplification oligomer comprises: (i) an oligomer comprising a target hybridizing sequence comprising at least the sequence of SEQ ID NO: 26, which is about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 63, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 26; and (ii) an oligomer comprising a target hybridizing sequence of about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 16, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 16; (b) A combination of at least two oligomers, wherein at least one amplification oligomer is about 17 to about 28 consecutive nucleotides contained in the sequence of SEQ ID NO: 47, and comprises a sequence that hybridizes to a target, including at least the sequence of SEQ ID NO: 25, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 25. (Item 2) 2. A combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (a) comprises a target-hybridizing sequence of about 14 to about 20 nucleotides contained in the sequence of SEQ ID NO: 13. (Item 3) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer in (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66. (Item 4) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. (Item 5) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 64, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29 and SEQ ID NO: 64. (Item 6) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (a) comprises a target hybridizing sequence comprising at least the sequence of SEQ ID NO: 27, which is 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16, including the RNA equivalent of SEQ ID NO: 27 and DNA / RNA chimeras. (Item 7) 7. The combination of at least two oligomers according to item 6, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32. (Item 8) 7. A combination of at least two oligomers according to item 6, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to the target of SEQ ID NO: 28, including RNA equivalents of SEQ ID NO: 28 and DNA / RNA chimeras. (Item 9) 9. The combination of at least two oligomers according to item 8, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29 and SEQ ID NO: 32. (Item 10) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 31, SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 66. (Item 11) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer in (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:56. (Item 12) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (b) comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 56. (Item 13) 13. A combination of at least two oligomers according to item 11 or 12, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 14) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (b) comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 46, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 46. (Item 15) 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 51. (Item 16) 16. A combination of at least two oligomers according to any of items 1 to 15, comprising an amplification oligomer according to (a)(i) and an amplification oligomer according to (a)(ii). (Item 17) 17. The combination of at least two oligomers according to item 16, wherein the amplification oligomer according to (a)(i) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66. (Item 18) 18. A combination of at least two oligomers according to item 17, wherein the amplification oligomer according to (a)(i) comprises a target-hybridizing sequence of SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64. (Item 19) 18. The combination of at least two oligomers according to item 17, wherein the amplification oligomer according to (a)(i) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 66. (Item 20) 20. The combination of at least two oligomers according to any one of items 16 to 19, wherein the amplification oligomer according to (a)(ii) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54. (Item 21) 20. The combination of at least two oligomers according to any of items 16 to 19, wherein the amplification oligomer according to (a)(ii) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32. (Item 22) the amplification oligomer according to (a)(i) comprises a sequence that hybridizes to the target SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64; 17. A combination of at least two oligomers according to item 16, wherein the amplification oligomer according to (a)(ii) comprises a target-hybridizing sequence of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29. (Item 23) the amplification oligomer according to (a)(i) comprises a sequence that hybridizes to the target SEQ ID NO: 65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 65; 17. A combination of at least two oligomers according to item 16, wherein the amplification oligomer according to (a)(ii) comprises a target-hybridizing sequence of SEQ ID NO: 29 or SEQ ID NO: 31, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29 or SEQ ID NO: 31. (Item 24) 2. A combination of at least two oligomers according to item 1, comprising a first amplification oligomer according to (a)(ii) and a second amplification oligomer according to (a)(ii). (Item 25) 25. The combination of at least two oligomers according to item 24, wherein each of the first and second amplification oligomers according to (a)(ii) comprises a target hybridizing sequence comprising at least the sequence of SEQ ID NO: 27, which is 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16, including the RNA equivalent of SEQ ID NO: 27 and DNA / RNA chimeras. (Item 26) 26. The combination of at least two oligomers according to item 25, wherein each of the first and second amplification oligomers of (a)(ii) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32. (Item 27) 26. The combination of at least two oligomers described in item 25, wherein each of the first and second amplification oligomers of (a)(ii) comprises a sequence that hybridizes to the target of SEQ ID NO: 28, including RNA equivalents of SEQ ID NO: 28 and DNA / RNA chimeras. (Item 28) the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; 28. The combination of at least two oligomers according to item 27, wherein the second amplification oligomer according to (a)(ii) comprises a target-hybridizing sequence of SEQ ID NO: 32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 32. (Item 29) 29. A combination of at least two oligomers according to any of items 1 to 28, comprising a first amplification oligomer according to (b) and a second amplification oligomer according to (b). (Item 30) 30. The combination of at least two oligomers described in item 29, wherein the first amplification oligomer described in (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 56. (Item 31) 30. The combination of at least two oligomers according to item 29, wherein the first amplification oligomer according to (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 51. (Item 32) the first amplification oligomer according to (b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 30. A combination of at least two oligomers according to item 29, wherein the second amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. (Item 33) 33. A combination of at least two oligomers according to item 30 or 32, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 34) the first amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO:23 or SEQ ID NO:51, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:23 or SEQ ID NO:51; 30. The combination of at least two oligomers according to item 29, wherein the second amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO: 45, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 45. (Item 35) 16. A combination of at least two oligomers according to any of items 1 to 15, comprising an amplification oligomer according to (a)(i), an amplification oligomer according to (a)(ii), a first amplification oligomer according to (b), and a second amplification oligomer according to (b). (Item 36) the amplification oligomer according to (a)(i) comprises a sequence that hybridizes to the target SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64; The amplification oligomer according to (a)(ii) above comprises a sequence that hybridizes to the target SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29. ; the first amplification oligomer according to (b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 36. A combination of at least two oligomers according to item 35, wherein the second amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. (Item 37) The amplification oligomer according to (a)(i) above comprises a sequence that hybridizes to a target of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29; and the amplification oligomer according to (a)(ii) above comprises a sequence that hybridizes to a target of SEQ ID NO: 65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 65. ; the first amplification oligomer according to (b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 36. A combination of at least two oligomers according to item 35, wherein the second amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. (Item 38) 38. A combination of at least two oligomers according to item 36 or 37, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 39) 16. A combination of at least two oligomers according to any of items 1 to 15, comprising a first amplification oligomer according to (a)(ii), a second amplification oligomer according to (a)(ii), a first amplification oligomer according to (b), and a second amplification oligomer according to (b). (Item 40) the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; the second amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO: 32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 32; the first amplification oligomer according to (b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 40. The combination of at least two oligomers according to item 39, wherein the second amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46. (Item 41) at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66; 2. The combination of at least two oligomers according to item 1, wherein at least one amplification oligomer of (b) comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 56. (Item 42) and first, second and third sets of amplification oligomers comprising first, second and third sets of target-hybridizing sequences, respectively, wherein the sets of target-hybridizing sequences comprise: (i) SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:24, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:24; (ii) SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:56; (iii) SEQ ID NO:29, SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:24 and SEQ ID NO:56; (iv) SEQ ID NO:66, SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:66, SEQ ID NO:24 and SEQ ID NO:56; (v) SEQ ID NO: 65, SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 65, SEQ ID NO: 24 and SEQ ID NO: 56; and (vi) A combination of at least two oligomers according to item 41, selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 29 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 62, SEQ ID NO: 29 and SEQ ID NO: 56. (Item 43) a first set of amplification oligomers and a second set of amplification oligomers, each of which comprises a first (A) and a second (B) set of sequences that hybridize to the target, wherein the set of sequences that hybridize to the target comprises: (i) (A) SEQ ID NO: 54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 54, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 53, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 53, and (B) SEQ ID NO: 23, 24, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 52, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (iv) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xiii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or its RNA equivalent or DNA / RNA chimera; and (xiv) 2. A combination of at least two oligomers according to item 1, selected from the group consisting of: (A) SEQ ID NO: 61, or an RNA equivalent or a DNA / RNA chimera thereof, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50 or 51, or an RNA equivalent or a DNA / RNA chimera thereof. (Item 44) a first set of amplification oligomers and a second set of amplification oligomers, each of which comprises a first (A) and a second (B) set of sequences that hybridize to the target, wherein the set of sequences that hybridize to the target comprises: (i) (A) SEQ ID NO: 54, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (ii) (A) SEQ ID NO: 53, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera; (iii) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45, 45, 46, or 47, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 56, 48, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 24, 45, 56 or 50, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 24, 45 or 56, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; and (xiii) (A) SEQ ID NO: 61, or its RNA equivalent or DNA / RNA chimera, and (B) A combination of at least two oligomers according to item 1, selected from the group consisting of SEQ ID NO: 22, 45 or 56, or RNA equivalents or DNA / RNA chimeras thereof. (Item 45) a first set of amplification oligomers and a second set of amplification oligomers, each of which comprises a first (A) and a second (B) set of sequences that hybridize to the target, wherein the set of sequences that hybridize to the target comprises: (i) (A) SEQ ID NO: 48, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 52, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (x) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) A combination of at least two oligomers according to item 1, selected from the group consisting of SEQ ID NOs: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65 or 66, or RNA equivalents or DNA / RNA chimeras thereof. (Item 46) a first and second set of amplification oligomers comprising a set of sequences that hybridize to a first (A) and a second (B) target, respectively, wherein the sets of sequences that hybridize to the targets are: (i) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 49, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 31, 61, 62, 64 or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 33, 34, 35, 62, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 54, 62, or 64, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (ix) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) A combination of at least two oligomers according to item 1, selected from the group consisting of SEQ ID NOs: 31, 33, 34, 35, 53, 61, 62, 64, 65 or 66, or RNA equivalents or DNA / RNA chimeras thereof. (Item 47) 47. A combination of at least two oligomers according to any of items 41 to 46, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 48) At least one amplification oligomer according to (b) is a promoter primer that further comprises a promoter sequence located 5' to the sequence that hybridizes to the target. 48. A combination of at least two oligomers according to any of items 1 to 47. (Item 49) 49. A combination of at least two oligomers according to item 48, wherein the promoter sequence is a T7 promoter sequence. (Item 50) 50. A combination of at least two oligomers according to item 49, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 73. (Item 51) 51. The combination of at least two oligomers according to any of items 1 to 50, further comprising at least one detection probe oligomer comprising a target-hybridizing sequence that is about 14 to about 28 nucleotides in length and configured to specifically hybridize to a target sequence contained in SEQ ID NO: 39 or its complement. (Item 52) 52. The combination of at least two oligomers according to item 51, wherein the sequence that hybridizes to the target of the detection probe is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67 and SEQ ID NO: 71, including complements, DNA equivalents and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67 and SEQ ID NO: 71. (Item 53) 52. The combination of at least two oligomers according to item 51, wherein the sequence that hybridizes to the target of the detection probe is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 67 and SEQ ID NO: 71, including complements, DNA equivalents and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 67 and SEQ ID NO: 71. (Item 54) 54. A combination of at least two oligomers according to any of items 51 to 53, wherein the at least one detection probe oligomer contains a 2'-methoxy backbone at one or more linkages of its nucleic acid backbone. (Item 55) 51. The combination of at least two oligomers according to any of items 1 to 50, further comprising at least two detection probe oligomers comprising target-hybridizing sequences that are about 14 to about 28 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 39 or its complement. (Item 56) 56. The combination of at least two oligomers according to item 55, wherein the sequences that hybridize to the targets of each detection probe are individually selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67 and SEQ ID NO: 71, including complements, DNA equivalents and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 67 and SEQ ID NO: 71. (Item 57) the at least two detection probe oligomers comprising: a first detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 55 or its complement, or an RNA equivalent or DNA / RNA chimera thereof; and 56. A combination of at least two oligomers according to item 55, comprising a second detection probe oligomer comprising a sequence that hybridizes to the target of SEQ ID NO: 67 or its complement, or its RNA equivalent or DNA / RNA chimera. (Item 58) 57. A combination of at least two oligomers according to item 55 or 56, comprising at least three detection probe oligomers. (Item 59) the at least three detection probe oligomers a first detection probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 37 or its complement, or its RNA equivalent or DNA / RNA chimera; a second detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 67 or its complement, or its RNA equivalent or DNA / RNA chimera; and 59. A combination of at least two oligomers according to item 58, comprising a third detection probe oligomer comprising a sequence that hybridizes to the target of SEQ ID NO: 71 or its complement, or its RNA equivalent or DNA / RNA chimera. (Item 60) 60. A combination of at least two oligomers according to any of items 55 to 59, wherein each detection probe oligomer contains a 2'-methoxy backbone at one or more linkages of its nucleic acid backbone. (Item 61) 61. The combination of at least two oligomers according to any of items 1 to 60, further comprising a capture probe oligomer comprising a target-hybridizing sequence covalently linked to an immobilized probe-binding sequence or moiety, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 4 and SEQ ID NO: 42. (Item 62) 62. A combination of at least two oligomers according to item 61, wherein the nucleobase at position 20 of SEQ ID NO: 4 is adenine (A). (Item 63) 63. A combination of at least two oligomers according to item 62, wherein the nucleobase at position 19 of SEQ ID NO: 4 is cytosine (C) or uracil (U). (Item 64) 62. The combination of at least two oligomers according to item 61, wherein the capture probe oligomers have a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 43. (Item 65) 61. The combination of at least two oligomers according to any of items 1 to 60, further comprising at least two capture probe oligomers comprising a target-hybridizing sequence covalently linked to an immobilized probe-binding sequence or moiety, wherein each target-hybridizing sequence is individually selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 4 and SEQ ID NO: 42. (Item 66) 66. A combination of at least two oligomers according to item 65, wherein the nucleobase at position 20 of SEQ ID NO: 4 is adenine (A). (Item 67) 67. A combination of at least two oligomers according to item 66, wherein the nucleobase at position 19 of SEQ ID NO: 4 is cytosine (C) or uracil (U). (Item 68) A combination of at least two oligomers according to Item 65, comprising at least three capture probe oligomers. (Item 69) the three capture probe oligomers are a first capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 2 or its complement, or its DNA equivalent or DNA / RNA chimera; a second capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO:6 or its complement, or a DNA equivalent or DNA / RNA chimera thereof; and 69. The combination of at least two oligomers according to item 68, comprising a third capture probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 42 or its complement, or a DNA equivalent or DNA / RNA chimera thereof. (Item 70) 70. A combination of at least two oligomers according to item 69, wherein the first, second and third capture probe oligomers have the sequences of SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 43, respectively. (Item 71) 71. A kit comprising a combination of at least two oligomers according to any of items 1 to 70. (Item 72) 71. A reaction mixture comprising a combination of at least two oligomers according to any one of items 1 to 70. (Item 73) 1. A method for determining the presence or absence of Hepatitis E virus (HEV) in a sample, comprising: (1) contacting a sample suspected of containing HEV with at least two oligomers for amplifying a target region of an HEV target nucleic acid, the combination of oligomers comprising: (a) At least one amplification oligomer selected from the group consisting of: (i) an oligomer comprising a target hybridizing sequence comprising at least the sequence of SEQ ID NO: 26, which is about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 63, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 26; and (ii) an oligomer comprising a target hybridizing sequence comprising about 14 to about 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 16, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 16; and (b) comprising at least one amplification oligomer comprising a target-hybridizing sequence comprising at least the sequence of SEQ ID NO:25, which is about 17 to about 28 contiguous nucleotides contained in the sequence of SEQ ID NO:47, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:25; (2) performing an in vitro nucleic acid amplification reaction, in which any HEV target nucleic acid present in the sample is used as a template to generate an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of HEV in the sample. (Item 74) 74. The method according to Item 73, wherein the at least one amplification oligomer of (a) comprises a sequence that hybridizes to the target, the sequence being about 14 to about 20 nucleotides contained in the sequence of SEQ ID NO: 13. (Item 75) At least one amplification oligomer of (1)(a) is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30 74. The method of claim 73, comprising a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66. (Item 76) 74. The method of claim 73, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. (Item 77) 74. The method of claim 73, wherein at least one amplification oligomer of (1)(a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 64, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29 and SEQ ID NO: 64. (Item 78) 74. The method of Item 73, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target comprising at least the sequence of SEQ ID NO: 27, which is 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 27. (Item 79) 79. The method of claim 78, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32. (Item 80) 79. The method of claim 78, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to the target of SEQ ID NO: 28, including RNA equivalents of SEQ ID NO: 28 and DNA / RNA chimeras. (Item 81) 81. The method of claim 80, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29 and SEQ ID NO: 32. (Item 82) 74. The method of claim 73, wherein at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 62, SEQ ID NO: 65, and SEQ ID NO: 66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 31, SEQ ID NO: 62, SEQ ID NO: 65, and SEQ ID NO: 66. (Item 83) 74. The method of claim 73, wherein at least one amplification oligomer of (1)(b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:56. (Item 84) 74. The method of claim 73, wherein at least one amplification oligomer of (1)(b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 56. (Item 85) 85. The method of item 83 or 84, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 86) 74. The method of claim 73, wherein at least one amplification oligomer of (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 46, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 46. (Item 87) 74. The method of claim 73, wherein at least one amplification oligomer of (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 51. (Item 88) 88. The method of any of items 73 to 87, wherein the at least two oligomers for amplifying the target region of HEV comprise an amplification oligomer according to (1)(a)(i) and an amplification oligomer according to (1)(a)(ii). (Item 89) 89. The method of claim 88, wherein the amplification oligomer described in (1)(a)(i) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. (Item 90) Item 90. The method of Item 89, wherein the amplification oligomer described in (1)(a)(i) comprises a sequence that hybridizes to the target of SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64. (Item 91) 90. The method of claim 89, wherein the amplification oligomer described in (a)(i) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 65, and SEQ ID NO: 66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 62, SEQ ID NO: 65, and SEQ ID NO: 66. (Item 92) 92. The method of any of Items 88 to 91, wherein the amplification oligomer according to (1)(a)(ii) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54. . (Item 93) 93. The method of claim 92, wherein the amplification oligomer described in (1)(a)(ii) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 32. (Item 94) the amplification oligomer according to (1)(a)(i) comprises a sequence that hybridizes to the target SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64; 89. The method of claim 88, wherein the amplification oligomer described in (1)(a)(ii) comprises a sequence that hybridizes to the target of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29. (Item 95) the amplification oligomer according to (a)(i) comprises a sequence that hybridizes to the target SEQ ID NO: 65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 65; 89. The method of claim 88, wherein the amplification oligomer described in (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO: 29 or SEQ ID NO: 31, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29 or SEQ ID NO: 31. (Item 96) 74. The method of claim 73, wherein the at least two oligomers for amplifying the HEV target region comprise a first and second amplification oligomer described in (a)(ii). (Item 97) 97. The method of Item 96, wherein each of the first and second amplification oligomers described in (a)(ii) comprises a sequence that hybridizes to a target comprising at least the sequence of SEQ ID NO: 27, which is 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16, including the RNA equivalent and DNA / RNA chimera of SEQ ID NO: 27. (Item 98) 98. The method of claim 97, wherein each of the first and second amplification oligomers of (a)(ii) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32. (Item 99) 98. The method of claim 97, wherein each of the first and second amplification oligomers of (a)(ii) comprises a sequence that hybridizes to the target of SEQ ID NO: 28, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 28. (Item 100) the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; 99. The method of claim 99, wherein the second amplification oligomer described in (a)(ii) comprises a sequence that hybridizes to the target of SEQ ID NO: 32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 32. (Item 101) 101. The method of any of items 73 to 100, wherein the at least two oligomers for amplifying the HEV target region comprise the first and second amplification oligomers described in (1)(b). (Item 102) Item 102. The method of item 101, wherein the first amplification oligomer described in (1)(b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 56. (Item 103) The method described in Item 101, wherein the first amplification oligomer described in (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 51, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 51. (Item 104) the first amplification oligomer according to (1)(b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 102. The method of claim 101, wherein the second amplification oligomer described in (1)(b) comprises a sequence that hybridizes to the target of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. (Item 105) 105. The method of claim 102 or 104, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 106) the first amplification oligomer according to (b) comprises a target-hybridizing sequence of SEQ ID NO:23 or SEQ ID NO:51, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:23 or SEQ ID NO:51; 102. The method of claim 101, wherein the second amplification oligomer described in (b) comprises a sequence that hybridizes to the target of SEQ ID NO: 45, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 45. (Item 107) 88. The method of any of items 73 to 87, wherein the at least two oligomers for amplifying the HEV target region comprise an amplification oligomer described in (1)(a)(i), an amplification oligomer described in (1)(a)(ii), a first amplification oligomer described in (1)(b), and a second amplification oligomer described in (1)(b). (Item 108) the amplification oligomer according to (1)(a)(i) comprises a sequence that hybridizes to the target SEQ ID NO: 64, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 64; the amplification oligomer according to (1)(a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29; the first amplification oligomer according to (1)(b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 108. The method of claim 107, wherein the second amplification oligomer described in (1)(b) comprises a sequence that hybridizes to the target of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. (Item 109) The amplification oligomer according to (a)(i) above comprises a sequence that hybridizes to a target of SEQ ID NO: 29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 29; and the amplification oligomer according to (a)(ii) above comprises a sequence that hybridizes to a target of SEQ ID NO: 65, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 65. ; Item 108. The method of item 107, wherein the first amplification oligomer described in (b) comprises a sequence that hybridizes to a target of SEQ ID NO: 24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 24; and the second amplification oligomer described in (b) comprises a sequence that hybridizes to a target of SEQ ID NO: 56, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 56. (Item 110) 109. The method of claim 108, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 111) 88. The method of any of items 73 to 87, wherein the at least two oligomers for amplifying the HEV target region comprise a first amplification oligomer described in (a)(ii), a second amplification oligomer described in (a)(ii), a first amplification oligomer described in (b), and a second amplification oligomer described in (b). (Item 112) the first amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO:29, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:29; the second amplification oligomer of (a)(ii) comprises a sequence that hybridizes to the target SEQ ID NO: 32, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 32; the first amplification oligomer according to (b) comprises a sequence that hybridizes to the target SEQ ID NO:24, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO:24; 112. The method of claim 111, wherein the second amplification oligomer described in (b) comprises a sequence that hybridizes to the target of SEQ ID NO: 46, or an RNA equivalent or DNA / RNA chimera of SEQ ID NO: 46. (Item 113) at least one amplification oligomer of (a) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66; 74. The method of claim 73, wherein at least one amplification oligomer of (b) comprises a sequence that hybridizes to a target selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 24 and SEQ ID NO: 56. (Item 114) a first, second and third set of amplification oligomers comprising a first, second and third set of target-hybridizing sequences, wherein the sets of target-hybridizing sequences are: (i) SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:24, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:24; (ii) SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:65, SEQ ID NO:29 and SEQ ID NO:56; (iii) SEQ ID NO:29, SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:29, SEQ ID NO:24 and SEQ ID NO:56; (iv) SEQ ID NO:66, SEQ ID NO:24 and SEQ ID NO:56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO:66, SEQ ID NO:24 and SEQ ID NO:56; (v) SEQ ID NO: 65, SEQ ID NO: 24 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 65, SEQ ID NO: 24 and SEQ ID NO: 56; and (vi) The method of claim 113, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 29 and SEQ ID NO: 56, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 62, SEQ ID NO: 29 and SEQ ID NO: 56. (Item 115) The combination comprises a first and a second set of amplification oligomers, each comprising a first (A) and a second (B) set of sequences that hybridize to the target, wherein the set of sequences that hybridize to the target is: (i) (A) SEQ ID NO: 54, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 54, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 53, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 53, and (B) SEQ ID NO: 23, 24, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 52, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (iv) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (xiii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or its RNA equivalent or DNA / RNA chimera; and (xiv) 74. The method of claim 73, wherein the nucleic acid sequence is selected from the group consisting of: (A) SEQ ID NO: 61, or an RNA equivalent or DNA / RNA chimera thereof; and (B) SEQ ID NO: 21, 22, 23, 24, 45, 56, 48, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof. (Item 116) The combination comprises first and second sets of amplification oligomers, each comprising a first (A) and second (B) set of target-hybridizing sequences, wherein the sets of target-hybridizing sequences are: (i) (A) SEQ ID NO: 54, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera; (ii) (A) SEQ ID NO: 53, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera; (iii) (A) SEQ ID NO: 31, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45, 49, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 30, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 21, 56, 48, 50, or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 66, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 65, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, 56 or 51, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 64, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 24, 45, 56 or 50, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 62, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 23, 24, 45 or 56, or an RNA equivalent or DNA / RNA chimera thereof; (x) (A) SEQ ID NO: 35, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xi) (A) SEQ ID NO: 34, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; (xii) (A) SEQ ID NO: 33, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 23, 45, or 56, or an RNA equivalent or DNA / RNA chimera thereof; and (xiii) (A) SEQ ID NO: 61, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 22, 45 or 56, or an RNA equivalent or a DNA / RNA chimera thereof. (Item 117) The combination comprises first and second sets of amplification oligomers, each comprising a first (A) and second (B) set of target-hybridizing sequences, wherein the sets of target-hybridizing sequences are: (i) (A) SEQ ID NO: 48, including RNA equivalents and DNA / RNA chimeras (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ii) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras; and (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66. or its RNA equivalent or DNA / RNA chimera; (iii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (v) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 52, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (ix) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (x) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 31, 33, 34, 35, 53, 54, 61, 62, 64, 65 or 66, or an RNA equivalent or DNA / RNA chimera thereof. (Item 118) The combination comprises a first and a second set of amplification oligomers comprising a set of sequences that hybridize to a first (A) and a second (B) target, respectively, wherein the sets of sequences that hybridize to the targets are: (i) (A) SEQ ID NO: 49, including RNA equivalents and DNA / RNA chimeras of SEQ ID NO: 49, and (B) SEQ ID NO: 29 or 31, or RNA equivalents or DNA / RNA chimeras thereof; (ii) (A) SEQ ID NO: 50, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29 or 31, or an RNA equivalent or DNA / RNA chimera thereof; (iii) (A) SEQ ID NO: 51, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 31, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (iv) (A) SEQ ID NO: 21, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, or its RNA equivalent or DNA / RNA chimera; (v) (A) SEQ ID NO: 22, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 31, 61, 62, 64 or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vi) (A) SEQ ID NO: 23, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 33, 34, 35, 62, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; (vii) (A) SEQ ID NO: 24, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 54, 62, or 64, or an RNA equivalent or DNA / RNA chimera thereof; (viii) (A) SEQ ID NO: 56, or its RNA equivalent or DNA / RNA chimera, and (B) SEQ ID NO: 29, 30, 33, 34, 35, 61, 62, 64, 65, or 66, or an RNA equivalent or DNA / RNA chimera thereof; and (ix) (A) SEQ ID NO: 45, or its RNA equivalent or DNA / RNA chimera, and (B) The method of item 73, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 31, 33, 34, 35, 53, 61, 62, 64, 65, or 66, or an RNA equivalent or a DNA / RNA chimera thereof. (Item 119) 119. The method according to any one of items 113 to 118, wherein the nucleobase at position 1 of SEQ ID NO: 56 is guanine (G). (Item 120) 119. The method according to any of items 73 to 119, wherein the at least one amplification oligomer according to (1)(b) is a promoter primer further comprising a promoter sequence located 5' to the sequence that hybridizes to the target. (Item 121) The method described in Item 120, wherein the promoter sequence is a T7 promoter sequence. (Item 122) Item 122. The method of item 121, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 73. (Item 123) 123. The method according to any one of items 73 to 122, further comprising, prior to step (1), purifying the HEV target nucleic acid from other components in the sample. (Item 124) 124. The method of claim 123, wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to an immobilized probe-binding sequence or moiety, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 4 and SEQ ID NO: 42. (Item 125) Item 125. The method of item 124, wherein the nucleobase at position 20 of SEQ ID NO: 4 is adenine (A). (Item 126) Item 126. The method of item 125, wherein the nucleobase at position 19 of SEQ ID NO: 4 is cytosine (C) or uracil (U). (Item 127) Item 125. The method of item 124, wherein the purifying step comprises contacting the sample with at least three capture probe oligomers. (Item 128) the three capture probe oligomers are a first capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:2, or its DNA equivalent or DNA / RNA chimera; a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:6, or its DNA equivalent or DNA / RNA chimera; and 128. The method of claim 127, comprising a third capture probe oligomer comprising a sequence that hybridizes to the target of SEQ ID NO: 42, or a DNA equivalent or DNA / RNA chimera thereof. (Item 129) 129. The method of claim 128, wherein the first, second, and third capture probe oligomers have the sequences of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 43, respectively. (Item 130) 129. The method of any of items 73 to 129, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer configured to specifically hybridize to the amplification product under conditions that determine the presence or absence of the amplification product, thereby determining the presence or absence of HEV in the sample. (Item 131) 131. The method of claim 130, further comprising at least one detection probe oligomer comprising a target-hybridizing sequence that is about 14 to about 28 nucleotides in length and configured to specifically hybridize to a target sequence contained in SEQ ID NO: 39 or its complement. (Item 132) 132. The method of claim 131, wherein the sequence that hybridizes to the target of the detection probe is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 55, SEQ ID NO: 67, and SEQ ID NO: 71. (Item 133) The method described in Item 131, wherein the sequence that hybridizes to the target of the detection probe is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 67 and SEQ ID NO: 71, including complements, DNA equivalents and DNA / RNA chimeras of SEQ ID NO: 37, SEQ ID NO: 67 and SEQ ID NO: 71. (Item 134) 134. The method according to any of items 131 to 133, wherein the at least one detection probe oligomer contains a 2'-methoxy backbone at one or more linkages of its nucleic acid backbone. (Item 135) 131. The method of claim 130, wherein the detecting step comprises contacting the in vitro nucleic acid amplification reaction with at least two detection probe oligomers comprising target-hybridizing sequences about 14 to about 28 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 39 or its complement. (Item 136) 136. The method of claim 135, wherein the target-hybridizing sequence of each detection probe is individually selected from the group consisting of SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:55, SEQ ID NO:67, and SEQ ID NO:71, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:55, SEQ ID NO:67, and SEQ ID NO:71. (Item 137) the at least two detection probe oligomers are a first detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 55 or its complement, or its RNA equivalent or DNA / RNA chimera; and 136. The method of claim 135, comprising a second detection probe oligomer comprising a sequence that hybridizes to the target of SEQ ID NO: 67 or its complement, or its RNA equivalent or DNA / RNA chimera. (Item 138) 137. The method of claim 135 or 136, wherein the detecting step comprises contacting the in vitro nucleic acid amplification reaction with at least three detection probe oligomers. (Item 139) the at least three detection probe oligomers a first detection probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 37 or its complement, or its DNA equivalent or DNA / RNA chimera; a second detection probe oligomer comprising a target-hybridizing sequence of SEQ ID NO: 67 or its complement, or a DNA equivalent or DNA / RNA chimera thereof; and 139. The method of claim 138, comprising a third detection probe oligomer comprising a sequence that hybridizes to the target of SEQ ID NO: 71, or its DNA or DNA / RNA equivalent. (Item 140) 139. The method of any of items 135 to 139, wherein each detection probe oligomer contains a 2'-methoxy backbone at one or more linkages of its nucleic acid backbone. (Item 141) the at least one detection probe oligomer is (a) Chemiluminescent label; (b) fluorescent labeling; (c) a quencher; and (d) The method according to any of items 130 to 140, comprising a label selected from the group consisting of one or more combinations of (a), (b) and (c). (Item 142) 142. The method of claim 141, wherein the at least one detection probe oligomer comprises the chemiluminescent label. (Item 143) Item 142. The method of Item 141, wherein the detecting step (3) detects hybridization of the at least one labeled detection probe oligomer to the amplification product in a homogeneous detection system. (Item 144) 144. The method of claim 143, wherein the label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the at least one detection probe oligomer. (Item 145) 145. The method according to any one of items 73 to 144, wherein the amplification reaction in step (2) is an isothermal amplification reaction. (Item 146) 146. The method of claim 145, wherein the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0043] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions being described relate. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0044] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "nucleic acid," as used herein, is understood to refer to one or more nucleic acids. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0045] A "sample" includes any specimen that may contain hepatitis E virus (HEV) (e.g., any one of HEV genotypes 1, 2, 3, or 4) or a component thereof, e.g., nucleic acid or nucleic acid fragment. A sample also includes a "biological sample," including any tissue or material from a living or deceased human that may contain HEV or a target nucleic acid therefrom, including, for example, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue (e.g., liver), or other bodily fluids or materials. A biological sample may be treated to physically or mechanically disrupt tissue or cellular structure, thereby releasing intracellular components into solutions that may further contain enzymes, buffers, salts, detergents, etc., which are used to prepare the biological sample for analysis using standard methods. A sample may also include processed samples, such as those obtained by passing the sample on or through a filtration device, or by subsequent centrifugation or adherence to a medium, matrix, or support.

[0046] "Nucleic acid" refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogen-containing heterocyclic bases or base analogs, linked together by phosphodiester or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and their analogs. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages (in "peptide nucleic acids" or PNAs, see, for example, International Patent Application Publication No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of a nucleic acid can be ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy and 2'-halide substitutions (e.g., 2'-F).Nitrogenous bases include the conventional bases (A, G, C, T, U), their analogs (e.g., inosine, 5-methylisocytosine, isoguanine; see, e.g., The Biochemistry of the Nucleic Acids, pp. 5-36, Adams et al., eds., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43:617-24), including derivatives of purine or pyrimidine bases (e.g., N4-methyldeoxyguanosine, deazapurines or azapurines, deazapyrimidines or azapyrimidines, pyrimidine bases having substituents at the 5- or 6-positions, purine bases having altered or replaced substituents at the 2-, 6-, and / or 8-positions, such as 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O4-alkyl-pyrimidines, and pyrazolo compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines; U.S. Pat. Nos. 5,378,825, 6,949,367, and International Patent Application Publication No. WO 93 / 13121, each of which is incorporated herein by reference). Nucleic acids may include "abasic" residues in which the backbone does not contain a nitrogenous base for one or more residues (see, e.g., U.S. Pat. No. 5,585,481, incorporated herein by reference). Nucleic acids may contain only conventional sugars, bases, and linkages, as found in RNA and DNA, or may contain conventional building blocks and substitutions (e.g., nucleic acids containing conventional bases linked by a 2'-methoxy backbone, or mixtures of conventional bases and one or more base analogs).Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA-mimicking sugar conformation, which enhances hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., Biochemistry 43:13233-41, 2004, incorporated herein by reference). Nucleic acids may also include modified bases that alter the function or behavior of the nucleic acid, for example, dideoxynucleotides may be added to the 3' end to block the addition of additional nucleotides to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art, but nucleic acids may also be purified from natural sources using routine techniques.

[0047] The term "polynucleotide," as used herein, refers to a nucleic acid strand. Throughout this application, nucleic acids are referred to from the 5' to the 3' end. Synthetic nucleic acids, e.g., DNA, RNA, and DNA / RNA chimerics (including those containing unnatural nucleotides or analogs), are typically synthesized "3' to 5'," i.e., by the addition of nucleotides to the 5' end of a growing nucleic acid.

[0048] As used herein, a "nucleotide" is a nucleic acid subunit consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base (also referred to herein as a "nucleobase"). The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of ribose (also referred to herein as "2'-O-Me" or "2'-methoxy"). As used herein, a methoxy oligonucleotide containing a "T" residue has a methoxy group at the 2' position of the ribose moiety and uracil at the base position of the nucleotide.

[0049] As used herein, a "non-nucleotide unit" is a unit that does not significantly participate in the hybridization of a polymer. Such a unit, for example, must not participate in any significant hydrogen bonding with a nucleotide, and excludes units having one of the five nucleotide bases or analogs thereof as constituent elements.

[0050] As used herein, a "target nucleic acid" is a nucleic acid containing a target sequence to be amplified. The target nucleic acid may be DNA or RNA as described herein, and may be single-stranded or double-stranded. In addition to the target sequence, the target nucleic acid may also contain other sequences that do not need to be amplified.

[0051] The term "target sequence" as used herein refers to the specific nucleotide sequence of the target nucleic acid to be amplified and / or detected. "Target sequence" includes the complex formation sequence with which an oligonucleotide (e.g., a priming oligonucleotide and / or a promoter oligonucleotide) forms a complex during the amplification process (e.g., TMA). If the target nucleic acid is originally single-stranded, the term "target sequence" also refers to the sequence that is complementary to the "target sequence" as present in the target nucleic acid. If the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) strand and the antisense (-) strand.

[0052] "Target-hybridizing sequence" is used herein to refer to the portion of an oligomer configured to hybridize with a target nucleic acid sequence. Preferably, the target-hybridizing sequence is configured to specifically hybridize with the target nucleic acid sequence. Target-hybridizing sequences may, but are not necessarily, 100% complementary to the portion of the target sequence to which they are configured to hybridize. Target-hybridizing sequences may also contain inserted, deleted, and / or substituted nucleotide residues relative to the target sequence. Less than 100% complementarity of a target-hybridizing sequence to a target sequence may occur, for example, when the target nucleic acid is multiple strains within a species, such as in the case of oligomers configured to hybridize to various genotypes of HEV. It is understood that there are other reasons for configuring a target-hybridizing sequence to have less than 100% complementarity to the target nucleic acid.

[0053] The term "targeting a sequence," as used herein with reference to a region of HEV nucleic acid, refers to the process by which an oligonucleotide hybridizes to a target sequence in a manner that allows for amplification and detection as described herein. In a preferred embodiment, the oligonucleotide is complementary to the targeted HEV nucleic acid sequence and contains no mismatches. In another preferred embodiment, the oligonucleotide is complementary to the targeted HEV nucleic acid sequence but contains one, two, three, four, or five mismatches with the sequence. Preferably, an oligonucleotide that hybridizes to an HEV nucleic acid sequence contains at least 10 to a maximum of 50 nucleotides that are complementary to the target sequence. At least 10 and a maximum of 50 are understood to be inclusive ranges, including 10, 50, and any integer therebetween. Preferably, the oligomer specifically hybridizes to the target sequence.

[0054] The term "configured to" refers to the actual arrangement of the polynucleotide sequence of a sequence that hybridizes to a referenced oligonucleotide target. For example, an amplification oligomer configured to generate a specific amplicon from a target sequence has a polynucleotide sequence that hybridizes to the target sequence and can be used in an amplification reaction to generate the amplicon. Also, as an example, an oligonucleotide configured to specifically hybridize to a target sequence has a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0055] As used herein, the term "configured to specifically hybridize to" means that the target-hybridizing region of an amplification oligonucleotide, detection probe, or other oligonucleotide is designed to have a polynucleotide sequence capable of targeting the sequence of the referenced HEV target region. Such oligonucleotides are not limited to targeting only that sequence, but rather are useful as compositions, in kits, or in methods for targeting HEV target nucleic acids. The oligonucleotides are designed to function as components of assays for amplifying and detecting HEV from a sample, and thus are designed to target HEV in the presence of other nucleic acids commonly found in test samples. "Specifically hybridizing to" does not mean exclusively hybridizing, as some small level of hybridization to non-target nucleic acids may occur, as is understood in the art. Rather, "specifically hybridizing to" means that the oligonucleotide is configured to function in an assay to hybridize primarily to the target, so that accurate detection of the target nucleic acid in a sample can be determined.

[0056] The term "fragment," as used herein in reference to an HEV-targeting nucleic acid, refers to a small piece of contiguous nucleic acid. In certain embodiments, the fragment includes contiguous nucleotides from the HEV RNA corresponding to SEQ ID NO: 1, where the number of contiguous nucleotides in the fragment is less than that for the entire sequence corresponding to SEQ ID NO: 1.

[0057] The term "region" as used herein refers to a portion of a nucleic acid that is smaller than the entire nucleic acid.For example, if the nucleic acid referred to is an oligonucleotide promoter primer, the term "region" can be used to refer to a smaller promoter portion of the entire oligonucleotide.Similarly and by way of example only, if the nucleic acid is HEV RNA, the term "region" can be used to refer to a smaller region of the nucleic acid, where the smaller region is targeted by one or more oligonucleotides of the present invention.As another non-limiting example, if the nucleic acid referred to is an amplicon, the term region can be used to refer to a smaller nucleotide sequence that is identified for hybridization by the sequence that hybridizes to the target of the probe.

[0058] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" generally refer to nucleic acids having less than 1,000 nucleotide (nt) residues, including polymers ranging from about 5 nt residues at the lower end and about 500-900 nt residues at the upper end. In some embodiments, oligonucleotides range in size from about 12-15 nt at the lower end and about 50-600 nt at the upper end, and in other embodiments from about 15-20 nt at the lower end and about 22-100 nt at the upper end. Oligonucleotides may be purified from naturally occurring sources or synthesized using any of a variety of well-known enzymatic or chemical methods. Preferably, oligonucleotides are synthesized using, for example, a DNA synthesizer and associated chemistry (e.g., ABI 3900, Life Technologies, Foster City, CA). Synthesized amplification oligomers, in one aspect, may be oligonucleotides synthesized using an automated synthesizer and phosphoramidite chemistry. In one embodiment, synthesized oligonucleotides are produced using phosphoramidite chemistry, and the hydroxyl group at the 3' end of most oligonucleotides is covalently attached to a solid support. Solid supports include, but are not limited to, controlled pore glass (CPG) and porous polystyrene (MPPS). In one embodiment, synthesized oligonucleotides are produced using phosphoramidite chemistry, and the phosphoramidite building blocks have protecting groups attached to their functional groups. Reactive groups include, but are not limited to, dimethoxytrityl (DMT), t-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and 2-cyanoethyl. The term "oligonucleotide" does not indicate any specific function for the reagent; rather, it is used generally to encompass all such reagents described herein. Oligonucleotides can perform a variety of different functions.For example, an oligonucleotide can function as a primer if it is specific for and can hybridize to a complementary strand and can be extended in the presence of a nucleic acid polymerase; an oligonucleotide can function as a primer and provide a promoter if it contains a sequence recognized by an RNA polymerase and allows transcription (e.g., a T7 primer); an oligonucleotide can function to detect a target nucleic acid if it can hybridize to a target nucleic acid or its amplicon and further provides a detectable moiety (e.g., an acridinium-ester compound).

[0059] As used herein, an oligonucleotide "substantially corresponding" to a particular reference nucleic acid sequence means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence so that it has similar hybridization properties to the reference nucleic acid sequence, in that it hybridizes to the same target nucleic acid sequence under stringent hybridization conditions. Those skilled in the art will understand that a "substantially corresponding oligonucleotide" may differ from the reference sequence but still hybridize to the same target nucleic acid sequence. It is also understood that a first nucleic acid corresponding to a second nucleic acid includes its RNA or DNA equivalent, as well as its DNA / RNA chimera, and its complement unless the context clearly dictates otherwise. This variation from the nucleic acid may be described in terms of the percentage of identical bases within the sequence or the percentage of perfectly complementary bases between a probe or primer and its target sequence. Thus, in certain embodiments, an oligonucleotide "substantially corresponds" to a reference nucleic acid sequence if these percentages of base identity or complementarity are between 100% and about 80%. In a preferred embodiment, the percentage is between 100% and about 85%. In more preferred embodiments, this percentage is 100% to about 90%; in other preferred embodiments, this percentage is 100% to about 95%. Similarly, a region of a nucleic acid or amplified nucleic acid may be referred to herein as corresponding to a reference nucleic acid sequence. Those skilled in the art will understand the various modifications to hybridization conditions that may be required for different percentages of complementarity that allow hybridization to specific target sequences without causing unacceptable levels of non-specific hybridization.

[0060] As used herein, the phrase "or its complement, or its RNA equivalent or DNA / RNA chimera," when referring to a DNA sequence, includes (in addition to the DNA sequence referred to) the complement of the DNA sequence, the RNA equivalent of the DNA sequence referred to, the RNA equivalent of the complement of the DNA sequence referred to, DNA / RNA chimera of the DNA sequence referred to, and DNA / RNA chimera of the complement of the DNA sequence referred to. Similarly, the phrase "or its complement, or its DNA equivalent or DNA / RNA chimera," when referring to an RNA sequence, includes (in addition to the RNA sequence referred to) the complement of the RNA sequence, the DNA equivalent of the RNA sequence referred to, the DNA equivalent of the complement of the RNA sequence referred to, DNA / RNA chimera of the RNA sequence referred to, and DNA / RNA chimera of the complement of the RNA sequence referred to.

[0061] As used herein, a "blocking moiety" is a substance used to "block" the 3' end of an oligonucleotide or other nucleic acid so that it cannot be efficiently extended by a nucleic acid polymerase. Oligomers not intended for extension by a nucleic acid polymerase can contain a blocker group that replaces the 3' OH to prevent enzyme-mediated extension of the oligomer in an amplification reaction. For example, blocked amplification oligomers and / or detection probes present during amplification may not have a functional 3' OH but instead contain one or more blocking groups located at or near the 3' end. In some embodiments, the blocking group near the 3' end may be within five residues of the 3' end and is sufficiently large to limit polymerase binding to the oligomer. In other embodiments, the blocking group is covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, such as alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin.

[0062] An "amplification oligomer" is an oligomer that has at least its 3' end complementary to a target nucleic acid, hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase during the amplification process. Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (e.g., because it has a 3'-blocked end) but participates in or facilitates amplification. For example, the 5' region of an amplification oligonucleotide may contain a promoter sequence (also referred to as a "promoter primer" or "promoter provider") that is not complementary to the target nucleic acid. Those skilled in the art will understand that an amplification oligomer that functions as a primer may be modified to include a 5' promoter sequence and thus function as a promoter primer. The incorporation of a 3'-blocked end further modifies the promoter primer, where the promoter primer hybridizes to the target nucleic acid and can provide an upstream promoter sequence that serves to initiate transcription, but does not provide a primer for oligo extension. Such modified oligos are referred to herein as "promoter provider" oligomers. Amplification oligonucleotides range in size from about 10 to about 70 nt in length (not including any promoter sequence or poly-A tail) and contain at least about 10 contiguous bases, or even at least 12 contiguous bases, that are complementary to a region of the target nucleic acid sequence (or its complementary strand). The contiguous bases are at least 80%, or at least 90%, or fully complementary to the target sequence to which the amplification oligomer binds. Amplification oligomers may optionally contain modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but are not complementary to and not contained in the target nucleic acid or template sequence. When referring to a range for the length of an oligonucleotide, amplicon, or other nucleic acid, it is understood that the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0063] As used herein, a "promoter" is a specific nucleic acid sequence that binds to a nucleic acid at a particular site and is recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to initiate transcription of RNA.

[0064] As used herein, "promoter provider" or "provider" refers to an oligonucleotide containing a first and a second region and modified to prevent initiation of DNA synthesis from its 3' end. The "first region" of a promoter provider oligonucleotide contains a base sequence that hybridizes to a DNA template, and the hybridizing sequence is located 3' of the promoter region, but is not necessarily adjacent to it. The hybridizing portion of a promoter oligonucleotide is typically at least 10 nucleotides in length and can be extended up to 50 or more nucleotides in length. The "second region" contains a promoter sequence for an RNA polymerase. The promoter oligonucleotide is engineered to be non-extendable by an RNA- or DNA-dependent DNA polymerase, e.g., reverse transcriptase, preferably containing a blocking moiety at its 3' end as described above. As referred to herein, a "T7 provider" is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequence recognized by T7 RNA polymerase.

[0065] A "terminating oligonucleotide" is an oligonucleotide containing a base sequence substantially complementary to a sequence within a target nucleic acid near the 5' end of the target region, such that it "terminates" primer extension of a nascent nucleic acid containing a priming oligonucleotide, thereby providing a defined 3' end of the nascent nucleic acid strand. The terminating oligonucleotide is designed to hybridize to the target nucleic acid at a position sufficient to provide the desired 3' end of the nascent nucleic acid strand. The positioning of the terminating oligonucleotide is flexible depending on its design. The terminating oligonucleotide may be modified or unmodified. In certain embodiments, a terminating oligonucleotide is synthesized containing at least one or more 2'-O-ME ribonucleotides. These modified nucleotides have been shown to exhibit higher thermal stability of complementary duplexes. 2'-O-ME ribonucleotides also function to increase the resistance of the oligonucleotide to exonucleases, thereby prolonging the half-life of the modified oligonucleotide. (See, e.g., Majlessi et al., Nucleic Acids Res. 26:2224-9, 1988, incorporated herein by reference.) Other modifications described elsewhere herein may be utilized in addition to or in place of 2'-O-Me ribonucleotides. For example, a terminating oligonucleotide may comprise a PNA or LNA. (See, e.g., Petersen et al., J. Mol. Recognit. 13:44-53, 2000, incorporated herein by reference.) A terminating oligonucleotide of the invention typically comprises a blocking moiety at its 3' end that prevents extension. A terminating oligonucleotide may also comprise a protein or peptide tethered to the oligonucleotide to terminate further extension of the nascent nucleic acid chain by a polymerase. A terminating oligonucleotide of the invention is typically at least 10 bases in length and may extend up to 15, 20, 25, 30, 35, 40, 50, or more nucleotides in length.A terminating oligonucleotide typically or necessarily includes a 3' blocking moiety, although a "3' blocked" oligonucleotide is not necessarily a terminating oligonucleotide.

[0066] "Amplification" refers to any known technique for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments. The multiple copies can be referred to as amplicons or amplification products. Amplification of "fragments" refers to the production of amplified nucleic acids that contain less than the entire target nucleic acid or its complement, for example, by using amplification oligonucleotides that hybridize to and initiate polymerization at an internal position of the target nucleic acid. Known amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification. Replicase-mediated amplification uses self-replicating RNA molecules and replicases such as QB-replicase (see, for example, U.S. Patent No. 4,786,600, which is incorporated herein by reference). PCR amplification uses DNA polymerase, primer pairs, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or from cDNA (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159, each of which is incorporated herein by reference). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (see, e.g., U.S. Patent Nos. 5,427,930 and 5,516,663, each of which is incorporated herein by reference). SDA uses primers containing recognition sites for restriction endonucleases and endonucleases that nick one strand of a hemi-modified DNA duplex containing the target sequence, thereby causing amplification in a series of primer extension and strand displacement steps (see, e.g., U.S. Pat. Nos. 5,422,252; 5,547,861; and 5,648,211, each of which is incorporated herein by reference).

[0067] "Transcription-associated amplification" or "transcription-mediated amplification" (TMA) refers to nucleic acid amplification using an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally use an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template-complementary oligonucleotide containing a promoter sequence, and may optionally contain one or more other oligonucleotides. TMA and single-primer transcription-associated amplification are embodiments of amplification methods used to detect HEV target sequences as described herein. The variant of transcription-associated amplification is well known in the art, as previously disclosed in detail (see, for example, United States Patent Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,554,516; and 7,374,885; and International Patent Application Publication No. WO88 / 01302; WO88 / 10315 and WO95 / 03430, each of which is incorporated herein by reference).Those skilled in the art will understand that disclosed compositions can also be used in amplification methods that are based on the extension of oligomer sequence by polymerase.

[0068] As used herein, the term "real-time TMA" refers to single-primer transcription-mediated amplification ("TMA") of a target nucleic acid monitored by a real-time detection means.

[0069] The term "amplicon" or "amplification product" as used herein refers to a nucleic acid molecule generated during an amplification procedure that is complementary or homologous to a sequence contained within a target sequence. The complementary or homologous sequence of an amplicon is sometimes referred to herein as a "target-specific sequence." Amplicons generated using the amplification oligomers of the present invention may contain non-target-specific sequences. Amplicons may be double-stranded or single-stranded and may contain DNA, RNA, or both. For example, DNA-dependent RNA polymerase transcribes single-stranded amplicons from double-stranded DNA during transcription-mediated amplification procedures. These single-stranded amplicons are RNA amplicons, and depending on how the amplification oligomer is configured, they may be either strand of a double-stranded complex. Thus, amplicons may be single-stranded RNA. RNA-dependent DNA polymerase synthesizes a DNA strand complementary to an RNA template. In this way, amplicons may be a hybrid of double-stranded DNA and RNA. RNA-dependent DNA polymerases often contain RNase activity or are used together with RNase to degrade RNA strands. Thus, an amplicon can be single-stranded DNA. RNA-dependent DNA polymerases and DNA-dependent DNA polymerases synthesize complementary DNA strands from DNA templates. Thus, an amplicon can be double-stranded DNA. RNA-dependent RNA polymerases synthesize RNA from RNA templates. Thus, an amplicon can be double-stranded RNA. DNA-dependent RNA polymerases synthesize RNA from double-stranded DNA templates, also known as transcription. Thus, an amplicon can be single-stranded RNA. Methods for generating amplicons and amplicons are known to those skilled in the art. For convenience herein, single-stranded RNA or single-stranded DNA can refer to amplicons generated by the combination of amplification oligomers of the present invention. Such terms are not intended to limit the amplicon to the terms shown. Those skilled in the art, in possession of this disclosure, will use amplification oligomers and polymerase enzymes to generate any of numerous types of amplicons, all of which are within the spirit and scope of the present invention.

[0070] As used herein, "non-target-specific sequence" refers to a region of an oligomer sequence that does not stably hybridize to a target sequence under standard hybridization conditions. Oligomers with non-target-specific sequences include, but are not limited to, promoter-primers and molecular beacons. Amplification oligomers may contain sequences that are not complementary to the target or template sequence; for example, the 5' region of a primer may contain a promoter sequence that is non-complementary to the target nucleic acid (referred to as a "promoter-primer"). Those skilled in the art will understand that an amplification oligomer that functions as a primer can be modified to include a 5' promoter sequence and thus function as a promoter-primer. Similarly, a promoter-primer can be modified by removing or synthesizing the promoter sequence without it and still function as a primer. A 3'-blocked amplification oligomer can provide a promoter sequence and serve as a template for polymerization (referred to as a "promoter provider"). In this way, the amplicon generated by an amplification oligomer member such as a promoter-primer contains target-specific and non-target-specific sequences.

[0071] "Detection probe," "detection oligonucleotide," and "detection probe oligomer" are used interchangeably and refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization and allow detection of the target sequence or amplified nucleic acid. Detection can be direct (e.g., a probe that hybridizes directly to its target sequence) or indirect (e.g., a probe that is linked to its target via an intermediate molecular structure). Detection probes can be DNA, RNA, analogs thereof, or combinations thereof (e.g., DNA / RNA chimeras), and they can be labeled or unlabeled. Detection probes can further include alternative backbone linkages, such as, for example, 2'-O-methyl linkages. The "target sequence" of a detection probe generally refers to a smaller nucleic acid sequence region within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through standard base pairing. Detection probes may contain target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (see, e.g., U.S. Patent Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Patent Application Publication No. 20060068417, each of which is incorporated herein by reference).

[0072] By "stable" or "stable for detection" is meant that the temperature of the reaction mixture is at least 2° C. below the melting temperature of the nucleic acid duplex.

[0073] As used herein, "label" refers to a moiety or compound directly or indirectly tethered to a probe that is detected or provides a detectable signal. Direct labeling can occur through a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonds, hydrophobic interactions, and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can occur through the use of a bridging moiety or "linker," such as a binding pair member, antibody, or additional oligomer, that can be labeled either directly or indirectly and amplify the detectable signal. Labels include any detectable moiety, such as a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group, or a chromophore (e.g., a dye, particle, or bead that provides a detectable color), a light-emitting compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label), or a fluorophore. The label can be detectable in a homogeneous assay, where the bound labeled probe in a mixture exhibits a detectable change that differs from that of the unbound labeled probe, such as instability or differential decomposition characteristics.The "homogeneous detectable label" can be detected without physically removing the bound form from the unbound form of the label or labeled probe (see, for example, U.S. Patent Nos. 5,283,174; 5,656,207; and 5,658,737, each of which is incorporated herein by reference).Labels include chemiluminescent compounds, such as standard acridinium esters ("AE") and AE compounds, including derivatives (see, for example, U.S. Patent Nos. 5,656,207; 5,658,737 and 5,639,604, each of which is incorporated herein by reference).Methods for synthesizing and attaching labels to nucleic acids and detecting labels are well known. (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10, which is incorporated herein by reference.See also U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333, each of which is incorporated herein by reference.) More than one label, and more than one label type, may be present on a particular probe, or detection may use a mixture of probes, each labeled with a compound that produces a detectable signal (see, e.g., U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference).

[0074] The terms "capture probe," "capture oligonucleotide," and "capture probe oligomer" are used interchangeably and refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid through standard base pairing, tethers to a binding partner on an immobilized probe, and captures the target nucleic acid to a support. An example of a capture oligomer typically contains two binding regions on the same oligomer: a sequence binding region (e.g., a target-specific portion) and an immobilized probe binding region, although the two regions can be present on two different oligomers connected together by one or more linkers. Another embodiment of a capture oligomer uses a target sequence binding region containing a random or non-random poly-GU, poly-GT, or poly-U sequence that nonspecifically binds to the target nucleic acid and links the nucleic acid to the immobilized probe on the support.

[0075] As used herein, "immobilized oligonucleotide," "immobilized probe," or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly tethers a capture oligomer to a support. Support-tethered immobilized probes facilitate separation of the target bound to the capture probe from unbound material in a sample. One embodiment of an immobilized probe is an oligomer tethered to a support, facilitating separation of the bound target sequence from unbound material in a sample. Supports may include known materials, such as matrices and particles free in solution, which may be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other compositions, one embodiment of which is a magnetically attractable particle. Supports may also be monodisperse magnetic spheres (e.g., uniform size ±5%) to which immobilized probes are tethered directly (via covalent bonds, chelation, or ionic interactions) or indirectly (via one or more linkers), where the linkage or interaction between the probe and the support is stable during hybridization conditions.

[0076] "Complementary" means that the nucleotide sequences of similar regions of two single-stranded nucleic acids or two different regions of the same single-stranded nucleic acid have a nucleotide base composition that allows the single-stranded regions to hybridize together to form a stable double-stranded hydrogen-bonded region under stringent hybridization or amplification conditions. The sequences that hybridize to each other may be fully complementary or partially complementary to the intended target sequence through standard nucleic acid base pairing (e.g., G:C, A:T, or A:U pairing). "Sufficiently complementary" refers to a contiguous sequence that can hybridize to another sequence through hydrogen bonding between a series of complementary bases, which may be complementary at each position in the sequence through standard base pairing, or may contain one or more residues, including non-complementary, abasic residues. A sufficiently complementary contiguous sequence is typically at least 80% or at least 90% complementary to the sequence to which the oligomer is intended to specifically hybridize. A "sufficiently complementary" sequence allows stable hybridization of the nucleic acid oligomer with its target sequence under appropriate hybridization conditions, even if the sequences are not perfectly complementary. Nucleotide sequences are "perfectly" complementary if a contiguous sequence of nucleotides in one single-stranded region can form a series of "canonical" hydrogen-bonded base pairs with a similar sequence of nucleotides in the other single-stranded region, such that A pairs with U or T and C pairs with G (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, especially §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57, which are incorporated herein by reference).It is understood that the percent identity ranges include all integers and partial numbers (eg, at least 90% includes 90, 91, 93.5, 97.687, etc.).

[0077] "Preferentially hybridize" or "specifically hybridize" means that under stringent hybridization assay conditions, probes hybridize to their target sequences or their replicas and form stable probe:target sequence hybrids, while at the same time minimizing the formation of stable probe:non-target hybrids. Thus, the probes hybridize to the target sequence or its replicas to a sufficiently higher degree than to non-target sequences, allowing one skilled in the art to accurately detect or quantify RNA copies or complementary DNA (cDNA) of the target sequence formed during amplification. Appropriate hybridization conditions are well known in the art and may be predictable based on sequence composition or may be determined by using routine testing (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, especially §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57, which are incorporated herein by reference).

[0078] "Nucleic acid hybrid," "hybrid," or "duplex" refers to a nucleic acid structure containing two-stranded, hydrogen-bonded regions, where each strand is complementary to the other, and which regions are sufficiently stable under stringent hybridization conditions to be detected by means including, but not limited to, chemiluminescent or fluorescent detection, autoradiography, or gel electrophoresis. Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

[0079] "Sample preparation" refers to any step or method of processing a sample for subsequent amplification and / or detection of HEV nucleic acids present in the sample. A sample may be a complex mixture of components in which the target nucleic acid is a minor component. Sample preparation may include any known method of concentrating components, such as microorganisms or nucleic acids, from a larger sample volume, such as by filtering airborne or waterborne particles from a larger sample volume, or by isolating microorganisms from a sample using standard microbiological methods. Sample preparation may include physical disruption and / or chemical lysis of cellular components, releasing the subcellular components substantially into an aqueous or organic phase, and removing debris, for example, by using filtration, centrifugation, or adsorption. Sample preparation may also include the use of nucleic acid oligonucleotides to selectively or nonspecifically capture target nucleic acids and separate them from other sample components (e.g., as described in U.S. Pat. No. 6,110,678 and International Patent Application Publication No. WO2008 / 016988, each of which is incorporated herein by reference).

[0080] "Separation" or "purification" means removing or separating one or more components of a sample from other sample components. Sample components typically include target nucleic acids in a generally aqueous solution phase, which may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids. "Separation" or "purification" does not include any degree of purification. Typically, separation or purification removes at least 70%, or at least 80%, or at least 95% of the target nucleic acids from other sample components.

[0081] As used herein, a "DNA-dependent DNA polymerase" is an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples include DNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNA polymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNA polymerases may be naturally occurring enzymes isolated from bacteria or bacteriophages, or may be recombinantly expressed, or may be modified or "evolved" forms engineered to possess certain desirable properties, such as thermostability or the ability to recognize or synthesize DNA strands from various modified templates. All known DNA-dependent DNA polymerases require a complementary primer to initiate synthesis. Under appropriate conditions, DNA-dependent DNA polymerases are known to be capable of synthesizing a complementary DNA copy from an RNA template. RNA-dependent DNA polymerases also typically possess DNA-dependent DNA polymerase activity.

[0082] As used herein, a "DNA-dependent RNA polymerase" or "transcriptase" is an enzyme that synthesizes multiple RNA copies from a double-stranded or partially double-stranded DNA molecule that contains a promoter sequence, which is usually double-stranded. RNA molecules ("transcripts") begin at specific positions immediately downstream of the promoter and are synthesized in a 5' to 3' direction. Examples of transcriptases are the DNA-dependent RNA polymerases from E. coli and bacteriophages T7, T3, and SP6.

[0083] As used herein, an "RNA-dependent DNA polymerase" or "reverse transcriptase" ("RT") is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; therefore, they are both RNA-dependent and DNA-dependent DNA polymerases. RTs may also have RNase H activity. Primers are required to initiate synthesis using both RNA and DNA templates.

[0084] As used herein, a "selective RNase" is an enzyme that degrades the RNA portion of an RNA:DNA duplex but does not degrade single-stranded RNA, double-stranded RNA, or DNA. An exemplary selective RNase is RNase H. Enzymes with the same or similar activity as RNase H can also be used. The selective RNase may be an endonuclease or exonuclease. Most reverse transcriptases have RNase H activity in addition to their polymerase activity. However, other sources of RNase H are available without associated polymerase activity. This degradation may result in separation of the RNA from the RNA:DNA complex. Alternatively, the selective RNase may simply cleave the RNA at various locations, such that portions of the RNA are melted or allow the enzyme to unwind portions of the RNA. Other enzymes that selectively degrade the RNA target sequence or RNA product of the present invention will be readily apparent to those skilled in the art.

[0085] The term "specificity" is used herein in the context of amplification and / or detection system, and refers to the characteristic of the system, which describes its ability to distinguish target sequence from non-target sequence, depending on sequence and assay conditions.In the context of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of by-products (for example, signal-to-noise ratio).In the context of detection, specificity generally refers to the ratio of the signal produced from target nucleic acid to the signal produced from non-target nucleic acid.

[0086] The term "sensitivity" is used herein to refer to the accuracy with which a nucleic acid amplification reaction can be detected or quantified. The sensitivity of an amplification reaction is generally a measure of the minimum copy number of the target nucleic acid that can be reliably detected in an amplification system, and depends, for example, on the detection assay used and the specificity of the amplification reaction, for example, the ratio of specific amplicon to by-product.

[0087] As used herein, the term "relative light unit" ("RLU") is an arbitrary unit of measurement that indicates the relative number of photons emitted by a sample at a given wavelength or band of wavelengths. RLUs vary with the characteristics of the detection means used in the measurement. [Brief explanation of the drawings]

[0088] [Figure 1A] FIG. 1 shows the reference sequence of the Hepatitis E Virus (HEV) genome (SEQ ID NO: 1), the complete sequence found in GenBank under accession numbers AB074918.2 and GI:2 1218075. [Figure 1B] FIG. 1 shows the reference sequence of the Hepatitis E Virus (HEV) genome (SEQ ID NO: 1), the complete sequence found in GenBank under accession numbers AB074918.2 and GI:2 1218075. [Figure 1C] FIG. 1 shows the reference sequence of the Hepatitis E Virus (HEV) genome (SEQ ID NO: 1), the complete sequence found in GenBank under accession numbers AB074918.2 and GI:2 1218075. [Figure 1D] FIG. 1 shows the reference sequence of the Hepatitis E Virus (HEV) genome (SEQ ID NO: 1), the complete sequence found in GenBank under accession numbers AB074918.2 and GI:2 1218075. DETAILED DESCRIPTION OF THE INVENTION

[0089] Detailed Description of the Invention The present invention provides compositions, kits, and methods for amplifying and detecting hepatitis C virus (HEV) nucleic acids from a sample. Preferably, the sample is a biological sample. The compositions, kits, and methods provide oligonucleotide sequences that recognize target sequences in the HEV genome, including target sequences of HEV genotypes 1, 2, 3, and 4, or their complementary sequences. Such oligonucleotides can be used as amplification oligonucleotides, and may include primers, promoter primers, blocked oligonucleotides, and promoter provider oligonucleotides, the functions of which have been previously described (see, e.g., U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 5,399,491; 5,554,516; 5,824,518; and 7,374,885, each of which is incorporated herein by reference). Other oligonucleotides can be used as probes to detect amplified sequences of HEV or to capture HEV target nucleic acids.

[0090] A method provides sensitive and specific detection of HEV nucleic acids. The method includes performing nucleic acid amplification of an HEV target region and detecting the amplification product, e.g., by specifically hybridizing the amplification product with a nucleic acid detection probe that provides a signal indicating the presence of HEV in the sample. The amplification step includes contacting the sample with one or more amplification oligomers specific to a target sequence in the HEV target nucleic acid to generate an amplification product if HEV nucleic acid is present in the sample. Amplification synthesizes additional copies of the target sequence or its complement by using at least one nucleic acid polymerase and the amplification oligomers to generate copies from the template strand (e.g., by extending sequences from primers using the template strand). One embodiment for detecting the amplification product uses a hybridization step that includes contacting the amplification product with at least one probe specific to a sequence amplified by selected amplification oligomers, e.g., a sequence contained in the target sequence flanked by a selected pair of amplification oligomers.

[0091] The detection step can be carried out using any of a variety of known techniques for detecting signals specifically associated with the amplified target sequence, such as by hybridizing a labeled detection probe to the amplification product and detecting the signal generated by the labeled probe. The detection step can also provide additional information about the amplified sequence, such as all or part of its nucleic acid base sequence. Detection can be carried out after the amplification reaction is completed, or can be carried out simultaneously with the amplification of the target region, e.g., in real time. In one embodiment, the detection step allows for homogeneous detection, e.g., detection of hybridized probes without removing unhybridized probes from the mixture (see, e.g., U.S. Patent Nos. 5,639,604 and 5,283,174, each of which is incorporated herein by reference).

[0092] In embodiments in which amplification products are detected near or at the end of the amplification step, linear detection probes may be used to provide a signal indicating hybridization of the probe to the amplification product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is achieved by chemiluminescence using a luminometer. (See, e.g., International Patent Application Publication No. WO 89 / 002476, incorporated herein by reference.) In other embodiments using real-time detection, the detection probe may be a hairpin probe, e.g., a molecular beacon, molecular torch, or hybridization switch probe labeled with a reporter moiety that is detected when the probe binds to the amplification product. Such probes may include a sequence that hybridizes to the target and a sequence that does not hybridize to the target. Various forms of such probes have been described previously (see, e.g., U.S. Patent Nos. 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Patent Application Publication Nos. 20060068417A1 and 20060194240A1, each of which is incorporated herein by reference).

[0093] Preferred compositions of the invention are configured to specifically hybridize to nucleic acids of all four major HEV genotypes (types 1, 2, 3, and 4) with minimal cross-reactivity to other non-HEV nucleic acids (e.g., other blood-borne pathogens) suspected to be present in a sample. In certain variations, compositions of the invention further enable detection of sequences tentatively designated as belonging to HEV genotype 6. In some embodiments, compositions of the invention are configured to specifically hybridize to HEV nucleic acids with minimal cross-reactivity to one or more of hepatitis C virus (HCV), human immunodeficiency virus 1 (HIV1), hepatitis B virus (HBV), and West Nile virus. In one embodiment, compositions of the invention are part of a multiplex system further including components and methods for detecting one or more of these organisms.

[0094] In certain aspects of the present invention, a combination of at least two oligomers is provided for determining the presence or absence of HEV in a sample. Typically, the combination of oligomers comprises at least two amplification oligomers for amplifying a target region of an HEV target nucleic acid corresponding to the sequence of SEQ ID NO: 1. In such embodiments, at least one amplification oligomer comprises a target-hybridizing sequence in a sense orientation ("sense THS"), and at least one amplification oligomer comprises a target-hybridizing sequence in an antisense orientation ("antisense THS"), where the sense THS and antisense THS are each configured to specifically hybridize to the HEV target sequence corresponding to the sequence contained in SEQ ID NO: 1, and where the target-hybridizing sequences are selected so that the HEV sequence targeted by the antisense THS is located downstream of the HEV sequence targeted by the sense THS (i.e., the at least two amplification oligomers are located so as to flank the target region to be amplified). In some variations, the oligomer combination includes (a)(i) an oligomer comprising a target-hybridizing sequence that is about 14 to about 23 contiguous nucleotides and substantially corresponds to or is identical to a sequence contained in SEQ ID NO: 63, including at least SEQ ID NO: 26 or its complement, or an RNA equivalent or DNA / RNA chimera thereof. In some variations, at least one amplification oligomer is (a)(ii) an oligomer comprising a target-hybridizing sequence that is about 14 to about 23 contiguous nucleotides and substantially corresponds to or is identical to a sequence contained in SEQ ID NO: 16 or its complement, or an RNA equivalent or DNA / RNA chimera thereof. In some variations, at least one amplification oligomer is (b) an oligomer comprising a target-hybridizing sequence of about 17 to about 28 contiguous nucleotides, contained in the sequence of SEQ ID NO: 47, and substantially corresponding to or identical to at least the sequence of SEQ ID NO: 25 or its complement, or its RNA equivalent or DNA / RNA chimera.In a more specific embodiment, providing at least one amplification oligomer for detecting HEV involves providing at least one amplification oligomer in an amplification reaction mixture. In one aspect, each of the at least one amplification oligomer is provided in the amplification reaction mixture at a concentration of about 4 pmol / reaction to about 12 pmol / reaction, including all integers and subnumbers within that range (e.g., 4, 4.5, 5, 6.75, 8, 10, 10.25, 11, 12.01). In some variations, the at least one amplification oligomer is a plurality of amplification oligomers, each of which is provided in the amplification reaction mixture at equal concentrations. In some variations, the at least one amplification oligomer is a plurality of amplification oligomers, each of which is not necessarily provided in the amplification reaction mixture at equal concentrations (e.g., one amplification oligomer is provided in the amplification reaction mixture at twice the concentration of another amplification oligomer).

[0095] In variations involving amplification oligomers described in (a)(i), (a)(ii), or (b) above, the combination of oligomers includes at least one amplification oligomer that contains a sequence that hybridizes to an HEV-specific target of opposite polarity (sense versus antisense, or vice versa) as the sequence that hybridizes to the target of the oligomer (a)(i), (a)(ii), or (b), such that the at least two amplification oligomers flank the target region to be amplified. In some such embodiments, the combination of oligomers includes at least one oligomer described in (a)(i) and / or (a)(ii) and at least one oligomer described in (b), such that the oligomer(s) in (a)(i) and / or (a)(ii) and the oligomer(s) in (b) flank the target region to be amplified. In some such embodiments, the oligomer combination comprises at least one amplification oligomer described in (a)(i), at least one amplification oligomer described in (a)(ii), and at least one amplification oligomer described in (b) (e.g., two amplification oligomers). In other such variations, the oligomer combination comprises at least two amplification oligomers described in (b) and at least one amplification oligomer described in either (a)(i) or (a)(ii).

[0096] In more specific embodiments of the invention, an oligomer combination for determining the presence or absence of HEV in a sample comprises (1) at least one amplification oligomer comprising a region that hybridizes to an HEV target substantially corresponding to at least one sense oligomer sequence set forth in Table 1 below, and (2) at least one amplification oligomer comprising a region that hybridizes to an HEV target substantially corresponding to at least one antisense oligomer sequence set forth in Table 1. In some such embodiments, the oligomer combination comprises at least two amplification oligomers of (1) above and / or at least two amplification oligomers of (2) above. In particular variations, the sequence(s) that hybridize to the sense and / or antisense target of the amplification oligomer combination comprise or consist of the sense and / or antisense sequence(s) selected from Table 1. [Table 1-1] [Table 1-2]

[0097] In certain embodiments, the amplification oligomers described herein are promoter primers or promoter providers that further comprise a promoter sequence 5' of the target-hybridizing sequence and that is non-complementary to the HEV target nucleic acid. For example, in some embodiments of the oligomer combinations described herein for amplifying an HEV target region, the amplification oligomer described in (b) above (e.g., an amplification oligomer comprising or consisting of an antisense target-hybridizing sequence shown in Table 1) is a promoter primer that further comprises a 5' promoter sequence. In certain embodiments, the promoter sequence is a T7 RNA polymerase promoter sequence, such as the T7 promoter sequence set forth in SEQ ID NO:73. In a specific variation, the amplification oligomer in (b) is a promoter primer that has the sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:18, or SEQ ID NO:20.

[0098] In some embodiments, the oligomer combinations described herein further comprise a terminating oligonucleotide (also referred to herein as a "blocker" oligonucleotide) that comprises a base sequence that is substantially complementary (e.g., perfectly complementary) to a sequence contained within the target nucleic acid near the 5' end of the target region. Terminating oligomers are typically used in combination with, for example, a promoter provider amplification oligomer, such as in certain embodiments described herein relating to transcription-mediated amplification (TMA).

[0099] In some embodiments, the oligomer combinations described herein further include at least one capture probe oligomer comprising a target-hybridizing sequence substantially corresponding to the sequence contained in the complement of SEQ ID NO:1, wherein the target-hybridizing sequence is covalently linked to the immobilized probe-binding sequence or moiety. In specific variations, the target-hybridizing sequence comprises or consists of a sequence substantially corresponding to or identical to a sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:42. In more specific variations, the capture probe oligomer has a sequence selected from SEQ ID NO:3, SEQ ID NO:7, and SEQ ID NO:43. The oligomer combination may include at least two (e.g., three) capture probe oligomers as described above. In more specific embodiments, providing at least one capture probe oligomer in a target capture reaction mixture includes providing at least one capture probe oligomer. In one embodiment, each of the at least one capture probe oligomer is provided in the target capture reaction mixture at a concentration of about 3 pmol / reaction to about 6 pmol / reaction, including all integers and subnumbers therein (e.g., 4, 4.75, 5.12, 5.98, 6). When multiple, at least one capture probe oligomers are used in the target capture reaction, the concentration of each capture probe oligomer may be equal to the concentration of the others or may vary, as described herein.

[0100] In certain variations, the oligomer combinations described herein further comprise at least one detection probe oligomer configured to specifically hybridize to an HEV target sequence amplifiable using the first and second amplification oligomers (e.g., an HEV target sequence flanked by sequences hybridizing to the targets of the first and second amplification oligomers). In certain embodiments, the detection probe oligomer comprises a target-hybridizing sequence that is about 14 to about 28 nucleotides in length and configured to specifically hybridize to the target sequence contained within SEQ ID NO:39 or its complement. Particularly suitable detection probe oligomers include oligomers comprising a target-hybridizing sequence that is substantially corresponding to or identical to a sequence selected from SEQ ID NO:37, SEQ ID NO:55, SEQ ID NO:67, and SEQ ID NO:71, including, for example, complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO:37, SEQ ID NO:55, SEQ ID NO:67, and SEQ ID NO:71. The detection probe oligomer may comprise a 2'-nucleotide sequence at one or more junctions in the nucleic acid backbone. -methoxy backbone. In some variations, the oligomer combination comprises at least two detection probe oligomers. In more specific embodiments, the at least one detection probe oligomer comprises providing at least one detection probe oligomer in the amplicon detection reaction mixture. In one aspect, each of the at least one detection probe oligomer is provided in the detection reaction mixture at about 2.0E+06 RLU / reaction to about 6.0E+06 RLU / reaction, including all integers and subnumbers therein (e.g., 2.0E+06, 2.138E+06, 3.385E+06 RLU). When multiple at least one detection probe oligomers are used in the detection reaction, the concentration of each detection oligomer may be equal to the concentration of the others or may vary, as described herein.

[0101] Typically, the detection probe oligomer according to the present invention further comprises a label. Particularly suitable labels include compounds that emit detectable light signals, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. More than one label and more than one type of label may be present on a specific probe, or detection may rely on the use of a mixture of probes, each of which is labeled with a compound that generates a detectable signal (see, for example, U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). Labels may be attached to probes by various means, including covalent bonding, chelation, and ionic interactions, but preferably, the labels are covalently attached. For example, in some embodiments, the detection probe has an attached chemiluminescent label, e.g., an acridinium ester (AE) compound, which, in typical variations, is attached to the probe by a non-nucleotide linker (see, e.g., U.S. Pat. Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744, each of which is incorporated herein by reference). (See, e.g., U.S. Pat. Nos. 5,585,481; 5,656,744; and 5,639,604, particularly column 10, line 6 to column 11, line 3, and Example 8, each of which is incorporated herein by reference.) In other embodiments, the detection probe includes both a fluorescent label and a quencher, a combination that is particularly useful in fluorescence resonance energy transfer (FRET) assays. Specific variations of such detection probes include, for example, TaqMan detection probes (Roche Molecular Diagnostics) and "molecular beacons" (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; U.S. Patent Nos. 5,118,801 and 5,312,728, each of which is incorporated herein by reference).

[0102] Detection probe oligomer according to the present invention can further comprise a sequence that does not hybridize to target.Specific embodiments of such detection probe include probes that form a three-dimensional structure that is maintained by intramolecular hybridization, such as the three-dimensional structure generally known as hairpin.Particularly suitable hairpin probes include "molecular torch" (see, for example, U.S. Patent No. 6,849,412; U.S. Patent No. 6,835,542; U.S. Patent No. 6,534,274; and U.S. Patent No. 6,361,945, each of which is incorporated herein by reference) and "molecular beacon" (see, for example, Tyagi et al., supra; U.S. Patent No. 5,118,801 and U.S. Patent No. 5,312,728, supra).Methods for using such hairpin probes are well known in the art.

[0103] In yet other embodiments, the detection probe is a linear oligomer that does not substantially form a conformation held together by intramolecular bonds. In a particular variation, the linear detection probe oligomer comprises a chemiluminescent compound, preferably an acridinium ester (AE) compound, as a label.

[0104] In yet another variation, the oligomer combination for detecting HEV nucleic acids further comprises a probe-protection oligomer substantially complementary to the detection probe oligomer. The probe-protection oligomer hybridizes to a substantially complementary labeled detection probe oligomer (e.g., a probe labeled with a chemiluminescent compound) and can stabilize the labeled probe during storage. In a specific embodiment, the probe-protection oligomer has a sequence substantially corresponding to or identical to a sequence selected from SEQ ID NO: 36 and SEQ ID NO: 40.

[0105] The invention also provides detection probe oligomers, capture probe oligomers and probe protection oligomers as described herein.

[0106] In another aspect, the present invention provides a method for determining the presence or absence of HEV in a sample using the oligomer combination described herein. Such a method generally includes the steps of: (1) contacting a sample with at least two oligomers for amplifying an HEV nucleic acid target region corresponding to an HEV target nucleic acid, the oligomers comprising at least two amplification oligomers described above; (2) performing an in vitro nucleic acid amplification reaction in which any HEV target nucleic acid present in the sample is used as a template to generate an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of HEV in the sample. Detection methods according to the present invention typically further include the step of obtaining a sample to be contacted with at least two oligomers. In certain embodiments, "obtaining" a sample used in steps (1) through (3) includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed and / or retrieving the sample from a location within the facility where one or more steps of the method are performed (e.g., from storage or other deposit).

[0107] In certain embodiments, the method further comprises purifying the HEV target nucleic acid from other components in the sample prior to the contacting step. Such purification may include methods for separating and / or concentrating organisms contained in the sample from other sample components. In certain embodiments, purifying the target nucleic acid comprises capturing the target nucleic acid to specifically or nonspecifically separate the target nucleic acid from other sample components. Nonspecific target capture methods may involve selective precipitation of the nucleic acid from a substantially aqueous mixture, attaching the nucleic acid to a support and washing the support to remove other sample components, or other means of physically separating the nucleic acid from a mixture containing HEV nucleic acid and other sample components.

[0108] In some embodiments, the HEV target nucleic acid is selectively separated from other sample components by specifically hybridizing the HEV target nucleic acid to a capture probe oligomer. The capture probe oligomer comprises a target-hybridizing sequence configured to specifically hybridize to the HEV target sequence, such that a target sequence:capture probe complex is formed that is separated from the sample components. Suitable capture probe target-hybridizing sequences include sequences that substantially correspond to or are identical to sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:42, including complements, DNA equivalents, and DNA / RNA chimeras of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:42. In a preferred variation, specific target capture occurs by binding the HEV target:capture probe complex to an immobilized probe, forming a target:capture probe:immobilized probe complex, which is then separated from the sample and optionally washed to remove non-target sample components (see, e.g., U.S. Patent Nos. 6,110,678; 6,280,952; and 6,534,273, each of which is incorporated herein by reference). In such a variation, the capture probe oligomer further comprises a sequence or moiety that binds or attaches the capture probe, allowing the target sequence to bind to the immobilized probe attached to the solid support, thereby separating the hybridized target nucleic acid from other sample components.

[0109] In more specific embodiments, the capture probe oligomer includes a tail portion (e.g., a 3' tail) that is not complementary to the HEV target sequence but specifically hybridizes to a sequence on the immobilized probe, thereby functioning as a moiety that allows the target nucleic acid to be separated from other sample components, as described, for example, in U.S. Patent No. 6,110,678, incorporated herein by reference. Any sequence can be used in the tail region, generally about 5-50 nt in length, and preferred embodiments include a substantially homopolymeric tail of about 10-40 nt (e.g., A10-A40), more preferably about 14-33 nt (e.g., A14-A30 or T3A14-T3A30), that binds to a complementary immobilized sequence (e.g., poly-T) attached to a solid support, e.g., a matrix or particle. For example, in specific embodiments of a capture probe that includes a 3' tail, the capture probe has a sequence selected from SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 43.

[0110] Target capture typically occurs in a solution-phase mixture containing one or more capture probe oligomers that specifically hybridize to the HEV target sequence under hybridizing conditions, usually at a temperature higher than the Tm of the tail-sequence:immobilized probe sequence duplex. For embodiments including a capture probe tail, the HEV target:capture probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to the immobilized probe, and then the entire complex on the solid support is separated from other sample components. The support with the bound immobilized probe:capture probe:HEV target sequence can be washed one or more times to further remove other sample components. A preferred embodiment uses a particulate solid support, such as paramagnetic beads, so that the particles with the bound HEV target:capture probe:immobilized probe complex are suspended in a wash solution and can be removed from the wash solution, preferably by the use of magnetic attraction. To limit the number of processing steps, the HEV target nucleic acid can be amplified by simply mixing the HEV target sequence in the complex on the support with amplification oligomers and proceeding with the amplification step.

[0111] Amplification of the HEV target sequence utilizes an in vitro amplification reaction using at least two amplification oligomers that flank the target region to be amplified. In certain embodiments, the target region to be amplified substantially corresponds to SEQ ID NO:1 from about nucleotide 5230 to about nucleotide 5379. Particularly suitable amplification oligomer combinations for amplifying these target regions are described herein (see, e.g., paragraphs [4]-

[14] and

[87] -

[90] , supra). Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA). Such amplification methods are well known in the art (see, e.g., paragraphs

[59] and

[60] , supra) and are readily used in accordance with the methods of the present invention.

[0112] For example, some amplification methods using TMA amplification include the following steps: Briefly, a target nucleic acid containing the sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art will understand that conventional melting of double-stranded nucleic acids (e.g., dsDNA) can be used to provide a single-stranded target nucleic acid. A promoter primer specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) uses the target strand as a template to extend the 3' end of the promoter primer, creating a cDNA copy of the target sequence strand, resulting in an RNA:DNA duplex. RNase digests the RNA strand of the RNA:DNA duplex, and a second primer specifically binds to its target sequence located on the cDNA strand downstream from the promoter primer end. RT uses the first cDNA template to synthesize a new DNA strand by extending the 3' end of the second primer, creating a dsDNA containing a functional promoter sequence. Next, RNA polymerase specific to the promoter sequence initiates transcription, producing RNA transcripts that are approximately 100-1000 amplified copies ("amplicons") of the initial target strand in the reaction. Amplification continues when a second primer specifically binds to its target sequence in each amplicon and RT creates DNA copies from the amplicon RNA template, generating an RNA:DNA duplex. RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA duplex, and the promoter primer specifically binds to its complementary sequence in the newly synthesized DNA. RT extends the 3' end of the promoter primer, creating dsDNA containing a functional promoter for RNA polymerase to bind and transcribe additional amplicons complementary to the target strand. This autocatalytic cycle, creating more amplicon copies, repeats over the course of the reaction, resulting in approximately one billion-fold amplification of the target nucleic acid present in the sample. The amplification products can be detected in real time during amplification or at the end of the amplification reaction using probes that specifically bind to the target sequence contained in the amplification product. Detection of a signal arising from the bound probe indicates the presence of the target nucleic acid in the sample.

[0113] In some embodiments, the method utilizes a "reverse" TMA reaction. In such variations, the first or "forward" amplification oligomer is a priming oligonucleotide that hybridizes to the target nucleic acid near the 3' end of the target region. Reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3' end of the primer using the target nucleic acid as a template. The second or "reverse" amplification oligomer is a promoter primer or promoter provider having a target-hybridizing sequence configured to hybridize to the target sequence contained within the synthesized cDNA strand. If the second amplification oligomer is a promoter primer, RT extends the 3' end of the promoter primer using the cDNA strand as a template to create a second cDNA copy of the target sequence strand, thereby creating a dsDNA containing a functional promoter sequence. Amplification then continues essentially as described above in paragraph

[0105] , with initiation of transcription from the promoter sequence using RNA polymerase. Alternatively, when the second amplification oligomer is a promoter provider, a terminating oligonucleotide hybridizing to the target sequence near the 5' end of the target region is typically used to terminate the extension of the priming oligomer at the 3' end of the terminating oligonucleotide, thereby providing a defined 3' end for the first cDNA strand synthesized by extension from the priming oligomer.The sequence hybridizing to the target of the promoter provider then hybridizes to the defined 3' end of the first cDNA strand, and the 3' end of the cDNA strand is extended to add a sequence complementary to the promoter sequence of the promoter provider, thereby forming a double-stranded promoter sequence.The first cDNA strand is then used as a template to transcribe multiple RNA transcripts complementary to the first cDNA strand without the promoter portion, using an RNA polymerase that recognizes and initiates transcription from the double-stranded promoter.Each of these RNA transcripts is then available to serve as a template for further amplification from the first priming amplification oligomer.

[0114] Detection of amplification products can be achieved by various methods. Nucleic acids can be associated with a surface, causing a physical change, such as a detectable electrical change. Amplified nucleic acids can be detected by concentrating the nucleic acids in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g., intercalating agents such as ethidium bromide or SYBR Green), or by detecting an increase in dyes associated with nucleic acids in the solution phase. Other methods of detection can use nucleic acid detection probes configured to specifically hybridize to the sequence of the amplification product to detect the presence of a probe:product complex, or by using a complex of probes that can amplify a detectable signal associated with the amplification product (e.g., U.S. Patent Nos. 5,424,413; 5,451,503; and 5,849,481, each of which is incorporated herein by reference). Directly or indirectly labeled probes that specifically associate with the amplification product provide a detectable signal indicating the presence of the target nucleic acid in the sample. Specifically, the amplification product contains a target sequence in the sequence of HEV genomic RNA or a target sequence complementary to the sequence of HEV genomic RNA, and the probe binds directly or indirectly to a sequence contained in the amplification product that indicates the presence of HEV nucleic acid in the sample being tested.

[0115] Preferred embodiments of detection probes that hybridize to complementary amplified sequences can be DNA or RNA oligomers, or oligomers containing a combination of DNA and RNA nucleotides, or oligomers synthesized with modified backbones, such as oligomers containing one or more 2'-methoxy-substituted ribonucleotides. Probes used to detect amplified HEV sequences can be unlabeled, can be detected indirectly (e.g., by attaching another binding partner to a moiety on the probe), or can be labeled with various detectable labels. Specific embodiments of detection probes suitable for use in accordance with the methods of the present invention are further described herein (see, e.g., paragraphs

[15] ,

[16] , and

[31] -

[33] , supra). In some preferred embodiments of methods for detecting HEV sequences, for example, certain embodiments using transcription-mediated amplification (TMA), the detection probe is a linear chemiluminescent-labeled probe, more preferably a linear acridinium ester (AE)-labeled probe.

[0116] Oligomers that are not intended to be extended by nucleic acid polymerase preferably include a blocker group that replaces the 3' OH to prevent enzyme-mediated extension of the oligomer during amplification reactions. For example, blocked amplification oligomers and / or detection probes present during amplification preferably do not have a functional 3' OH, but instead contain one or more blocking groups located at or near the 3' end. The blocking group near the 3' end is preferably within five residues of the 3' end and is large enough to restrict polymerase binding to the oligomer, and other preferred embodiments contain a blocking group covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, including alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin.

[0117] An example of an oligomer that is typically blocked at the 3' end and is particularly suitable in certain embodiments using transcription-mediated amplification is a promoter provider. As described above, the promoter provider comprises a first region that hybridizes to a target and a second region located 5' to the first region that comprises a promoter sequence for an RNA polymerase. The promoter provider oligonucleotide is modified to prevent the initiation of DNA synthesis from its 3' end, for example, by including a blocker group as discussed above.

[0118] Another example of a typically 3'-blocked oligomer is the terminating ("blocker") oligonucleotide previously described above. Terminating oligomers are typically used in combination with, for example, a promoter provider amplification oligomer, such as in certain embodiments described herein, e.g., in connection with transcription-mediated amplification (TMA). The terminating oligomer hybridizes to a sequence contained within the target nucleic acid near the 5' end of the target region to "terminate" primer extension of the nascent nucleic acid, including the priming oligonucleotide, thereby providing a defined 3' end for the nascent nucleic acid strand.

[0119] Other embodiments using transcription-mediated amplification utilize a promoter primer that includes a first target-hybridizing region and a second region located 5' to the first region that includes a promoter sequence for RNA polymerase, but that is not modified to prevent initiation of DNA synthesis from its 3' end. In some embodiments, a promoter primer for use in accordance with the detection method includes a target-hybridizing sequence that has a sequence substantially corresponding to or identical to a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:56. In certain variations of promoter primers that include a target-hybridizing sequence set forth in SEQ ID NO:56, the nucleobase at position 1 of SEQ ID NO:56 is guanine (G); in other variations, the promoter primer is degenerate at position 1 of SEQ ID NO:56, such that this position is occupied by either cytosine (C) or guanine (G) within a population of oligomers that include SEQ ID NO:56. In more particular variations, the promoter primer for use in accordance with the detection method has the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0120] Assays for detecting HEV nucleic acids may optionally include a non-HEV internal control (IC) nucleic acid that is amplified and detected in the same assay reaction mixture by using amplification oligomers and detection oligomers specific for the IC sequence. The IC nucleic acid sequence may be an RNA template sequence (e.g., an in vitro transcript), a synthetic nucleic acid sequence spiked into the sample, or the IC nucleic acid sequence may be a cellular component. Cellular component IC nucleic acid sequences may be derived from exogenous or endogenous cellular sources relative to the specimen. In these cases, the internal control nucleic acid is co-amplified with the HEV nucleic acid in the amplification reaction mixture. The internal control amplification product and the HEV target sequence amplification product can be detected independently. Two different internal control systems were used in the procedures described below.

[0121] The first arrangement for the internal control system was useful for monitoring the integrity of amplification and detection reactions using a paired set of primers and an oligonucleotide probe, or its complement, that hybridizes to the amplification product at a position between the primer binding sites. This arrangement was used in the assay described in the following examples. In a simple application, the internal control template nucleic acid can be distinguished from the analyte template nucleic acid by the sequence of bases that serve as probe binding sites. These bases can be scrambled, replaced with unrelated base sequences, or simply contain a sufficient number of point mutations to result in differential probe binding. In this way, the nucleic acid product resulting from amplification of the analyte nucleic acid can be detected by an analyte-specific probe but not by an internal control-specific probe. Similarly, the amplicon resulting from amplification of the internal control nucleic acid can be detected by an internal control-specific probe but not by an analyte-specific probe. This configuration allows both the analyte and internal control nucleic acid templates to be amplified using the same primers or primer sets.

[0122] In certain embodiments, amplification and detection of a signal from the amplified IC sequence clearly indicates that the performance of the assay reagents, conditions, and assay steps used in the assay are appropriate if no signal is obtained for the intended target HEV nucleic acid (e.g., a sample that tests negative for HEV). The IC may also be used as an internal calibrator for the assay if a quantitative result is desired; i.e., the signal obtained from amplification and detection of the IC is used to set parameters used in an algorithm to quantify the amount of HEV nucleic acid in a sample based on the signal obtained for the amplified HEV target sequence. The IC is also useful for monitoring the integrity of one or more steps in the assay. A preferred embodiment of a synthetic IC nucleic acid sequence is a randomized sequence derived from a naturally occurring source (e.g., an HIV sequence rearranged in a random manner). Another preferred IC nucleic acid sequence may be an RNA transcript isolated from a naturally occurring source, or an RNA transcript synthesized in vitro, for example, by generating transcripts from a cloned randomized sequence, so that the number of copies of the IC included in the assay can be accurately determined. Primers and probes for IC target sequences may be constructed and synthesized using any known method, provided that the primers and probes function for amplification of the IC target sequence and detection of the amplified IC sequence using substantially the same assay conditions used to amplify and detect the HEV target sequence. In preferred embodiments involving a target capture-based purification step, a target capture probe specific for the IC target is preferably included in the assay in the target capture step, such that the IC is processed in the assay in a manner similar to that for the intended HEV analyte throughout all of the assay steps.

[0123] In certain embodiments of the methods for determining the presence or absence of HEV in a sample, the methods further include the use of a probe protection oligomer described herein to adjust assay sensitivity.

[0124] The present invention also provides a reaction mixture for determining the presence or absence of HEV target nucleic acid in a sample.The reaction mixture according to the present invention comprises at least one or more of the oligomer combinations described herein for amplifying HEV target nucleic acid; the capture probe oligomers described herein for purifying HEV target nucleic acid; the detection probe oligomers described herein for determining the presence or absence of HEV amplification products; and the probe protection oligomers described herein for detuning the sensitivity of the assay for detecting HEV target nucleic acid.The reaction mixture may further comprise several optional components, such as an array of capture probe nucleic acids. For amplification reaction mixtures, the reaction mixture typically includes other reagents suitable for performing in vitro amplification, such as buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), and typically includes components of the test sample in which HEV target nucleic acid may or may not be present. Additionally, for reaction mixtures that include detection probes along with combinations of amplification oligomers, the selection of amplification oligomers and detection probe oligomers for the reaction mixture is linked by a common target region (i.e., the reaction mixture includes probes that bind to sequences that can be amplified by the combination of amplification oligomers in the reaction mixture).

[0125] The present invention also provides kits for carrying out the methods described herein. Kits according to the present invention include at least one or more of the amplification oligomer combinations described herein for amplifying HEV target nucleic acids; capture probe oligomers described herein for purifying HEV target nucleic acids; detection probe oligomers described herein for determining the presence or absence of HEV amplification products; and probe protection oligomers described herein for detuning the sensitivity of the assay for detecting HEV target nucleic acids. The kits may further include several optional components, such as an array of capture probe nucleic acids. Other reagents that may be present in the kit include reagents suitable for performing in vitro amplification, such as buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase). The oligomers described herein may be packaged in a variety of different embodiments, and those skilled in the art will understand that the present invention encompasses many different kit configurations. For example, a kit may contain amplification oligomers for only one target region of the HEV genome, or may contain amplification oligomers for multiple HEV target regions. Furthermore, for kits that include a detection probe along with a combination of amplification oligomers, the selection of amplification oligomers and detection probe oligomers for the kit is linked by a common target region (i.e., the kit includes a probe that binds to a sequence that can be amplified by the combination of amplification oligomers in the kit). In certain embodiments, the kit further includes a set of instructions for carrying out a method according to the invention, in which case the instructions may be associated with a package insert and / or packaging of the kit or its components.

[0126] The present invention is further illustrated by the following non-limiting examples. [Example]

[0127] Example 1 This example describes amplification reactions using various primer sets to amplify HEV target regions. Table 2 below lists all amplification oligomers used in this assay. [Table 2]

[0128] Each possible combination of T7 and non-T7 primers listed in Table 2 was tested. Primers were tested in transcription-mediated amplification (TMA) reactions using HEV in vitro transcripts (IVT) at 15 and 0 copies per reaction. TMA reactions were performed essentially as described by Kacian et al. in U.S. Patent No. 5,399,491, the disclosure of which is incorporated herein by reference. Amplification reactions were performed for various primer combinations using approximately 5-10 pmol of each T7 and non-T7 primer per reaction. Amplification products were detected by hybridization protection assay (HPA) using an AE-labeled detection probe (having the nucleobase sequence shown in SEQ ID NO: 67). The signal-to-noise ratio was calculated for each primer pair by dividing the RLU value observed with 15 copies of HEV IVT by the background RLU value observed with 0 copies of HEV IVT. The results are shown in Table 3 below. [Table 3]

[0129] Primer pairs that demonstrated a signal-to-background ratio of at least 10 or greater were considered successful in amplifying HEV target nucleic acid to as low as at least 15 copies per reaction, while those pairs that demonstrated a ratio below 10 were considered unsuccessful. Ratios greater than 10 are shown in bold in Table 3.

[0130] Example 2 This example describes HEV amplification and detection assays performed using various oligomer combinations. The reagents, oligonucleotides, and samples used in these experiments are listed in Tables 4-6 below. [Table 4] [Table 5-1] [Table 5-2] [Table 6]

[0131] Steps taken Principles of the method The HEV assay involved three major steps performed in a single tube: sample preparation; target amplification of HEV RNA by transcription-mediated amplification (TMA); and detection of the amplified product (amplicon) by hybridization protection assay (HPA).

[0132] During sample preparation, RNA was isolated from specimens through the use of target capture. The specimens were treated with detergent to solubilize viral particles, denature proteins, and release viral genomic RNA. Oligonucleotides homologous to highly conserved regions of HEV ("capture oligonucleotides") were hybridized to HEV RNA targets, if present, in the test specimen. The hybridized targets were then captured on magnetic microparticles and separated from the specimen in a magnetic field. Washing steps were used to remove excess components from the reaction tube. The magnetic separation and washing steps were performed using a target capture system.

[0133] Target amplification occurred via TMA, a transcription-based nucleic acid amplification method that utilizes two enzymes, MMLV reverse transcriptase and T7 RNA polymerase. Reverse transcriptase was used to generate a DNA copy of the target RNA sequence (containing a promoter sequence for T7 RNA polymerase). T7 RNA polymerase generates multiple copies of an RNA amplicon from the DNA copy template. The HEV assay utilized the TMA method to amplify a region of HEV RNA.

[0134] Detection was achieved by HPA using a single-stranded nucleic acid probe bearing a chemiluminescent label complementary to the amplicon. The labeled nucleic acid probe hybridized specifically to the amplicon. A selection reagent distinguished between hybridized and unhybridized probes by inactivating the label on the unhybridized probe. During the detection step, the chemiluminescent signal generated by the hybridized probe was measured in a luminometer and reported as relative light units (RLU).

[0135] An internal control was added to each test sample and assay calibrator via the working target capture reagent. The internal control (IC) in the HEV assay was controlled for the sample processing, amplification, and detection steps. The internal control signal was distinguished from the HEV signal by the differential kinetics of light emission from probes with different labels. The internal control-specific amplicon was detected using a probe with a rapid light emission (Flasher signal). The HEV-specific amplicon was detected using a probe with a relatively slow light emission kinetics (Glauer signal). Dual kinetic assay (DKA) is a method used to distinguish between signals from the Flasher and Glauer labels.

[0136] Step Order Target Capture: Nucleic acids underwent sample processing and target capture prior to amplification essentially according to the techniques disclosed in published International Patent Application No. PCT / US2000 / 18685, except that the template was captured using a Hepatitis E virus target capture oligonucleotide having the sequence provided herein. Notably, the capture oligonucleotide does not participate in the amplification or detection reactions of the assay. The virus-containing sample was combined with a target capture reagent to facilitate nucleic acid release and hybridization to the capture oligonucleotides located on the magnetic beads. Incubation was performed to capture HEV nucleic acids from the sample. After incubation, the magnetic beads and any captured target nucleic acids were transferred to a magnetic wash station for a 10-20 minute wash step. The captured target nucleic acids were then assayed in an amplification reaction.

[0137] Transcription-mediated amplification (TMA) reactions were performed essentially as described in Example 1. The isolated target nucleic acid was combined with primers (Table 2) in an amplification reagent, heated to 60°C for 10 minutes, and then cooled to 42°C to promote primer annealing. Enzyme reagents were then added to the mixture, and the amplification reaction was carried out, as is well known to those skilled in the art.

[0138] Detection: After incubation at 42°C for 1 hour, the amplification reaction volume was subjected to a hybridization assay using probes internally labeled with chemiluminescent compounds, using techniques familiar to those skilled in the art. Each probe was used in the hybridization reaction in an amount equivalent to approximately 2E+06 to approximately 6E+06 RLU. (See, e.g., U.S. Pat. Nos. 5,585,481 and 5,639,604, the disclosures of which are incorporated by reference.) Following the hybridization reaction, an aliquot of 0.15 M sodium tetraborate (pH 8.5) and 1% TRITON X-100 (Union Carbide Corporation; Danbury, CT) was added. The mixture was first incubated at 60°C for 10 minutes to inactivate the chemiluminescent labels bound to unhybridized probes, and then briefly cooled to room temperature (i.e., 15-30°C) before reading the hybridization signal. Chemiluminescence from the hybridized probe in each sample was assayed using a commercially available instrument (Gen-Probe Incorporated; San Diego, CA) configured to inject 1 mM nitric acid and 0.1% (v / v) hydrogen peroxide, followed by a solution containing 1 N sodium hydroxide. The results of the chemiluminescent reaction were measured in relative light units (RLU). In this procedure, the signal / noise value corresponded to the chemiluminescent signal (measured in RLU) generated by the label attached to the specifically hybridized probe divided by the background signal measured in the absence of target nucleic acid.

[0139] Results and Discussion Experiment I - Combinatorial Amplification System The goal was to test T7 and non-T7 pairs, as well as several individually, to determine which of the different primer combinations performed better and which individual primers performed best. Target capture reactions were performed using SEQ ID NO:76 as the target capture oligomer. Detection reactions were performed using SEQ ID NO:67 as the AE-labeled detection probe oligomer. The amplification oligomer combinations are shown in Table 7 below. After determining in previous experiments that SEQ ID NO:29 + SEQ ID NO:65 and SEQ ID NO:12 primers performed best, the majority of this experiment focused on these specific primers. Increasing concentrations of several primers were also tested to assess their performance and function in the system. A panel of 20 copies / mL HEV IVT (8 replicates) and BI0052-negative sera (2 replicates) was tested for each amplification system. [Table 7]

[0140] Table 8 shows a summary for Experiment I. When paired with SEQ ID NO: 12, SEQ ID NO: 65 performed better than SEQ ID NO: 29, as indicated by higher average RLU and lower %CV values ​​(Amplification Systems 1 and 2). When paired with SEQ ID NO: 29 + SEQ ID NO: 65, SEQ ID NO: 12 performed better than SEQ ID NO: 15, as indicated by higher average RLU and lower %CV values ​​(Amplification Systems 3 and 4). As can be seen in Amplification System 5, the addition of SEQ ID NO: 15 improved the RLU signal compared to Amplification System 3. Both at 5 pmol Retention of SEQ ID NO:29 + SEQ ID NO:65 in the 1000 μg / reaction yielded better RLU and %CV performance than increasing SEQ ID NO:65 to 10 pmol / reaction in Amplification System 6 (Amplification System 5). When comparing Amplification Systems 8 and 9, increasing SEQ ID NO:12 from 5 to 10 pmol / reaction yielded better performance than increasing SEQ ID NO:15. Amplification Systems 10-14 compared which non-T7s performed with higher RLU and lower %CV. Based on the criteria, SEQ ID NO:66 and SEQ ID NO:64 yielded the highest RLU and lowest %CV. Amplification Systems 15-17 increased the concentration of either non-T7 or T7 in the system by 5-10 pmol / reaction. Comparing Amplification Systems 15 and 16, these data indicate that increasing SEQ ID NO:65 improved performance (Amplification System 16), whereas increasing SEQ ID NO:15 reduced performance (Amplification System 17). [Table 8]

[0141] Experiment II - Identification of the best performing primer pair combinations The objective was to test T7 and non-T7 pairs and different primer combinations in the amplification reagent. A panel of 20 c / mL HEV IVT was tested in 5 replicates for each amplification system.

[0142] Table 9 shows the experimental design. All conditions remained the same except for the amplification system being tested. Target capture was performed using SEQ ID NO:76, and detection was performed using AE-labeled SEQ ID NO:67 as the detection probe. As shown in Experiment I, Amplification System 1 showed good performance and was set as the control. Amplification Systems 2-4 tested how each non-T7 compared to each other when paired only with SEQ ID NO:15. Because SEQ ID NO:66, SEQ ID NO:64, and SEQ ID NO:62 showed good RLU and %CV performance in Experiment I, each of these non-T7s was tested in the primer pairing combinations shown in Amplification Systems 5-7. [Table 9]

[0143] Table 10 shows a summary for Experiment II. Comparing Amplification Systems 2-4, SEQ ID NO:64 performed best with a higher average RLU compared to SEQ ID NO:66 and SEQ ID NO:62. Comparing Amplification Systems 1 and 6, SEQ ID NO:29 paired with SEQ ID NO:64 performed better than SEQ ID NO:29 + SEQ ID NO:65. Amplification System 5 showed the highest RLU performance compared to Amplification Systems 6 and 7, but Amplification System 6 was selected for further study based on sequence alignment. [Table 10]

[0144] Experiment III - Screening of novel probe pairs The objective was to test novel probe oligo pairs and evaluate their performance. Each probe was also tested individually to evaluate performance. Panels at 0 (IC buffer only) and 1,000 copies / mL HEV IVT were tested as negative and positive calibrators for the assay, respectively. Panels of 20 copies / mL HEV IVT and BI0052-negative serum were each tested in seven replicates as samples. Each panel type was tested for each probe condition.

[0145] Table 11 shows the experimental design. All conditions remained the same except for the seven probe system tested. An internal control (IC) probe was also added to each of the seven probes listed in the table. [Table 11]

[0146] Table 12 provides a summary for Experiment III. When the probe reagent containing the probe pair (probes 5, 6, and 7) was examined, probes 5 and 6 demonstrated low background (low analyte RLU in the negative calibrator and BI0052-negative serum) and high analyte RLU signal in the positive calibrator and 20 c / mL HEV IVT. Probe 7 demonstrated similar results. Of the three probes (probes 5, 6, and 7), probe 5 had the highest analyte RLU signal in the positive sample. Regarding the performance of the individual probes (probes 1-4), SEQ ID NO: 67 demonstrated the highest analyte RLU signal in the positive sample, which was accompanied by a relatively high background signal in the negative sample. [Table 12-1] [Table 12-2]

[0147] Table 13 shows a summary of the mean analyte S / CO values, including reactivity and efficacy, for Experiment III. [Table 13-1] [Table 13-2]

[0148] Example 3 This example describes the evaluation of analytical sensitivity, cross-reactivity, specificity, and further probe formulation for HEV amplification and detection assays. Reagents are as previously described in Example 2. The oligonucleotides and samples used in these experiments are listed in Tables 14 and 15 below. [Table 14-1] [Table 14-2] [Table 15]

[0149] Steps taken The assay steps were carried out as described in Example 2.

[0150] Results and Discussion Analytical Sensitivity The analytical sensitivity of the HEV assay was determined using the World Health Organization (WHO) International Standard (IS) (PEI code 6329 / 10) for an HEV RNA nucleic acid amplification technique (NAT)-based assay. The WHO IS is based on HEV genotype 3a from a clinical isolate (GenBank accession number AB630970) originally obtained by the Paul Ehrlich Institute (Langen, Germany) from the Hokkaido Red Cross Society (part of the Japanese Red Cross Society (JRC)).

[0151] Sensitivity was determined to be 8.4 international units (IU) / mL at a 95% detection level for the HEV WHO IS. [Table 16]

[0152] Analytical sensitivity of the HEV assay across various HEV genotype IVTs. RNA IVTs were prepared for each of the major known HEV genotypes 1–4, including the three subgenotypes of HEV-3: HEV-3a, HEV-3b, and HEV-3f. HEV-3a IVTs were used as HEV assay positive calibrators. In addition, RNA IVTs were also prepared for putative HEV genotype 6. The sensitivity for each of the major HEV genotype / subgenotype IVTs was determined to range from 10–19 c / mL at the 95% detection level. The exception was putative HEV genotype 6, where the sensitivity of the HEV assay was approximately 5-fold lower compared to the average sensitivity of HEV genotype 1–4 IVTs. The lower detection of putative HEV genotype 6 strains in the HEV assay is mitigated by the fact that there is only one putative genotype 6-related strain (wbJOY_06; GenBank accession number AB602441), which was found in a single wild boar in Japan and was not associated with any known human HEV cases (Takahashi M et al., J. Gen. Virol. 92:902-908, 2011). [Table 17]

[0153] Cross-reactivity In silico BLAST analysis of the HEV-specific oligos revealed no sequence matches to other blood-borne pathogens, with the exception of hepatitis C virus (HCV). HEV oligo SEQ ID NO:64 showed 100% identity to a portion of the HCV envelope gene (positions 199-213 in GenBank accession number JQ063881). Because the remainder of the HEV-specific oligo sequence (whole or part) is not found in the HCV genome sequence, this is not expected to cause any false-positive issues. This is supported by testing of HCV-positive samples that were nonreactive in the HEV assay. Other blood-borne viruses tested (human immunodeficiency virus 1, hepatitis B virus, and West Nile virus) were also nonreactive with the HEV assay. [Table 18]

[0154] specificity The specificity of the HEV assay was determined to be 99.95% (95% CI: 99.73%-99.99%) for 2,100 unrelated frozen plasma specimens. The data demonstrated excellent specificity of the HEV assay in frozen plasma specimens. [Table 19]

[0155] Probe Design The HEV probe oligo SEQ ID NO:71 showed increased detection signal for HEV-3f compared to SEQ ID NO:55 [12, 13], increasing HEV 3f sensitivity from a 95% LOD of 12.4 c / mL to 10.2 c / mL and increasing the RLU signal to levels more comparable to the other HEV genotypes tested.

[0156] Example 4 This example describes the determination of HEV RNA prevalence in blood donors and the performance characteristics of an HEV amplification and detection assay using oligonucleotides as described above.

[0157] method The study was conducted to demonstrate the analytical sensitivity and clinical specificity of the HEV assay described above ("HEV assay") for the HEV WHO International Reference Standard (Paul Ehrlich Institute (PEI) code 6329 / 10) and RNA transcripts of all four clinically relevant HEV genotypes (1-4). Plasma (for nucleic acid testing) was collected from approximately 10,000 unrelated volunteer whole blood donors. Samples were tested for HEV RNA using the HEV assay on an automated Panther system (Hologic, Inc., cat. no. 303095). Samples repeatedly reactive using the TMA were confirmed by PCR and sequence analysis.

[0158] result The HEV assay demonstrated a 95% limit of detection (LOD) of 7.9 IU / mL using the WHO standard and 14.4 copies / mL using the HEV 3a RNA transcript, which has the same sequence as the HEV WHO standard (see Tables 20 and 21). The assay detected all four HEV genotypes with a 95% LOD ranging from 7.9 to 17.7 copies / mL using RNA transcripts for HEV1, 2, 3a, 3b, 3f, and 4c (see Table 23). A total of 9,998 blood donations were screened for HEV RNA using the TMA assay. Three TMA repeat-reactive donations were identified and confirmed as positive by testing independent aliquots using PCR. One sample was determined to be genotype 3f by sequence analysis. Based on this study, the prevalence of HEV RNA in these blood donations was estimated to be 1 in 3,333 or 0.03%, and the clinical specificity of the HEV assay was determined to be 99.99% (see Table 22 and Table 24). [Table 20] [Table 21] [Table 22] [Table 23] [Table 24]

[0159] Discussion / Conclusion Results showed that the HEV assay was highly sensitive and specific, detecting all four clinically relevant HEV genotypes. Testing of nearly 10,000 individual blood donors for this study yielded three HEV RNA-confirmed positive donations, yielding an HEV RNA prevalence of 0.03%. Based on the performance demonstrated in this study, the HEV assay may be useful for screening blood donations for HEV RNA. array [Table 25-1] [Table 25-2] [Table 25-3] [Table 25-4] Although amplicon and partial amplicon sequences are exemplified herein and in the sequence listing as DNA, it should be noted that those skilled in the art will understand that the amplification product generated during the TMA reaction is either RNA or DNA depending on the stage in the amplification cycle.DNA designation is provided herein for convenience only and is not limiting.N at position 11 is C or absent, N at position 16 is G or absent, and N at position 17 is G or absent.In some embodiments, if N at position 16 is G and N at position 17 is absent, then N at position 1 is C.

[0160] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A combination of oligomers for determining the presence or absence of hepatitis E virus (HEV) in a sample, wherein the combination of oligomers is The invention comprises at least two amplification oligomers and at least one detection probe oligomer for amplifying the target region of an HEV target nucleic acid, (a) At least one amplified oligomer comprises a sequence that hybridizes to a target, which includes 15 to 17 nucleotides contained in the sequence of SEQ ID NO: 16 and at least the sequence of SEQ ID NO: 27, including its RNA equivalent and DNA / RNA chimeric form. (b) At least one amplified oligomer, including its RNA equivalent and DNA / RNA chimeric form, comprises a sequence that hybridizes to a target, comprising 17 to 28 consecutive nucleotides contained in the sequence of SEQ ID NO: 47 and at least the sequence of SEQ ID NO:

25. A combination of oligomers.

2. The oligomer combination according to claim 1, comprising one detection probe oligomer containing a target hybridizing sequence, wherein the sequence is 10 to 28 nucleotides in length and is configured to specifically hybridize to the target sequence or its complement contained in SEQ ID NO:

39.

3. The oligomer combination according to claim 1 or 2, wherein the oligomer combination comprises at least two detection probe oligomers, each containing a sequence that hybridizes to a target individually selected from SEQ ID NOs. 37, SEQ ID NOs. 55, SEQ ID NOs. 67 and SEQ ID NOs. 71, including its complement, DNA equivalent, and DNA / RNA chimera.

4. A combination of oligomers according to any one of claims 1 to 3, further comprising at least one, at least two, or at least three capture probe oligomers, each containing a target hybridizing sequence covalently bound to a sequence or portion bound to an immobilized probe, wherein the target hybridizing sequence is selected from the group consisting of SEQ ID NOs. 4 and SEQ ID NOs. 42, which include its complement, DNA equivalent, and DNA / RNA chimera.

5. The oligomer combination according to claim 4, wherein the first, second, and third capture probe oligomers each have the sequences of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 43, respectively.

6. A combination of oligomers according to any one of claims 1 to 5, comprising the first amplified oligomer of (b) and the second amplified oligomer of (b), wherein the first and second amplified oligomers of (b) are sequences of SEQ ID NOs. 12 and SEQ ID NOs. 15, including their RNA equivalents and DNA / RNA chimeric forms.

7. It comprises at least three detection probe oligomers, and in particular, at least three detection probe oligomers, A first detection probe oligomer containing a sequence that hybridizes to a target of Sequence ID No. 37 or its complement, or its RNA equivalent or DNA / RNA chimeric form; A second detection probe oligomer containing a sequence that hybridizes to the target of Sequence ID No. 67 or its complement, or its RNA equivalent or DNA / RNA chimeric form; and A third detection probe oligomer containing a sequence that hybridizes to the target of Sequence ID No. 71 or its complement, or its RNA equivalent or DNA / RNA chimeric form. Includes, Each detection probe oligomer optionally contains a 2'-methoxy backbone at one or more linkages of its nucleic acid backbone. A combination of oligomers according to any one of claims 1 to 6.

8. A kit comprising a combination of oligomers described in any one of claims 1 to 7.

9. A method for determining the presence or absence of hepatitis E virus (HEV) in a sample, (1) A step of contacting a sample suspected of containing HEV with at least two amplification oligomers for amplifying a target region of an HEV target nucleic acid, wherein the combination of amplification oligomers is as defined in claim 1; (2) A step of 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) A step of detecting the presence or absence of the amplification product using at least one detection probe oligomer as defined in any one of claims 1 to 8, thereby determining the presence or absence of HEV in the sample. Methods that include...

10. Step (1) further includes the step of purifying the HEV target nucleic acid from other components in the sample; In particular, the method according to claim 9, wherein the purification step includes contacting the sample with at least one capture probe oligomer containing a target hybridizing sequence covalently bound to a sequence or portion bound to an immobilized probe, and the target hybridizing sequence is selected from the group consisting of SEQ ID NOs. 4 and 42, which include its complement, DNA equivalent, and DNA / RNA chimera.

11. The method according to claim 10, wherein the nucleic acid base at position 20 of sequence number 4 is adenine (A), and optionally, the nucleic acid base at position 19 of sequence number 4 is cytosine (C) or uracil (U).

12. The method according to claim 10, wherein the purification step includes a step of contacting the sample with at least three capture probe oligomers.

13. The method according to claim 12, wherein the first, second, and third capture probe oligomers each have the sequence of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 43, respectively.

14. The detection step (3) includes contacting the in vitro nucleic acid amplification reaction product with at least three detection probe oligomers, The above-mentioned at least three detection probe oligomers, A first detection probe oligomer containing a sequence that hybridizes to a target of Sequence ID No. 37 or its complement, or its DNA equivalent or DNA / RNA chimeric form; A second detection probe oligomer containing a sequence that hybridizes to the target of Sequence ID No. 67 or its complement, or its DNA equivalent or DNA / RNA chimeric form; and A third detection probe oligomer containing a sequence that hybridizes to the target of Sequence ID No. 71 or its complement, or its DNA equivalent or DNA / RNA chimeric form. Includes, At least one detection probe, (a) Chemiluminescent labeling; (b) Fluorescent labeling; (c) Quencher; and (d) One or more combinations of (a), (b), and (c) The method according to any one of claims 10 to 13, comprising optionally including a label selected from the group consisting of the following.

15. The method according to any one of claims 9 to 14, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

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