Compositions and method for detecting human parvovirus nucleic acid and for detecting hepatitis a virus nucleic acids

Abstract

The present application relates to compositions and methods for detecting human parvovirus nucleic acid and for detecting hepatitis A virus nucleic acid in single or multiplex assays. In particular, the present application discloses nucleic acid oligomers specific for human parvovirus genomic DNA. Also disclosed is an assay for amplifying and detecting nucleic acid of human parvovirus genotypes 1, 2 and 3 in a biological specimen. Also disclosed are compositions for amplifying and detecting the presence of genomic DNA of human parvovirus genotypes 1, 2 and 3 in a human biological specimen.

Description

[0001] This application is a divisional of application 201280035195.2, filed July 13, 2012, entitled "Compositions and Methods for Detecting Human Parvovirus Nucleic Acid and for Detecting Hepatitis A Virus Nucleic Acid in Single or Multiplex Assays."

[0002] Cross Reference to Related Patent Applications

[0003] This application claims priority under 35 U.S.C. § 119 to U.S. Patent Application No. 61 / 508,597, filed July 15, 2011, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0004] The present invention relates to diagnostic methods and compositions for detecting human infectious agents, and in particular to methods and compositions for detecting in vitro nucleic acid of human parvovirus genotypes 1, 2 and 3 and / or hepatitis A virus. BACKGROUND

[0005] A variety of therapeutic proteins, including coagulation factors, immunoglobulin (IVIG), and albumin, are purified from human plasma by companies such as Grifols, Baxter, and CSL. Testing for parvovirus B19 and HAV is important because they are non-enveloped viruses, which makes these viruses resistant to inactivation during the purification (fractionation) process. Relatively low levels of B19 are allowed to exist in plasma fractions (current regulations require less than 10,000 IU in a manufacturing pool that can contain 4,000 to 5,000 individual plasma units). Rather than risk pooling into a large manufacturing pool and then finding it contaminated with B19, smaller pools are often screened with plasma fractionators to identify individual plasma units that contain high titers of B19. There are currently no regulations regarding HAV, but testing is generally performed as well because it has become an industry standard to do so.

[0006] Human parvovirus (Erythrovirus) is a blood-borne, nonenveloped virus with a single-stranded DNA (ssDNA) genome of about 5.5 kb (Shade et al., 1986, J. Virol. 58(3):921-936; Brown et al., 1997, Ann. Rev. Med. 48:59-67). Individual virions contain one copy of the genome either in the positive or negative strand, with approximately equal numbers of each. The ssDNA genome has an inverted terminal repeat that forms a 5' and 3' hairpin of about 350 nt, which is essential for viral replication. The genome includes two open reading frames on the positive strand, which encode structural proteins (VP1 and VP2) and a nonstructural protein (NS1).

[0007] Human parvovirus was once thought to be highly conserved, with less than 2% genetic diversity. However, recently, a human erythrovirus isolate (originally designated V9) has been found to have greater than 11% divergence in genomic sequence compared to B19, with the most striking DNA dissimilarity >20% observed within the p6 promoter region. The V9 isolate has also been determined to have greater than 11% clinical prevalence. The human erythrovirus population is now divided into three distinct viral genotypes: genotype 1 (B19), genotype 2 (A6-like and LaLi-like), and genotype 3 (V9-like). (Servant et al., 2002, J. Virol. 76(18):9124-34; Ekman et al., 2007, J. Virol. 81(13):6927-35). Servant et al. refer to genotype 1 as viruses corresponding to parvovirus B19 and genotypes 2 and 3 as viruses corresponding to parvovirus V9-related viruses. Ekman et al. refer to genotypes 1-3 as all corresponding to parvovirus B19. For convenience herein, genotypes 1, 2, and 3 are referred to as parvovirus genotypes 1, 2, and 3 or human parvovirus genotypes 1, 2, and 3. Nucleic acid testing assays that cannot accurately detect all parvovirus genotypes result in many plasma pools remaining contaminated with human parvovirus. Thus, there is a need for a nucleic acid test that can detect human parvovirus genotypes 1, 2, and 3.

[0008] Human parvovirus infection can occur through respiratory transmission or through infected blood or blood products. Infected individuals can exhibit no symptoms or symptoms of erythema infectiosum, which include mild flu-like symptoms, a rash ("fifth disease"), transient arthritic joint pain (arthralgia), aplastic crisis in patients with hemolytic anemia, persistent parvovirus infection, and approximately 10% of early miscarriages due to fetal death. Thus, failure to detect parvovirus in pooled plasma samples or failure to diagnose infection has serious consequences.

[0009] Furthermore, there is a need for detection assays that provide detection sensitivity that enables detection of low titers of virus as can occur early in infection or in diluted or pooled samples. A parvovirus nucleic acid detection assay that can detect appropriate levels of contaminating parvovirus nucleic acid can aid in the removal of infected donation units or contaminated lots of pooled plasma from the blood supply prior to use.

[0010] Many immuno-diagnostic methods detect anti-parvovirus antibodies (IgM or IgG) present in the serum or plasma of an individual (see, e.g., Wolf et al. PCT No. WO 96 / 09391 and Hedman et al. PCT No. WO 96 / 27799). These methods have limitations in detecting recent or current infection because they rely on detection of the body's response to the infectious agent. Rapid increases in viremia following infection result in the presence of high levels of parvovirus in the blood of an individual without a corresponding detectable level of anti-parvovirus antibodies (see, e.g., Example 4 of Brentano et al. U.S. Patent No. 7,094,541). Thus, immuno-based detection assays are prone to false negative results. Furthermore, viremia is often rapidly cleared, however, a person can remain antibody positive in the absence of these infectious particles, thus producing false positive results. Up to 90% of adults are seropositive for parvovirus, making accurate immuno-diagnostic detection of recent or current infection difficult. Other similar assays detect the presence of parvovirus by detecting virus or empty viral capsids bound to purified cellular receptors (Young et al. U.S. Patent 5,449,608), and these immuno-based assays suffer from similar problems.

[0011] DNA hybridization and amplification methods have also been used to detect human parvovirus, but these assays are generally directed only to genotype 1. However, the U.S. and European regulatory agencies have issued standards that specify that plasma pools used to manufacture anti-D immunoglobulin and other plasma-derived products can contain no more than 10,000 IU / ml (10 IU / microliter in Europe) of any human parvovirus. As discussed above, therapeutic plasma pools and diagnostic assays likewise require reliable identification of human parvovirus types 1, 2, and 3. Thus, there is a need in the art for compositions, kits, and methods useful for in vitro nucleic acid detection of human parvovirus types 1, 2, and 3.

[0012] Hepatitis A virus (HAV) is the causative agent of a form of hepatitis that can produce symptoms including fever, fatigue, nausea, abdominal pain, diarrhea, loss of appetite, and jaundice in less than two months. About 10% to 15% of HAV infected individuals have long-term or recurrent symptoms within six to nine months after infection. Immunity to HAV is based on the production of anti-HAV immunoglobulin G (IgG) in an individual after symptomatic and asymptomatic infection.

[0013] Although the incidence of HAV infection has dramatically decreased in parts of the world where HAV vaccination has been widely used since the late 1990s, epidemics of HAV infection (per 100,000 population > 700, and increasing to per 100,000 population > 20 for children living in areas with high hepatitis A rates) can occur in non-immune populations where poor sanitary conditions exist, even temporarily, such as after an earthquake. Transmission can also result from contact with HAV-contaminated serum or blood products. Even in the United States, about 100 people die each year from acute liver failure due to hepatitis A (a mortality rate of about 0.015%). Even in non-fatal cases of hepatitis A, the costs associated with HAV infection are substantial, including costs incurred by patient hospitalization, outpatient visits, and lost work hours.

[0014] HAV is a 27-nm RNA virus (picornavirus) that contains a positive single-stranded RNA genome of about 7.5 kb. The virus replicates in the liver during the acute phase of infection, is excreted in the bile and shed in the feces (e.g., up to 108viruses per milliliter). The incubation period before the onset of symptoms is usually two to six weeks. A single serotype of HAV has been found worldwide. Diagnosis of hepatitis A cannot be distinguished from other types of viral hepatitis by symptoms or other clinical features (e.g., elevated serum transaminase levels). Typically, a diagnosis of hepatitis A is confirmed by a serological test that provides a positive result for the presence of anti-HAV immunoglobulin (Ig). Anti-HAV IgM is generally present five to ten days before the onset of symptoms and is not detectable in most patients after six months, whereas anti-HAV IgG appears early during infection and is detectable for the lifetime of the individual. HAV RNA can be detected in the blood and stool of most people during the acute phase of infection by using nucleic acid testing methods (e.g., by polymerase chain reaction (PCR) amplification), and nucleic acid sequencing has been used to identify genetic relatedness of HAV after community-wide infection (Dato et al., Morbidity Mortality Wkly. Rpt., 2003, 52(47): 1155-57; LaPorte et al., Morbidity Mortality Wkly. Rpt., 2003, 52(24): 565-67). However, these methods are generally not used for diagnostic purposes.

[0015] Accordingly, there is a need for accurately detecting the presence of HAV in biological and environmental samples. There is also a need for detecting the presence of HAV contamination in products that can be used for medical treatment (e.g., blood or serum for transfusions, or factors derived from human blood or serum). There is a further need for detecting the presence of HAV in potentially contaminated substances (e.g., water or food) to prevent community-wide disease outbreaks or epidemics due to consumption of the contaminated substances.

[0016] The inventions disclosed herein address these needs by describing oligonucleotide sequences that can be used in nucleic acid testing methods to detect the presence of HAV nucleic acid (HAV RNA or a sequence derived therefrom, e.g., cDNA). The present application also describes nucleic acid testing methods that detect the presence of HAV RNA present in a sample. SUMMARY

[0017] The present invention relates to compositions, kits, and methods for detecting hepatitis A virus and / or human parvovirus genotypes 1, 2, and 3. These compositions, kits, and methods are configured to amplify target sequences of hepatitis A virus and / or human parvovirus nucleic acids and are configured to detect target sequences of hepatitis A virus and / or human parvovirus nucleic acids or amplified nucleic acids. In certain embodiments and aspects, specific regions within target sequences of hepatitis A virus and specific regions within human parvovirus have been identified as preferred targets for nucleic acid amplification reactions of samples, including biological specimens derived from infected humans, such as plasma samples. Amplification oligomers or detection oligomers targeting these regions can have a common core sequence, and thus provide a number of particularly preferred amplification oligomers or detection oligomers. Amplification products produced using such particularly preferred amplification oligomers will contain target-specific sequences that can be used for specific detection of human parvovirus or HAV from a sample. Detection of amplification products can include any of a variety of methods, including but not limited to probe-based detection, hybridization protection assays, assays based on molecular torches, molecular beacons, or molecular switches, mass spectrometry, MALDI-TOF mass spectrometry, ESI-TOF mass spectrometry, real-time detection assays, gel electrophoresis, SDS-PAGE electrophoresis, Sanger sequencing, Next Generation Sequencing, and the like. These preferred regions of target sequences can provide improvements in specificity, sensitivity, or speed of detection, as well as high sensitivity detection. Using these amplification and / or detection oligomers, the methods include steps of amplifying target sequences within human parvovirus genomes or HAV genomes and detecting amplification products. Detection oligomers are preferred for detection of amplification products.

[0018] One embodiment is an oligomer combination for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the oligomer combination comprising: (I) a first amplification oligomer and a second amplification oligomer for amplifying a parvovirus nucleic acid target region, wherein (a) the first parvovirus amplification oligomer comprises a first target-hybridizing sequence that is from about 14 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 181 and includes at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) the second parvovirus amplification oligomer comprises a second target-hybridizing sequence selected from the group consisting of: (i) a sequence that is from about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 189 and includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is from about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 193 and includes at least the sequence of SEQ ID NO: 192; and / or (II) a first amplification oligomer and a second amplification oligomer for amplifying a HAV nucleic acid target region, wherein (a) the first HAV amplification oligomer comprises a first target-hybridizing sequence that is from about 14 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 174 and includes at least the sequence of SEQ ID NO: 173; and (b) the second HAV amplification oligomer comprises a second target-hybridizing sequence that has from about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 177 and includes at least the sequence of SEQ ID NO: 175.

[0019] In one aspect, the oligomer combination comprises a first parvovirus amplification oligomer of (I) and a second parvovirus amplification oligomer. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 182 and comprises at least the sequence of SEQ ID NO: 179. In one aspect, the first parvovirus target hybridization sequence of (I)(a) comprises at least the sequence of SEQ ID NO: 183 or SEQ ID NO: 117. In one aspect, the first target hybridization sequence of (I)(a) has a sequence selected from the group consisting of SEQ ID NOs: 75-80. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 184. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 81-84. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 185 and comprises at least the sequence of SEQ ID NO: 180. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 82-84. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is comprised in the sequence of SEQ ID NO: 187 and comprises at least the sequence of SEQ ID NO: 188. In one aspect, the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 108-113. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is comprised in the sequence of SEQ ID NO: 191. In one aspect, the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121. In one aspect, the second parvovirus amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

[0020] In one aspect, the oligomer set described above further comprises (III) a third amplification oligomer and a fourth amplification oligomer for amplifying a human parvovirus nucleic acid target region, wherein: (a) the third parvovirus amplification oligomer comprises a third target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and that includes at least the sequence of SEQ ID NO: 179, SEQ ID NO: 117, or SEQ ID NO: 180; and (b) the fourth parvovirus amplification oligomer comprises a fourth target-hybridizing sequence selected from the group consisting of: (i) a sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and that includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and that includes at least the sequence of SEQ ID NO: 192; wherein the third target-hybridizing sequence of (III)(a) is different from the first parvovirus target-hybridizing sequence of (I)(a); and wherein the fourth target-hybridizing sequence of (III)(b) is different from the second parvovirus target-hybridizing sequence of (I)(b).

[0021] In one aspect, the third parvovirus amplification oligomer is as described above and the first parvovirus amplification oligomer is as described below: the first parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 182 and includes at least the sequence of SEQ ID NO: 179; the first parvovirus target-hybridizing sequence of (I)(a) includes at least the sequence of SEQ ID NO: 117 or SEQ ID NO: 183; the first target-hybridizing sequence of (I)(a) has a sequence selected from the group consisting of SEQ ID NOs: 75-80; the first parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 184; the first parvovirus target-hybridizing sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 81-84; the first parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 185 and includes at least the sequence of SEQ ID NO: 180, or the first parvovirus target-hybridizing sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 82-84.

[0022] In one aspect, the fourth parvovirus amplification oligomer is as described above and the second amplification oligomer is as described below: the second parvovirus target hybridization sequence of (I)(b) is contained within the sequence of SEQ ID NO: 187 and comprises at least the sequence of SEQ ID NO: 188; the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186; the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 108-113; the second parvovirus target hybridization sequence of (I)(b) is contained within the sequence of SEQ ID NO: 191; the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190; the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121; the second parvovirus amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' of the target hybridization sequence; the second parvovirus amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence that is a T7 promoter sequence; the second parvovirus amplification oligomer is a promoter primer or promoter provider that further comprises a T7 promoter sequence and the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196; and the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

[0023] In one embodiment, there is provided an oligomer combination consisting of any of the amplification oligomers described herein and the oligomer combination further comprises at least one parvovirus specific capture probe oligomer comprising a target hybridization sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target hybridization sequence is selected from the group consisting of SEQ ID NOs: 132-135. In one aspect, the parvovirus specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

[0024] In one embodiment, there is provided an oligomer combination consisting of any of the amplification oligomers described herein and further comprising at least one parvovirus-specific detection probe oligomer comprising a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067. In one aspect, the parvovirus-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 194 or 195. In one aspect, the parvovirus-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NO: 137-169. In one aspect, the oligomer combination further comprises a displacer oligomer comprising a target-hybridizing sequence configured for hybridization to a parvovirus target nucleic acid upstream of the first parvovirus amplification oligomer or the second parvovirus amplification oligomer. In one aspect, the oligomer combination further comprises at least one parvovirus-specific capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NO: 132-135. In one aspect, the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NO: 128-131.

[0025] In one embodiment, there is provided an oligomer combination consisting of any of the amplification oligomers described herein and further comprising at least one parvovirus-specific detection probe oligomer comprising a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067. In one aspect, the parvovirus-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 194 or 195. In one aspect, the parvovirus-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NO: 137-169. In one aspect, the oligomer combination further comprises a displacer oligomer comprising a target-hybridizing sequence configured for hybridization to a parvovirus target nucleic acid upstream of the first parvovirus amplification oligomer or the second parvovirus amplification oligomer. In one aspect, the oligomer combination further comprises at least one parvovirus-specific capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NO: 132-135. In one aspect, the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NO: 128-131.

[0026] In one embodiment, the oligomer combination is any of the above parvovirus oligomer combinations further comprising (II) a first HAV amplification oligomer and a second HAV amplification oligomer. In one aspect, the first target hybridization sequence of (II)(a) is comprised in the sequence of SEQ ID NO: 172. In one aspect, the first target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 170. In one aspect, the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6 and 11. In one aspect, the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 171. In one aspect, the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6. In one aspect, the second HAV target hybridization sequence of (II)(b) is comprised in the sequence of SEQ ID NO: 176. In one aspect, the second HAV target hybridization sequence of (II)(b) is selected from the group consisting of SEQ ID NOs: 29-38 and 45. In one aspect, the second HAV amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the second HAV amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 12-21 and 28.

[0027] In one embodiment, the oligomer combination is any of the above parvovirus oligomer combinations and HAV oligomer combinations further comprising (IV) a third amplification oligomer and a fourth amplification oligomer for amplifying a HAV nucleic acid target region, wherein (a) the third HAV amplification oligomer comprises a third target hybridization sequence that is about 14 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 174 and comprises at least the sequence of SEQ ID NO: 173; and (b) the fourth HAV amplification oligomer comprises a fourth target hybridization sequence that is about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 177 and comprises at least the sequence of SEQ ID NO: 175; wherein the third target hybridization sequence of (IV)(a) is different from the first HAV target hybridization sequence of (II)(a); and wherein the fourth target hybridization sequence of (IV)(b) is different from the second HAV target hybridization sequence of (II)(b). In one aspect, the third HAV amplification oligomer is an oligomer as set forth in any of claims 30-34 with respect to a first HAV amplification oligomer. In one aspect, the fourth HAV amplification oligomer is an oligomer as set forth in any of claims 35-40 with respect to a second HAV amplification oligomer.

[0028] In one embodiment, there is provided an oligo combination consisting of any of the amplification oligomers described herein and further comprising at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57. In one aspect, the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

[0029] In one embodiment, there is provided an oligo combination consisting of any of the amplification oligomers described herein and further comprising a displacer oligomer comprising a target-hybridizing sequence configured for hybridization to a HAV target nucleic acid upstream of a first HAV amplification oligomer or a second HAV amplification oligomer. In one aspect, the oligo combination further comprises at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57. In one aspect, the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

[0030] In one embodiment, there is provided an oligo combination consisting of any of the amplification oligomers described herein and further comprising at least one parvovirus-specific HAV-specific detection probe oligomer comprising a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from about nucleotide position 5965 to about nucleotide position 6028. In one aspect, the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51. In one aspect, the HAV-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 178. In one aspect, the HAV-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 58-74. In one aspect, the oligo combination consists of any of the amplification oligomers described herein and further comprising a displacer oligomer comprising a target-hybridizing sequence configured for hybridization to a HAV target nucleic acid upstream of a first HAV amplification oligomer or a second HAV amplification oligomer. In one aspect, the oligo combination further comprises at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57.

[0031] One embodiment is a kit comprising any of the oligomer combinations described herein. One embodiment is a reaction mixture comprising any of the oligomer combinations described herein. One embodiment is a singleplex amplification assay for amplifying hepatitis A virus using at least one amplification oligomer described herein. In one aspect, the hepatitis A virus singleplex amplification assay is for amplifying and detecting hepatitis A virus using at least one detection probe oligomer described herein. One embodiment is a singleplex amplification assay for amplifying parvovirus using at least one amplification oligomer described herein. In one aspect, the parvovirus singleplex amplification assay is for amplifying and detecting parvovirus using at least one detection probe oligomer described herein. One embodiment is a multiplex amplification assay for amplifying hepatitis A virus and parvovirus using at least one amplification oligomer described herein. In one aspect, the hepatitis A virus and parvovirus multiplex amplification assay is for amplifying and detecting hepatitis A virus and parvovirus using at least one detection probe oligomer described herein.

[0032] One embodiment is a method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the method comprising: (A) providing a sample, wherein the sample is suspected of containing at least one of a human parvovirus and HAV; (B) contacting the sample with an oligomer combination for amplifying at least one of a parvovirus nucleic acid target region and a HAV nucleic acid target region, the oligomer combination comprising: (I) for the parvovirus acid target region, (a) a first parvovirus amplification oligomer comprising a first target-hybridizing sequence that is from about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and that includes at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) a second parvovirus amplification oligomer comprising a second target-hybridizing sequence selected from the group consisting of: (i) a sequence that is from about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and that includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is from about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and that includes at least the sequence of SEQ ID NO: 192; and / or (II) for the HAV target region, (a) a first HAV amplification oligomer comprising a first target-hybridizing sequence that is from about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 174 and that includes at least the sequence of SEQ ID NO: 173; and (b) a second HAV amplification oligomer comprising a second target-hybridizing sequence that is from about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 177 and that includes at least the sequence of SEQ ID NO: 175; (C) performing an in vitro nucleic acid amplification reaction, wherein any parvovirus and / or HAV target nucleic acid present in the sample is used as a template to generate parvovirus and / or HAV amplification products; and (D) detecting the presence or absence of the parvovirus and / or HAV amplification products, thereby indicating the presence or absence of parvovirus and / or HAV in the sample.

[0033] In one aspect, the method is for detecting a human parvovirus target nucleic acid and the sample is contacted with a first parvovirus amplification oligomer and a second parvovirus amplification oligomer of (I). In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 182 and comprises at least the sequence of SEQ ID NO: 179. In one aspect, the first parvovirus target hybridization sequence of (I)(a) comprises at least the sequence of SEQ ID NO: 117 or SEQ ID NO: 183. In one aspect, the first parvovirus target hybridization sequence of (I)(a) has a sequence selected from the group consisting of SEQ ID NOs: 75-80. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 184. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 75, 76, 77, and 81-84. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 185 and comprises at least the sequence of SEQ ID NO: 180. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 82-84. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is comprised in the sequence of SEQ ID NO: 187 and comprises at least the sequence of SEQ ID NO: 188. In one aspect, the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 108-113. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is comprised in the sequence of SEQ ID NO: 191. In one aspect, the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121. In one aspect, the second parvovirus amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

[0034] In one embodiment, step (B) further comprises contacting the sample with (III) a third amplification oligomer and a fourth amplification oligomer for amplifying a human parvovirus nucleic acid target region, wherein (a) the third parvovirus amplification oligomer comprises a third target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and that includes at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) the fourth parvovirus amplification oligomer comprises a fourth target-hybridizing sequence selected from the group consisting of: (i) a sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and that includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and that includes at least the sequence of SEQ ID NO: 192; wherein the third target-hybridizing sequence of (III)(a) is different from the first parvovirus target-hybridizing sequence of (I)(a); and wherein the fourth target-hybridizing sequence of (III)(b) is different from the second parvovirus target-hybridizing sequence of (I)(b). In one aspect, the third parvovirus amplification oligomer is an oligomer contained in the sequence of SEQ ID NO: 182 and that includes at least the sequence of SEQ ID NO: 179. In one aspect, the third parvovirus target-hybridizing sequence of (I)(a) includes at least the sequence of SEQ ID NO: 117 or SEQ ID NO: 183. In one aspect, the third parvovirus target-hybridizing sequence of (I)(a) has a sequence selected from the group consisting of SEQ ID NOs: 75-80. In one aspect, the third parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 184. In one aspect, the third parvovirus target-hybridizing sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 75-77 and 81-84. In one aspect, the third parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 185 and includes at least the sequence of SEQ ID NO: 180. In one aspect, the third parvovirus target-hybridizing sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 82-84. In one aspect, the fourth parvovirus amplification oligomer is contained in the sequence of SEQ ID NO: 187 and includes at least the sequence of SEQ ID NO: 188. In one aspect, the fourth parvovirus target-hybridizing sequence of (I)(b) includes at least the sequence of SEQ ID NO: 186. In one aspect, the fourth parvovirus target-hybridizing sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 108-113.In one aspect, the fourth parvovirus target hybridization sequence of (I)(b) is contained within the sequence of SEQ ID NO: 191. In one aspect, the fourth parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190. In one aspect, the fourth parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121. In one aspect, the fourth parvovirus amplification oligomer is a promoter primer or a promoter provider that further comprises a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the fourth parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

[0035] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises purifying the parvovirus target nucleic acid from other components in the sample prior to step (B). In one aspect, the purification step comprises contacting the sample with at least one parvovirus-specific capture probe oligomer comprising a target hybridization sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target hybridization sequence is selected from the group consisting of SEQ ID NOs: 132-135. In one aspect, the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

[0036] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, step (B) further comprises contacting the sample with a displacer oligomer comprising a target hybridization sequence configured for hybridization to the parvovirus target nucleic acid upstream of the first parvovirus amplification oligomer or the second parvovirus amplification oligomer.

[0037] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the detecting step (D) comprises contacting the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer configured to specifically hybridize to a parvovirus amplification product under conditions whereby the presence or absence of the parvovirus amplification product is determined, thereby indicating the presence or absence of parvovirus in the sample. In one aspect, the parvovirus-specific detection probe oligomer comprises a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067. In one aspect, the parvovirus-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 194 or 195. In one aspect, the parvovirus-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 137-169. In one aspect, the parvovirus-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

[0038] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises contacting the sample with a false target oligomer that can be amplified in an in vitro nucleic acid amplification reaction using the first parvovirus amplification oligomer and the second parvovirus amplification oligomer to produce a second amplification product that does not specifically hybridize to the parvovirus-specific detection probe under the detection reaction conditions.

[0039] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises contacting the sample with a cold probe oligomer that competes for hybridization to the parvovirus amplification product with the parvovirus-specific detection probe oligomer.

[0040] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises contacting the sample with a tuner oligomer configured to specifically hybridize to both the first parvovirus amplification oligomer and the second parvovirus amplification oligomer.

[0041] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the detecting step (D) occurs during the amplifying step (C). In one aspect, the parvovirus-specific detection probe comprises a fluorescent label, a quencher, or both. In one aspect, the parvovirus-specific detection probe is a TaqMan detection probe or a molecular beacon. In one aspect, the parvovirus-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c). In one aspect, the parvovirus-specific detection probe further comprises a non-target hybridizing sequence. In one aspect, the parvovirus-specific detection probe is a hairpin detection probe. In one aspect, the hairpin detection probe is a molecular beacon or a molecular torch.

[0042] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the amplification reaction of step (C) is an isothermal amplification reaction. In one aspect, the amplification reaction is a real-time amplification reaction.

[0043] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the amplification reaction of step (C) is a PCR amplification reaction. In one aspect, the amplification reaction is a real-time amplification reaction.

[0044] In one embodiment of a method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, wherein the method is for detecting a HAV target nucleic acid, the sample is contacted with a first HAV amplification oligomer and a second HAV amplification oligomer of (II). In one aspect, the first HAV target hybridization sequence of (II)(a) is comprised in the sequence of SEQ ID NO: 172. In one aspect, the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 170. In one aspect, the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6 and 11. In one aspect, the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 171. In one aspect, the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6. In one aspect, the second HAV target hybridization sequence of (II)(b) is comprised in the sequence of SEQ ID NO: 176. In one aspect, the second HAV target hybridization sequence of (II)(b) is selected from the group consisting of SEQ ID NOs: 29-38 and 45. In one aspect, the second HAV amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the second HAV amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 12-21 and 28.

[0045] In one embodiment, step (B) further comprises contacting the sample with (IV) a third amplification oligomer and a fourth amplification oligomer for amplifying a target region of the HAV nucleic acid, wherein (a) the third HAV amplification oligomer comprises a third target hybridization sequence that is from about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 174 and that includes at least the sequence of SEQ ID NO: 173; and (b) the fourth HAV amplification oligomer comprises a fourth target hybridization sequence that is from about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 177 and that includes at least the sequence of SEQ ID NO: 175; wherein the third target hybridization sequence of (IV)(a) is different from the first HAV target hybridization sequence of (II)(a); and wherein the fourth target hybridization sequence of (IV)(b) is different from the second HAV target hybridization sequence of (II)(b). In one aspect, the third HAV amplification oligomer is an oligomer as described in any one of claims 96 to 100 with respect to the first HAV amplification oligomer. In one aspect, the fourth HAV amplification oligomer is an oligomer as described in claims 101 to 106 with respect to the second HAV amplification oligomer.

[0046] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises purifying the HAV target nucleic acid from other components in the sample prior to step (B). In one aspect, the purification step comprises contacting the sample with at least one HAV-specific capture probe oligomer comprising a target hybridization sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target hybridization sequence is selected from the group consisting of SEQ ID NOs: 52-57. In one aspect, the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

[0047] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises using a displacer oligomer in the amplification step, the displacer oligomer comprising a target hybridization sequence configured for hybridization to the HAV target nucleic acid upstream of the first HAV amplification oligomer or the second HAV amplification oligomer.

[0048] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the detecting step (D) comprises contacting the in vitro nucleic acid amplification reaction with an HAV-specific detection probe oligomer configured to specifically hybridize to the HAV amplification product under conditions whereby the presence or absence of the HAV amplification product is determined, thereby indicating the presence or absence of HAV in the sample. In one aspect, the HAV-specific detection probe oligomer comprises a target hybridization sequence of about 14 to about 40 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 198 from about nucleotide position 5965 to about nucleotide position 6028. In one aspect, the HAV-specific detection probe target hybridization sequence is contained within the sequence of SEQ ID NO: 178. In one aspect, the HAV-specific detection probe target hybridization sequence is selected from the group consisting of SEQ ID NOs: 58-74. In one aspect, the HAV-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

[0049] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises contacting the sample with a false target oligomer that can be amplified in an in vitro nucleic acid amplification reaction using the first HAV amplification oligomer and the second HAV amplification oligomer to produce a second amplification product that does not specifically hybridize to the HAV-specific detection probe under the detection reaction conditions.

[0050] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises contacting the sample with a cold probe oligomer that competes for hybridization to the HAV amplification product with the HAV-specific detection probe oligomer.

[0051] In one embodiment, the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample further comprises contacting the sample with a tuner oligomer configured to specifically hybridize to the first HAV amplification oligomer and the second HAV amplification oligomer.

[0052] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the detecting step (D) occurs during the amplifying step (C). In one aspect, the HAV-specific detection probe comprises a fluorescent label, a quencher, or both. In one aspect, the HAV-specific detection probe is a TaqMan detection probe or a molecular beacon. In one aspect, the HAV-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c). In one aspect, the HAV-specific detection probe further comprises a non-target hybridizing sequence. In one aspect, the HAV-specific detection probe is a hairpin detection probe. In one aspect, the hairpin detection probe is a molecular beacon or a molecular torch.

[0053] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the amplification reaction of step (C) is an isothermal amplification reaction.

[0054] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the amplification reaction of step (C) is a PCR amplification reaction.

[0055] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the amplification reaction is a real-time amplification reaction.

[0056] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the sample is from an individual patient. In one aspect, the sample is pooled. In one aspect, the pooled sample is a pooled plasma sample. In one aspect, the sample is a plasma sample used to derive a therapeutic compound. In one aspect, the sample is a plasma sample used to derive a compound that is a human thrombin, a human antibody or portion thereof, a human protein, a human cytokine receptor, a human cytokine ligand, or other compound derived from plasma.

[0057] In one embodiment of the method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, wherein the method is for detecting a human parvovirus target nucleic acid and a HAV target nucleic acid, and wherein the detecting step (D) comprises contacting the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer and a HAV-specific detection probe oligomer configured for specific hybridization to a parvovirus amplification product and a HAV amplification product, respectively, under conditions whereby the presence or absence of the parvovirus amplification product and the HAV amplification product is determined, thereby indicating the presence or absence of parvovirus and HAV in the sample. In one aspect, the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are differentially labeled. In one aspect, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a label, the label is independently selected from the group consisting of (a) a chemiluminescent label and (b) a fluorescent label. In one aspect, the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a chemiluminescent label. In one aspect, the chemiluminescent labels for the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are characterized by different luminescence kinetics sufficient to distinguish a parvovirus-specific chemiluminescent signal from a HAV-specific chemiluminescent signal. In one aspect, the chemiluminescent labels for the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise acridinium ester (AE). In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 182 and includes at least the sequence of SEQ ID NO: 117 or SEQ ID NO: 179. In one aspect, the first parvovirus target hybridization sequence of (I)(a) includes at least the sequence of SEQ ID NO: 183. In one aspect, the first parvovirus target hybridization sequence of (I)(a) has a sequence selected from the group consisting of SEQ ID NO: 75-80. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 184. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is selected from the group consisting of SEQ ID NO: 81-84. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 185 and includes at least the sequence of SEQ ID NO: 180. In one aspect, the first parvovirus target hybridization sequence of (I)(a) is selected from the group consisting of SEQ ID NO: 82-84. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is comprised in the sequence of SEQ ID NO: 187 and includes at least the sequence of SEQ ID NO: 188.In one aspect, the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 108-113. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is contained within the sequence of SEQ ID NO: 191. In one aspect, the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190. In one aspect, the second parvovirus target hybridization sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121. In one aspect, the second parvovirus amplification oligomer is a promoter primer or a promoter provider that further comprises a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101. In one aspect, the first HAV target hybridization sequence of (II)(a) is contained within the sequence of SEQ ID NO: 172. In one aspect, the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 170. In one aspect, the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6 and 11. In one aspect, the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 171. In one aspect, the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6. In one aspect, the second HAV target hybridization sequence of (II)(b) is contained within the sequence of SEQ ID NO: 176. In one aspect, the second HAV target hybridization sequence of (II)(b) is selected from the group consisting of SEQ ID NOs: 29-38 and 45. In one aspect, the second HAV amplification oligomer is a promoter primer or a promoter provider that further comprises a promoter sequence located 5' of the target hybridization sequence. In one aspect, the promoter sequence is a T7 promoter sequence. In one aspect, the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196. In one aspect, the second HAV amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 12-21 and 28. In one aspect, the parvovirus-specific detection probe oligomer comprises a target hybridization sequence of about 14 to about 40 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067.In one aspect, the parvovirus-specific detection probe target hybridization sequence is contained within the sequence of SEQ ID NO: 194 or 195. In one aspect, the parvovirus-specific detection probe target hybridization sequence is selected from the group consisting of SEQ ID NOs: 137-169. In one aspect, the HAV-specific detection probe oligomer comprises a target hybridization sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from about nucleotide position 5965 to about nucleotide position 6028. In one aspect, the HAV-specific detection probe target hybridization sequence is contained within the sequence of SEQ ID NO: 178. In one aspect, the HAV-specific detection probe target hybridization sequence is selected from the group consisting of SEQ ID NOs: 58-74.

[0058] In one embodiment, a method is provided for multiplex amplification and detection of human parvovirus genotype 1, 2, and 3 target nucleic acids and hepatitis A virus target nucleic acids from a sample. In one aspect of this embodiment, the amplification oligomers for amplification and detection of human parvovirus genotype 1, 2, and 3 comprise one or more of the amplification oligomers described in Table 3. In another aspect, two or more of the amplification oligomers described in Table 3. In another aspect, three or more of the amplification oligomers described in Table 3. In another aspect, four or more of the amplification oligomers described in Table 3. In another aspect, five or more of the amplification oligomers described in Table 3. In another aspect, six or more of the amplification oligomers described in Table 3. In another aspect, seven or more of the amplification oligomers described in Table 3. In one aspect of this embodiment, the amplification oligomers for amplification and detection of HAV comprise one or more of the amplification oligomers described in Table 3. In another aspect, two or more of the amplification oligomers described in Table 3. In another aspect, three or more of the amplification oligomers described in Table 3. In another aspect, four or more of the amplification oligomers described in Table 3. In another aspect, five or more of the amplification oligomers described in Table 3. In another aspect, six or more of the amplification oligomers described in Table 3. In another aspect, seven or more of the amplification oligomers described in Table 3. In one aspect of this embodiment, the detection probe oligomers for detection of amplification products produced by human parvovirus genotype 1, 2, and 3 and for detection of amplification products produced by HAV comprise one or more of the parvovirus detection probes described in Table 3 and one or more of the HAV detection probes described in Table 3. In another aspect, the detection probe oligomers are present during amplification for real-time detection. In another aspect, the detection probe oligomers are combined with amplification products after the amplification reaction for end-point detection. In one aspect of this embodiment, the multiplex amplification and detection of human parvovirus genotype 1, 2, and 3 target nucleic acids, HAV target nucleic acids is a quantitative multiplex amplification and detection reaction. In one aspect of this embodiment, the human parvovirus genotype 1, 2, and 3 target nucleic acids are separated from other sample components. In another aspect, the separation is performed using a target capture oligomer. In one aspect of this embodiment, the HAV target nucleic acids are separated from other sample components. In another aspect, the separation is performed using a target capture oligomer. In one aspect of this embodiment, the amplification and detection reaction comprises an internal control.

[0059] In particular, the present application provides the following:

[0060] 1. An oligomer combination for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the oligomer combination comprising:

[0061] (I) a first amplification oligomer and a second amplification oligomer for amplifying a human parvovirus nucleic acid target region, wherein

[0062] (a) the first parvovirus amplification oligomer comprises a first target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and that includes at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and

[0063] (b) the second parvovirus amplification oligomer comprises a second target-hybridizing sequence selected from the group consisting of:

[0064] (i) a sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and that includes at least the sequence of SEQ ID NO: 188; and

[0065] (ii) a sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and that includes at least the sequence of SEQ ID NO: 192;

[0066] and / or

[0067] (II) a first amplification oligomer and a second amplification oligomer for amplifying a HAV nucleic acid target region, wherein

[0068] (a) the first HAV amplification oligomer comprises a first target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 174 and that includes at least the sequence of SEQ ID NO: 173; and

[0069] (b) the second HAV amplification oligomer comprises a second target-hybridizing sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 177 and that includes at least the sequence of SEQ ID NO: 175.

[0070] 2. The oligomer combination according to item 1, wherein the oligomer combination comprises the first parvovirus amplification oligomer and the second parvovirus amplification oligomer of (I).

[0071] 3. The oligomer combination according to item 2, wherein the first parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 182 and includes at least the sequence of SEQ ID NO: 179.

[0072] 4. The oligomer combination of item 3, wherein the first parvovirus target-hybridizing sequence of (I)(a) comprises at least the sequence of SEQ ID NO: 183 or SEQ ID NO: 117.

[0073] 5. The oligomer combination of item 4, wherein the first target-hybridizing sequence of (I)(a) has a sequence selected from the group consisting of SEQ ID NOs: 75-80.

[0074] 6. The oligomer combination of item 2, wherein the first parvovirus target-hybridizing sequence of (I)(a) is contained within the sequence of SEQ ID NO: 184.

[0075] 7. The oligomer combination of item 6, wherein the first parvovirus target-hybridizing sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 81-84.

[0076] 8. The oligomer combination of item 2, wherein the first parvovirus target-hybridizing sequence of (I)(a) is contained within the sequence of SEQ ID NO: 185 and comprises at least the sequence of SEQ ID NO: 180.

[0077] 9. The oligomer combination of item 8, wherein the first parvovirus target-hybridizing sequence of (I)(a) is selected from the group consisting of SEQ ID NOs: 82-84.

[0078] 10. The oligomer combination of any one of items 2-9, wherein the second parvovirus target-hybridizing sequence of (I)(b) is contained within the sequence of SEQ ID NO: 187 and comprises at least the sequence of SEQ ID NO: 188.

[0079] 11. The oligomer combination of item 10, wherein the second parvovirus target-hybridizing sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186.

[0080] 12. The oligomer combination of item 11, wherein the second parvovirus target-hybridizing sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 108-113.

[0081] 13. The oligomer combination of any one of items 2-9, wherein the second parvovirus target-hybridizing sequence of (I)(b) is contained within the sequence of SEQ ID NO: 191.

[0082] 14. The oligomer combination of item 13, wherein the second parvovirus target-hybridizing sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190.

[0083] 15. The oligomer combination of item 14, wherein the second parvovirus target-hybridizing sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121.

[0084] 16. The oligomer combination of any one of items 2-15, wherein the second parvovirus amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5’ of the target-hybridizing sequence.

[0085] 17. The oligomer combination of item 16, wherein the promoter sequence is a T7 promoter sequence.

[0086] 18. The oligomer combination of item 17, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196.

[0087] 19. The oligomer combination of item 18, wherein the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

[0088] 20. The oligomer combination of any one of items 2-19, further comprising

[0089] (III) a third amplification oligomer and a fourth amplification oligomer for amplifying the human parvovirus nucleic acid target region, wherein

[0090] (a) the third parvovirus amplification oligomer comprises a third target-hybridizing sequence having about 14 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 181 and including at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and

[0091] (b) the fourth parvovirus amplification oligomer comprises a fourth target-hybridizing sequence selected from the group consisting of:

[0092] (i) a sequence that is about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 189 and including at least the sequence of SEQ ID NO: 188; and

[0093] (ii) a sequence that is about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 193 and including at least the sequence of SEQ ID NO: 192;

[0094] wherein the third target-hybridizing sequence of (III)(a) is different from the first parvovirus target-hybridizing sequence of (I)(a); and

[0095] wherein the fourth target hybridizing sequence of (III)(b) is different from the second parvovirus target hybridizing sequence of (I)(b).

[0096] 21. The oligomer combination of item 20, wherein the third parvovirus amplification oligomer is an oligomer as described in any one of items 3 to 9 with respect to the first parvovirus amplification oligomer.

[0097] 22. The oligomer combination of item 20 or 21, wherein the fourth parvovirus amplification oligomer is an oligomer as described in any one of items 10 to 19 with respect to the second parvovirus amplification oligomer.

[0098] 23. The oligomer combination of any one of items 2 to 22, further comprising at least one parvovirus-specific capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 132-135.

[0099] 24. The oligomer combination of item 23, wherein the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

[0100] 25. The oligomer combination of any one of items 2 to 24, further comprising a displacer oligomer comprising a target hybridizing sequence configured for hybridizing to the parvovirus target nucleic acid upstream of the first parvovirus amplification oligomer or the second parvovirus amplification oligomer.

[0101] 26. The oligomer combination of any one of items 2 to 25, further comprising at least one parvovirus-specific detection probe oligomer comprising a target hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067.

[0102] 27. The oligomer combination of item 26, wherein the parvovirus-specific detection probe target hybridizing sequence is comprised in the sequence of SEQ ID NO: 194 or 195.

[0103] 28. The oligomer combination of item 27, wherein the parvovirus-specific detection probe target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 137-169.

[0104] 29. The oligomer combination of any one of items 1 to 22, wherein the oligomer combination comprises the first and second HAV amplification oligomers of (II).

[0105] 30. The oligomer combination of item 29, wherein the first target hybridization sequence of (II)(a) is comprised in the sequence of SEQ ID NO: 172.

[0106] 31. The oligomer combination of item 30, wherein the first target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 170.

[0107] 32. The oligomer combination of item 31, wherein the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6 and 11.

[0108] 33. The oligomer combination of item 30, wherein the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 171.

[0109] 34. The oligomer combination of item 33, wherein the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6.

[0110] 35. The oligomer combination of any one of items 29 to 34, wherein the second HAV target hybridization sequence of (II)(b) is comprised in the sequence of SEQ ID NO: 176.

[0111] 36. The oligomer combination of item 35, wherein the second HAV target hybridization sequence of (II)(b) is selected from the group consisting of SEQ ID NOs: 29-38 and 45.

[0112] 37. The oligomer combination of any one of items 29 to 36, wherein the second HAV amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence.

[0113] 38. The oligomer combination of item 37, wherein the promoter sequence is a T7 promoter sequence.

[0114] 39. The oligomer combination of item 38, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196.

[0115] 40. The oligomer combination of item 39, wherein the second HAV amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 12-21 and 28.

[0116] 41. The oligomer combination of any one of items 29-40, further comprising

[0117] (IV) a third amplification oligomer and a fourth amplification oligomer for amplifying a target region of a HAV nucleic acid, wherein

[0118] (a) the third HAV amplification oligomer comprises a third target-hybridizing sequence that is about 14 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 174 and includes at least the sequence of SEQ ID NO: 173; and

[0119] (b) the fourth HAV amplification oligomer comprises a fourth target-hybridizing sequence that is about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 177 and includes at least the sequence of SEQ ID NO: 175;

[0120] wherein the third target-hybridizing sequence of (IV)(a) is different from the first HAV target-hybridizing sequence of (II)(a); and

[0121] wherein the fourth target-hybridizing sequence of (IV)(b) is different from the second HAV target-hybridizing sequence of (II)(b).

[0122] 42. The oligomer combination of item 41, wherein the third HAV amplification oligomer is an oligomer as described in any one of items 30-34 with respect to the first HAV amplification oligomer.

[0123] 43. The oligomer combination of item 41 or 42, wherein the fourth HAV amplification oligomer is an oligomer as described in items 35-40 with respect to the second HAV amplification oligomer.

[0124] 44. The oligomer combination of any one of items 29-43, further comprising at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57.

[0125] 45. The oligomer combination of item 44, wherein the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

[0126] 46. The oligomer combination of any one of items 29 to 45, further comprising a displacer oligomer comprising a target-hybridizing sequence configured for hybridizing to the HAV target nucleic acid upstream of the first HAV amplification oligomer or the second HAV amplification oligomer.

[0127] 47. The oligomer combination of any one of items 29 to 46, further comprising at least one HAV-specific detection probe oligomer comprising a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from about nucleotide position 5965 to about nucleotide position 6028.

[0128] 48. The oligomer combination of item 47, wherein the HAV-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 178.

[0129] 49. The oligomer combination of item 48, wherein the HAV-specific detection probe target-hybridizing sequence is selected from SEQ ID NOs: 58-74.

[0130] 50. A kit comprising the oligomer combination of any one of items 1 to 49.

[0131] 51. A reaction mixture comprising the oligomer combination of any one of items 1 to 49.

[0132] 52. A method for detecting at least one of a human parvovirus target nucleic acid and a hepatitis A virus (HAV) target nucleic acid in a sample, the method comprising:

[0133] (A) providing a sample, wherein the sample is suspected of containing at least one of a human parvovirus and HAV;

[0134] (B) contacting the sample with an oligomer combination for amplifying at least one of a human parvovirus nucleic acid target region and a HAV nucleic acid target region, the oligomer combination comprising:

[0135] (I) for the parvovirus target region, (a) a first parvovirus amplification oligomer comprising a first target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and that includes at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) a second parvovirus amplification oligomer comprising a second target-hybridizing sequence selected from the group consisting of: (i) a sequence that has about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and that includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that has about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and that includes at least the sequence of SEQ ID NO: 192;

[0136] and / or

[0137] (II) for the HAV target region, (a) a first HAV amplification oligomer comprising a first target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 174 and that includes at least the sequence of SEQ ID NO: 173; and (b) a second HAV amplification oligomer comprising a second target-hybridizing sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 177 and that includes at least the sequence of SEQ ID NO: 175;

[0138] (C) performing an in vitro nucleic acid amplification reaction in which any parvovirus and / or HAV target nucleic acid present in the sample is used as a template to generate parvovirus and / or HAV amplification products; and

[0139] (D) detecting the presence or absence of the parvovirus and / or the HAV amplification products, thereby indicating the presence or absence of parvovirus and / or HAV in the sample.

[0140] 53. The method of item 52, wherein the method is for detecting the human parvovirus target nucleic acid and the sample is contacted with the first parvovirus amplification oligomer and the second parvovirus amplification oligomer of (I).

[0141] 54. The method of item 53, wherein the first parvovirus target-hybridizing sequence of (I)(a) is contained in the sequence of SEQ ID NO: 182 and includes at least the sequence of SEQ ID NO: 117 or SEQ ID NO: 179.

[0142] 55. The method of item 54, wherein the first parvovirus target-hybridizing sequence of (I)(a) comprises at least the sequence of SEQ ID NO: 183.

[0143] 56. The method of item 55, wherein the first parvovirus target-hybridizing sequence of (I)(a) has a sequence selected from SEQ ID NOs: 75-80.

[0144] 57. The method of item 53, wherein the first parvovirus target-hybridizing sequence of (I)(a) is contained within the sequence of SEQ ID NO: 184.

[0145] 58. The method of item 57, wherein the first parvovirus target-hybridizing sequence of (I)(a) is selected from SEQ ID NOs: 81-84.

[0146] 59. The method of item 53, wherein the first parvovirus target-hybridizing sequence of (I)(a) is contained within the sequence of SEQ ID NO: 185 and comprises at least the sequence of SEQ ID NO: 180.

[0147] 60. The method of item 59, wherein the first parvovirus target-hybridizing sequence of (I)(a) is selected from SEQ ID NOs: 82-84.

[0148] 61. The method of any one of items 53-60, wherein the second parvovirus target- hybridizing sequence of (I)(b) is contained within the sequence of SEQ ID NO: 187 and comprises at least the sequence of SEQ ID NO: 188.

[0149] 62. The method of item 61, wherein the second parvovirus target-hybridizing sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186.

[0150] 63. The method of item 62, wherein the second parvovirus target-hybridizing sequence of (I)(b) is selected from SEQ ID NOs: 108-113.

[0151] 64. The method of any one of items 53-60, wherein the second parvovirus target- hybridizing sequence of (I)(b) is contained within the sequence of SEQ ID NO: 191.

[0152] 65. The method of item 64, wherein the second parvovirus target-hybridizing sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190.

[0153] 66. The method of item 65, wherein the second parvovirus target-hybridizing sequence of (I)(b) is selected from the group consisting of SEQ ID NOs: 118-121.

[0154] 67. The method of any one of items 53-66, wherein the second parvovirus amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target-hybridizing sequence.

[0155] 68. The method of item 67, wherein the promoter sequence is a T7 promoter sequence.

[0156] 69. The method of item 68, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196.

[0157] 70. The method of item 69, wherein the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

[0158] 71. The method of any one of items 53-70, wherein step (B) further comprises contacting the sample with:

[0159] (III) a third amplification oligomer and a fourth amplification oligomer for amplifying the human parvovirus nucleic acid target region, wherein (a) the third parvovirus amplification oligomer comprises a third target-hybridizing sequence that is about 14 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 181 and includes at least the sequence of SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and

[0160] (b) the fourth parvovirus amplification oligomer comprises a fourth target-hybridizing sequence selected from the group consisting of: (i) a sequence that is about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 189 and includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is about 14 to about 30 contiguous nucleotides contained in the sequence of SEQ ID NO: 193 and includes at least the sequence of SEQ ID NO: 192;

[0161] wherein the third target-hybridizing sequence of (III)(a) is different from the first parvovirus target-hybridizing sequence of (I)(a); and

[0162] wherein the fourth target-hybridizing sequence of (III)(b) is different from the second parvovirus target-hybridizing sequence of (I)(b).

[0163] 72. The method of item 71, wherein the third parvovirus amplification oligomer is an oligomer as described in any one of items 54 to 60 with respect to the first parvovirus amplification oligomer.

[0164] 73. The method of item 71 or 72, wherein the fourth parvovirus amplification oligomer is an oligomer as described in any one of items 61 to 70 with respect to the second parvovirus amplification oligomer.

[0165] 74. The method of any one of items 53 to 73, further comprising purifying the parvovirus target nucleic acid from other components of the sample prior to step (B).

[0166] 75. The method of item 74, wherein the purifying step comprises contacting the sample with at least one parvovirus-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 132-135.

[0167] 76. The method of item 75, wherein the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

[0168] 77. The method of any one of items 53 to 76, wherein step (B) further comprises contacting the sample with a displacer oligomer comprising a target-hybridizing sequence configured for hybridization to the parvovirus target nucleic acid upstream of the first parvovirus amplification oligomer or the second parvovirus amplification oligomer.

[0169] 78. The method of item 53, wherein the detecting step (D) comprises contacting the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer configured for specific hybridization to a parvovirus amplification product under conditions whereby the presence or absence of the parvovirus amplification product is determined, thereby indicating the presence or absence of parvovirus in the sample.

[0170] 79. The method of item 78, wherein the parvovirus-specific detection probe oligomer comprises a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067.

[0171] 80. The method of item 79, wherein the parvovirus-specific detection probe target hybridization sequence is comprised in the sequence of SEQ ID NO: 194 or 195.

[0172] 81. The method of item 80, wherein the parvovirus-specific detection probe target hybridization sequence is selected from the group consisting of SEQ ID NOs: 137-169.

[0173] 82. The method of any one of items 78-81, further comprising contacting the sample with a false target oligomer that is amplifiable in an in vitro nucleic acid amplification reaction using the first parvovirus amplification oligomer and the second parvovirus amplification oligomer to produce a second amplification product that does not specifically hybridize to the parvovirus-specific detection probe under the detection reaction conditions.

[0174] 83. The method of any one of items 78-82, further comprising contacting the sample with a cold probe oligomer that competes for hybridization to the parvovirus amplification product with the parvovirus-specific detection probe oligomer.

[0175] 84. The method of any one of items 78-81, further comprising contacting the sample with a tuner oligomer configured for specific hybridization to the first parvovirus amplification oligomer and the second parvovirus amplification oligomer.

[0176] 85. The method of any one of items 78-84, wherein the parvovirus-specific detection probe comprises a label selected from the group consisting of:

[0177] (a) a chemiluminescent label;

[0178] (b) a fluorescent label;

[0179] (c) a quencher; and

[0180] (d) a combination of one or more of (a), (b), and (c).

[0181] 86. The method of any one of items 78-85, wherein the detection step (D) occurs during the amplification step (C).

[0182] 87. The method of item 86, wherein the parvovirus-specific detection probe comprises a fluorescent label, a quencher, or both.

[0183] 88. The method of item 87, wherein the parvovirus-specific detection probe is a TaqMan detection probe or a molecular beacon.

[0184] 89. The method of any one of items 78-87, wherein the parvovirus-specific detection probe further comprises a non-target hybridization sequence.

[0185] 90. The method of item 89, wherein the parvovirus-specific detection probe is a hairpin detection probe.

[0186] 91. The method of item 90, wherein the hairpin detection probe is a molecular beacon or a molecular torch.

[0187] 92. The method of any one of items 53-91, wherein the amplification reaction of step (C) is an isothermal amplification reaction.

[0188] 93. The method of any one of items 53-91, wherein the amplification reaction of step (C) is a PCR amplification reaction.

[0189] 94. The method of item 92 or 93, wherein the amplification reaction is a real-time amplification reaction.

[0190] 95. The method of item 52, wherein the method is for detecting the HAV target nucleic acid and contacting the sample with the first HAV amplification oligomer and the second HAV amplification oligomer of (II).

[0191] 96. The method of item 95, wherein the first HAV target hybridization sequence of (II)(a) is comprised in the sequence of SEQ ID NO: 172.

[0192] 97. The method of item 96, wherein the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 170.

[0193] 98. The method of item 97, wherein the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6 and 11.

[0194] 99. The method of item 96, wherein the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 171.

[0195] 100. The method of item 99, wherein the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6.

[0196] 101. The method of any one of items 95-100, wherein the second HAV target hybridization sequence of (II)(b) is comprised in the sequence of SEQ ID NO: 176.

[0197] 102. The method of item 101, wherein the second HAV target-hybridizing sequence of (II)(b) is selected from the group consisting of SEQ ID NOs: 29-38 and 45.

[0198] 103. The method of any one of items 95-102, wherein the second HAV amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target-hybridizing sequence.

[0199] 104. The method of item 103, wherein the promoter sequence is a T7 promoter sequence.

[0200] 105. The method of item 104, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196.

[0201] 106. The method of item 105, wherein the second HAV amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 12-21 and 28.

[0202] 107. The method of any one of items 95-106, wherein step (B) further comprises contacting the sample with:

[0203] (IV) a third amplification oligomer and a fourth amplification oligomer for amplifying the HAV nucleic acid target region, wherein (a) the third HAV amplification oligomer comprises a third target-hybridizing sequence that is about 14 to about 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 174 and includes at least the sequence of SEQ ID NO: 173; and (b) the fourth HAV amplification oligomer comprises a fourth target-hybridizing sequence that is about 14 to about 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 177 and includes at least the sequence of SEQ ID NO: 175;

[0204] wherein the third target-hybridizing sequence of (IV)(a) is different from the first HAV target-hybridizing sequence of (II)(a); and

[0205] wherein the fourth target-hybridizing sequence of (IV)(b) is different from the second HAV target-hybridizing sequence of (II)(b).

[0206] 108. The method of item 107, wherein the third HAV amplification oligomer is an oligomer as described in any one of items 96-100 with respect to the first HAV amplification oligomer.

[0207] 109. The method of item 107 or 108, wherein the fourth HAV amplification oligomer is an oligomer as described in items 101-106 with respect to the second HAV amplification oligomer.

[0208] 110. The method of any one of items 95-109, further comprising purifying the HAV target nucleic acids from other components of the sample prior to step (B).

[0209] 111. The method of item 110, wherein the purification step comprises contacting the sample with at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57.

[0210] 112. The method of item 111, wherein the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

[0211] 113. The method of any one of items 95-112, further comprising a displacer oligomer comprising a target-hybridizing sequence configured for hybridization to the HAV target nucleic acid upstream of the first HAV amplification oligomer or the second HAV amplification oligomer.

[0212] 114. The method of item 95, wherein the detecting step (D) comprises contacting the in vitro nucleic acid amplification reaction with a HAV-specific detection probe oligomer configured for specific hybridization to the HAV amplification product under conditions whereby the presence or absence of the HAV amplification product is determined, thereby indicating the presence or absence of HAV in the sample.

[0213] 115. The method of item 114, wherein the HAV-specific detection probe oligomer comprises a target-hybridizing sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from about nucleotide position 5965 to about nucleotide position 6028.

[0214] 116. The method of item 115, wherein the HAV-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 178.

[0215] 117. The method of item 116, wherein the HAV-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 58-74.

[0216] 118. The method of any one of items 114 to 117, further comprising contacting the sample with a false target oligomer that is amplifiable in an in vitro nucleic acid amplification reaction using the first HAV amplification oligomer and the second HAV amplification oligomer to produce a second amplification product that does not specifically hybridize to the HAV-specific detection probe under the detection reaction conditions.

[0217] 119. The method of any one of items 114 to 118, further comprising contacting the sample with a cold probe oligomer that competes for hybridization to the HAV amplification product with the HAV-specific detection probe oligomer.

[0218] 120. The method of any one of items 114 to 117, further comprising contacting the sample with a tuner oligomer configured for specific hybridization to the first HAV amplification oligomer and the second HAV amplification oligomer.

[0219] 121. The method of any one of items 114 to 120, wherein the HAV-specific detection probe comprises a label selected from:

[0220] (a) a chemiluminescent label;

[0221] (b) a fluorescent label;

[0222] (c) a quencher; and

[0223] (d) a combination of one or more of (a), (b), and (c).

[0224] 122. The method of any one of items 114 to 121, wherein the detection step (D) occurs during the amplification step (C).

[0225] 123. The method of item 122, wherein the HAV-specific detection probe comprises a fluorescent label, a quencher, or both.

[0226] 124. The method of item 123, wherein the HAV-specific detection probe is a TaqMan detection probe or a molecular beacon.

[0227] 125. The method of any one of items 114 to 123, wherein the HAV-specific detection probe further comprises a non-target hybridization sequence.

[0228] 126. The method of item 125, wherein the HAV-specific detection probe is a hairpin detection probe.

[0229] 127. The method of item 126, wherein the hairpin detection probe is a molecular beacon or a molecular torch.

[0230] 128. The method of any one of items 95 to 127, wherein the amplification reaction of step (C) is an isothermal amplification reaction.

[0231] 129. The method of any one of items 95 to 127, wherein the amplification reaction of step (C) is a PCR amplification reaction.

[0232] 130. The method of item 128 or 129, wherein the amplification reaction is a real-time amplification reaction.

[0233] 131. The method of any one of items 52 to 130, wherein the sample is from an individual patient.

[0234] 132. The method of any one of items 52 to 130, wherein the sample is pooled.

[0235] 133. The method of item 132, wherein the pooled sample is a pooled plasma sample.

[0236] 134. The method of item 52, wherein the method is for detecting the human parvovirus target nucleic acid and the HAV target nucleic acid, and wherein the detecting step (D) comprises contacting the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer and a HAV-specific detection probe oligomer configured for specific hybridization to parvovirus amplification products and HAV amplification products, respectively, under conditions whereby the presence or absence of parvovirus amplification products and HAV amplification products is determined, thereby indicating the presence or absence of parvovirus and HAV in the sample.

[0237] 135. The method of item 135, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are differentially labeled.

[0238] 136. The method of item 135, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a label independently selected from the group consisting of (a) a chemiluminescent label and (b) a fluorescent label.

[0239] 137. The method of item 135, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a chemiluminescent label.

[0240] 138. The method of item 137, wherein the chemiluminescent labels for the parvovirus- specific detection probe oligomer and the HAV-specific detection probe oligomer are characterized by different luminescence kinetics sufficient to distinguish parvovirus-specific chemiluminescent signals from HAV-specific chemiluminescent signals.

[0241] 139. The method of item 138, wherein the chemiluminescent labels for the parvovirus- specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise acridinium ester (AE).

[0242] 140. The method of any one of items 134-139, wherein the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 182 and includes at least the sequence of SEQ ID NO: 179.

[0243] 141. The method of item 140, wherein the first parvovirus target hybridization sequence of (I)(a) includes at least the sequence of SEQ ID NO: 183.

[0244] 142. The method of item 141, wherein the first parvovirus target hybridization sequence of (I)(a) has a sequence selected from SEQ ID NOs: 75-80.

[0245] 143. The method of any one of items 134-139, wherein the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 184.

[0246] 144. The method of item 143, wherein the first parvovirus target hybridization sequence of (I)(a) is selected from SEQ ID NOs: 81-84.

[0247] 145. The method of any one of items 134-139, wherein the first parvovirus target hybridization sequence of (I)(a) is comprised in the sequence of SEQ ID NO: 185 and includes at least the sequence of SEQ ID NO: 180.

[0248] 146. The method of item 145, wherein the first parvovirus target hybridization sequence of (I)(a) is selected from SEQ ID NOs: 82-84.

[0249] 147. The method of any one of items 134-146, wherein the second parvovirus target hybridization sequence of (I)(b) is comprised in the sequence of SEQ ID NO: 187 and includes at least the sequence of SEQ ID NO: 188.

[0250] 148. The method of item 147, wherein the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 186.

[0251] 149. The method of item 148, wherein the second parvovirus target hybridization sequence of (I)(b) is selected from SEQ ID NOs: 108-113.

[0252] 150. The method of any one of items 134-146, wherein the second parvovirus target hybridization sequence of (I)(b) is contained within the sequence of SEQ ID NO: 191.

[0253] 151. The method of item 150, wherein the second parvovirus target hybridization sequence of (I)(b) comprises at least the sequence of SEQ ID NO: 190.

[0254] 152. The method of item 151, wherein the second parvovirus target hybridization sequence of (I)(b) is selected from SEQ ID NOs: 118-121.

[0255] 153. The method of any one of items 134-152, wherein the second parvovirus amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence.

[0256] 154. The method of item 153, wherein the promoter sequence is a T7 promoter sequence.

[0257] 155. The method of item 154, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196.

[0258] 156. The method of item 155, wherein the second parvovirus amplification oligomer has a sequence selected from SEQ ID NOs: 88-93 and 98-101.

[0259] 157. The method of any one of items 134-156, wherein the first HAV target hybridization sequence of (II)(a) is contained within the sequence of SEQ ID NO: 172.

[0260] 158. The method of item 157, wherein the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 170.

[0261] 159. The method of item 158, wherein the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6 and 11.

[0262] 160. The method of item 157, wherein the first HAV target hybridization sequence of (II)(a) comprises at least the sequence of SEQ ID NO: 171.

[0263] 161. The method of item 160, wherein the first HAV target hybridization sequence of (II)(a) is selected from the group consisting of SEQ ID NOs: 1-6.

[0264] 162. The method of any one of items 134-161, wherein the second HAV target hybridization sequence of (II)(b) is contained within the sequence of SEQ ID NO: 176.

[0265] 163. The method of item 162, wherein the second HAV target hybridization sequence of (II)(b) is selected from the group consisting of SEQ ID NOs: 29-38 and 45.

[0266] 164. The method of any one of items 134-163, wherein the second HAV amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence.

[0267] 165. The method of item 164, wherein the promoter sequence is a T7 promoter sequence.

[0268] 166. The method of item 165, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO: 196.

[0269] 167. The method of item 166, wherein the second HAV amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 12-21 and 28.

[0270] 168. The method of any one of items 134-167, wherein the parvovirus-specific detection probe oligomer comprises a target hybridization sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 199 from about nucleotide position 2921 to about nucleotide position 2966, or from about nucleotide position 2921 to about nucleotide position 3067.

[0271] 169. The method of item 168, wherein the parvovirus-specific detection probe target hybridization sequence is contained within the sequence of SEQ ID NO: 194 or 195.

[0272] 170. The method of item 169, wherein the parvovirus-specific detection probe target hybridization sequence is selected from the group consisting of SEQ ID NOs: 137-169.

[0273] 171. The method of any one of items 134-170, wherein the HAV-specific detection probe oligomer comprises a target hybridization sequence of about 14 to about 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from about nucleotide position 5965 to about nucleotide position 6028.

[0274] 172. The method of item 171, wherein the HAV-specific detection probe target hybridization sequence is contained within the sequence of SEQ ID NO: 178.

[0275] 173. The method of item 172, wherein the HAV-specific detection probe target hybridization sequence is selected from the group consisting of SEQ ID NOs: 58-74. DETAILED DESCRIPTION

[0276] The present application discloses oligonucleotide sequences configured as amplification oligomers and detection probe oligomers for use in detecting the presence of Hepatitis A Virus and / or Parvovirus Types 1, 2 and 3 nucleic acid sequences in a biological sample by an in vitro nucleic acid amplification assay. One embodiment of the method uses transcription-mediated nucleic acid amplification (as previously disclosed in detail in U.S. Patent Nos. 5,399,491 and 5,554,516 to Kacian et al.). The method for detecting the amplified nucleic acid uses a sequence-specific probe that specifically hybridizes to a portion of the amplified sequence. In one aspect, the method uses any known homogeneous detection step to detect the labeled probe bound to the amplified nucleic acid (e.g., as disclosed by Arnold et al., Clin. Chem. 35:1588-1594 (1989); U.S. Patent Nos. 5,658,737 to Nelson et al. and 5,118,801 and 5,312,728 to Lizardi et al.). The present application also discloses oligonucleotide sequences that can be used to capture Hepatitis A Virus target DNA or Parvovirus Types 1, 2 and 3 target DNA by using nucleic acid hybridization techniques. One embodiment of the capture step uses magnetic particles to isolate the captured target (see U.S. Patent No. 6,110,678 to Weisburg et al.).

[0277] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleic acid," is understood to represent one or more nucleic acids. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0278] "Sample" or "biological sample" means any material derived from a living or dead human that can contain parvovirus nucleic acid and / or hepatitis A virus nucleic acid, including, for example, sputum, peripheral blood, plasma, serum, biopsy tissue including lymph node, respiratory tissue or exudate, or other body fluids, tissues, or materials. A sample can be treated to disrupt tissue or cellular structure by physical, chemical, and / or mechanical means to release intracellular components. Sample preparation can use solutions containing buffers, salts, enzymes, detergents, etc. that are used to prepare the sample for analysis. A sample can be pooled from two or more sources (e.g., plasma samples pooled from two or more donors). A sample can be fractionated (e.g., a sample such as a fraction of a pooled sample). A sample can be a manufacturer's pool of plasma from which components are isolated.

[0279] A "nucleic acid" refers to a polymeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs consisting of a sugar moiety and a nitrogen-containing heterocyclic base or base analog. Nucleosides are linked together by phosphodiester bonds or other linkages to form RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs thereof. A nucleic acid "backbone" can be composed of a variety of linkages (see, e.g., International Patent Application Publication No. WO 95 / 32305). The sugar moiety of one or more residues in a nucleic acid can be ribose or deoxyribose, or an analogous compound with known substitutions, such as 2'-methoxy substitutions and 2'-halo substitutions (e.g., 2'-F). The nitrogen-containing base of one or more residues in a nucleic acid can be a conventional base (A, G, C, T, U), an analog thereof (see, e.g., The Biochemistry of the Nucleic Acids 5-36, Adams et al. eds., 11th ed. 1992; Abraham et al., 2007, BioTechniques 43:617-24), which includes derivatives of purine or pyrimidine bases (see, e.g., U.S. Patent Nos. 5,378,825, 6,949,367, and International Patent Application Publication No. WO 93 / 13121), or "abasic," in which the nucleoside unit lacks a nitrogen-containing base (see, e.g., U.S. Patent No. 5,585,481). A nucleic acid can include one or more "locked nucleic acid" (LNA) residues (Vester et al., Biochemistry 43:13233-41, 2004). A nucleic acid can include a 3' terminal dideoxynucleotide to prevent additional nucleotides from being added to the nucleic acid. Synthetic methods for preparing nucleic acids in vitro are well known in the art, but nucleic acids can be purified from natural sources using conventional techniques. The backbone of an oligomer can affect the stability of a hybridization complex (e.g., formed between a capture oligomer and its target nucleic acid). Such examples include peptide bonds, 2'-0-methoxy linkages, and sugar-phosphodiester type linkages. Peptide nucleic acids favor the formation of hybridization complexes with RNA. Oligomers with 2'-methoxy substituted RNA groups or 2'-fluoro substituted RNA can have enhanced hybridization complex stability relative to standard DNA or RNA and preferentially form hybridization complexes with complementary 2'-OH RNA. Linkages that join two sugar groups can affect hybridization complex stability by affecting overall charge or charge density, or by affecting steric interactions (e.g., bulky linkages can decrease hybridization complex stability). Preferred linkages include those with neutral groups (e.g., methylphosphonate groups) or charged groups (e.g., thiophosphonate groups) to affect complex stability.

[0280] The term "polynucleotide" as used herein denotes a nucleic acid strand. Throughout this application, nucleic acids are indicated 5' end to 3' end.

[0281] "Nucleotide" as used herein is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base. The 5-carbon sugar present in RNA is ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, e.g., methoxy (2'-0-Me) at the 2' position of ribose. As used herein, a methoxy oligonucleotide containing "T" residues has a methoxy group at the 2' position of the ribose moiety and a uracil at the base position of the nucleotide.

[0282] A "non-nucleotide unit" as used herein is a unit that does not significantly participate in hybridization of the polymer. Such a unit should not, for example, participate in any significant hydrogen bonding with nucleotides, and would exclude a unit having one of the five nucleotide bases or their analogs as a component.

[0283] The interchangeable terms "oligomer," "oligomer," and "oligonucleotide" refer to a polynucleotide having a length of contiguous nucleotide residues (nt) from 1,000 nt to as few as 5 nt. It is understood that the range from 1000 to as few as 5 is an inclusive range, thus 1000 nt, 5 nt, and every integer in between are included in the range. Oligonucleotides can be purified from naturally occurring sources or can be synthesized using any of a variety of well-known enzymatic or chemical methods. The term oligonucleotide does not denote any particular function of the reagent; rather, it is used generically to encompass all such reagents described herein.

[0284] By "amplification oligonucleotide" or "amplification oligomer" is meant an oligonucleotide whose 3' end is complementary to a target nucleic acid and hybridizes to the target nucleic acid or its complement and participates in nucleic acid amplification. Examples of amplification oligomers include primers and initiator primers. An amplification oligonucleotide preferably contains at least 10 contiguous bases, and more preferably at least about 12 contiguous bases but fewer than about 70 bases, that specifically hybridize to a region of a target nucleic acid sequence under standard hybridization conditions. The contiguous bases that hybridize to the target sequence are at least about 80%, preferably at least about 90%, and more preferably about 100% complementary to the sequence to which the amplification oligonucleotide hybridizes. At least about X% means the full range of all integers and fractions from X% to 100%. An amplification oligonucleotide can optionally include modified nucleotides.

[0285] An amplification oligomer can be referred to as a "primer" or a "promoter primer." A "primer" refers to an oligonucleotide that hybridizes to a template nucleic acid and has a 3' end that can be extended in a known polymerization reaction. The 5' region of the primer can be non-complementary to the target nucleic acid, for example, the 5' non-complementary region can include a promoter sequence and the oligomer is referred to as a "promoter primer" or it can include a tag sequence, or it can include an adapter sequence. As used herein, a "promoter" is a specific nucleic acid sequence recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to bind to the nucleic acid and begin RNA transcription at a specific site. In addition, a promoter primer can contain a blocked 3' end to prevent them from being used as primers, and in these cases the amplification oligomer is referred to as a promoter provider. In some embodiments, the blocking moiety replaces the 3' OH of the oligomer to prevent enzyme-mediated extension of the oligomer in an amplification reaction. In alternative embodiments, the blocking moiety can be within the five residues of the 3' end and large enough to limit binding of the polymerase to the oligomer. In other embodiments, the blocking moiety is covalently attached to the 3' end of the oligomer. The 3' end of the oligomer can be blocked using a number of different chemical groups, including but not limited to alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin. It will be further appreciated by those skilled in the art that any oligomer that can act as a primer (i.e., an amplification oligonucleotide that hybridizes specifically to a target sequence and has a 3' end that can be extended with a polymerase) can be modified to include a 5' promoter sequence and thus act as a promoter primer. Similarly, any promoter primer can be modified to act as a primer by removing the promoter sequence or synthesizing without the promoter sequence.

[0286] As used herein, a "target nucleic acid" is a nucleic acid comprising a "target sequence" to be amplified. The target nucleic acid can be DNA or RNA as described herein, and can be single- stranded or double-stranded. In addition to the target sequence, the target nucleic acid can include other sequences that can not be amplified. Target nucleic acids include genomic nucleic acids, gene products (e.g., mRNA), and amplification products thereof. The target nucleic acids herein are human parvovirus nucleic acids and HAV nucleic acids.

[0287] "Isolated" means that the sample containing the target nucleic acid is extracted from its natural environment, although the term does not imply any degree of purification.

[0288] As used herein, the term "target sequence" refers to a specific nucleotide sequence of a target nucleic acid to be amplified and / or detected. A "target sequence" includes a complex sequence that is complexed with an oligonucleotide (e.g., a guide oligonucleotide and / or a promoter oligonucleotide) during the amplification process. When the target nucleic acid is initially single-stranded, the term "target sequence" will also refer to a sequence that is complementary to the "target sequence" present in the target nucleic acid. When the target nucleic acid is initially double-stranded, the term "target sequence" refers to both the sense (+) and anti-sense (-) strands.

[0289] "Target binding sequence" is used herein to refer to the portion of an oligomer that is configured to hybridize to a target nucleic acid sequence. Preferably, the target binding sequence is configured to hybridize specifically to the target nucleic acid sequence. The target binding sequence can be 100% complementary to the portion of the target sequence to which they are configured to hybridize; but this is not a requirement. The target binding sequence can also include nucleotide residues that are inserted, deleted, and / or substituted with respect to the target sequence. Less than 100% complementarity of the target binding sequence to the target sequence can occur, for example, when the target nucleic acid is a plurality of strains within a species, such as oligomers configured to hybridize to different strains and genotypes of human parvovirus. It is understood that there are other reasons for configuring a target binding sequence to have less than 100% complementarity to a target nucleic acid.

[0290] The term "targeting a sequence" as used herein with respect to a region of a human parvovirus nucleic acid refers to the process by which an oligonucleotide is hybridized to a target sequence in a manner that allows amplification and detection as described herein. In one embodiment, the oligonucleotide is complementary to the human parvovirus nucleic acid sequence targeted and contains no mismatches. In another embodiment, the oligonucleotide is complementary to the human parvovirus nucleic acid sequence targeted but contains 1, or 2, or 3, or 4, or 5 mismatches. Preferably, the oligonucleotide hybridized to the human parvovirus nucleic acid sequence includes at least 10 and up to 50 nucleotides that are complementary to the target sequence. It is understood that at least 10 and up to 50 is an inclusive range, and thus 10, 50, and every integer in between is disclosed. Preferably, the oligomer hybridizes specifically to the target sequence. The term "configured to target a sequence" as used herein means that the target hybridization region of an amplification oligonucleotide is designed to have a polynucleotide sequence that can hybridize specifically to the referenced human parvovirus region or the referenced HAV region. Such an amplification oligonucleotide is not limited to only targeting the sequence, but can be used as a composition in a kit or method for targeting a human parvovirus target nucleic acid (including genotypes 1, 2, and / or 3) or a HAV target nucleic acid as described herein. The term "configured as" indicates the actual arrangement of the polynucleotide sequence of the target hybridization sequence of an amplification oligonucleotide.

[0291] The term "region" as used herein refers to a portion of a nucleic acid, wherein the portion is less than the entire nucleic acid. For example, when the nucleic acid in question is an oligonucleotide primer, the term "region" can be used to refer to the smaller primer portion of the entire oligonucleotide. Similarly, and again by way of example only, when the nucleic acid is a human parvovirus genome, the term "region" can be used to refer to a smaller region of the nucleic acid, wherein the smaller region is targeted by one or more oligonucleotides of the present application. The target binding sequence of an oligonucleotide can hybridize to all or a portion of a region. A target binding sequence that hybridizes to a portion of a region is one that hybridizes within the region in question. As another non-limiting example of the use of the term "region", when the nucleic acid in question is an amplicon, the term "region" can be used to refer to the smaller nucleotide sequence that is identified for hybridization by the target binding sequence of a probe.

[0292] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, or its complement, or a fragment thereof, and preferred embodiments specifically amplify the target by using sequence-specific methods. Known amplification methods include, for example, transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification (including RT-PCR), ligase chain reaction (LCR) amplification, and strand displacement amplification (SDA). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as QB-replicase (see, e.g., U.S. Patent No. 4,786,600 and PCT No. WO 90 / 14439 to Kramer et al.). PCR amplification is well known and uses a DNA polymerase, sequence-specific primers, and thermal cycling to synthesize multiple copies of DNA or cDNA of two complementary strands (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159, and Methods in Enzymology, 1987, vol. 155: 335-350, all to Mullis et al.). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complement by using multiple cycles of hybridization, ligation, and denaturation (European Patent No. 0 320 308). SDA amplification is performed by using primers containing recognition sites for a restriction endonuclease that cleaves one strand of a hemi-modified DNA duplex including the target sequence, followed by amplification in a series of primer extension and strand displacement steps (U.S. Patent No. 5,422,252 to Walker et al.). Preferred embodiments use transcription-associated amplification, as described below. It will be apparent to those skilled in the art that the method steps and amplification oligonucleotides of the present application can be readily adapted to a variety of nucleic acid amplification procedures based on primer extension using polymerase activity.

[0293] Amplification of a "fragment" or "portion" of a target sequence refers to the production of amplified nucleic acids comprising less than the entire target region nucleic acid sequence. Such fragments can be produced by amplifying a portion of a target sequence, for example, using an amplification oligonucleotide that hybridizes to the target sequence and initiates polymerization from an internal position in the target sequence.

[0294] By "transcription-mediated amplification" (TMA) or "transcription- associated amplification" is meant nucleic acid amplification using RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. Transcription-associated amplification generally uses RNA polymerase and DNA polymerase activity, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter primer, and optionally can include one or more additional amplification oligonucleotides, including "helper" oligomers. Variations of transcription-associated amplification are well known in the art and are described in detail elsewhere (see U.S. Patent Nos. 5,399,491 and 5,554,516 to Kacian et al., U.S. Patent No. 5,437,990 to Burg et al., U.S. Patent No. 5,130,238 to Malek et al., U.S. Patent Nos. 4,868,105 and 5,124,246 to Urdea et al., PCT No. WO 93 / 22461 to Kacian et al., PCT No. WO 88 / 01302 and WO 88 / 10315 to Gingeras et al., PCT No. WO 94 / 03472 to McDonough et al., and PCT No. WO 95 / 03430 to Ryder et al.). The procedures of U.S. Patent Nos. 5,399,491 and 5,554,516 are preferred amplification embodiments. As used herein, the term "real-time TMA" refers to single primer transcription-mediated amplification ("TMA") of a target nucleic acid monitored by real-time detection means.

[0295] By "probe," "detection probe," or "detection probe oligomer" is meant a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid, preferably an amplified nucleic acid, under conditions that allow hybridization, thereby allowing detection of the target or the amplified nucleic acid. Detection can be direct (i.e., by a probe that directly hybridizes to the sequence) or indirect (i.e., by a probe that hybridizes to an intermediate molecular structure that links the probe to the target). The "target" of a probe generally refers to a sequence within an amplified nucleic acid sequence or a subset thereof that specifically hybridizes by standard hydrogen bond bonding (i.e., base pairing) to at least a portion of a probe oligomer. Probes can comprise a target-specific sequence and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Patent Nos. 5,118,801 and 5,312,728 to Lizardi et al., and U.S. Patent No. 6,361,945 Bl to Becker et al.). Probes can be DNA, RNA, analogs thereof, or combinations thereof and they can be labeled or unlabeled. Probe sequences and their target sequences are sufficiently complementary if they are configured to allow stable hybridization of the probe oligomer to a target sequence that is not perfectly complementary to the target-specific sequence of the probe under appropriate hybridization conditions.

[0296] A "cold probe" refers to an oligonucleotide that has a substantially similar or identical oligonucleotide sequence as compared to a detection probe oligomer. The primary difference between a cold probe and a detection probe is that the cold probe lacks a detectable label, whereas the detection probe oligomer has a detectable label. The cold probe oligomer is used to compete with the detection probe oligomer in a detection reaction, thereby reducing the overall signal received in the detection step. Detection signal is often reduced for one target of a multiplex amplification and detection assay where one or more, but not all, target nucleic acids have robust amplification kinetics as compared to one or more other members of the multiplex assay. The cold probe is used to compete with the detection probe on the stronger amplification, thus in a sense "derailing" the robust amplification. The derailed amplification is thus brought into a range more comparable to the weaker amplification species in the multiplex assay. Similarly, a pseudo-target is a nucleic acid that is applied to a multiplex amplification reaction to deraii the stronger amplification species so that its reaction kinetics are more similar to the reaction kinetics of the weaker amplification species. A pseudo-target is a nucleic acid that typically contains the binding site of a primer for the stronger amplification species, but little additional sequence. The primer is thus diverted from producing the amplification product of the stronger amplification species.

[0297] By "complementary" is meant that the nucleotide sequence of similar regions of two single-stranded nucleic acids or different regions of the same single-stranded nucleic acid has a nucleotide base composition that allows the single-stranded regions to hybridize together in regions of stable double-stranded hydrogen bonding under stringent hybridization or amplification conditions. Sequences that hybridize to each other can be perfectly 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). By "substantially complementary" is meant a contiguous sequence that is capable of hybridizing to another sequence through a series of hydrogen bonds between complementary bases, which can be complementary at every position in the sequence through standard base pairing or can contain one or more non-complementary residues, including abasic residues. A substantially complementary contiguous sequence is typically at least 80%, or at least 90% complementary (including all integers and rational numbers up to and including 100%) to the sequence to which the oligomer is intended to specifically hybridize. A "substantially complementary" sequence allows stable hybridization of the nucleic acid oligomer to its target sequence under appropriate hybridization conditions, even though the sequences are not perfectly complementary. A sequence of nucleotides is "perfectly" complementary when the contiguous sequence of nucleotides of one single-stranded region is capable of forming a series of "typical" hydrogen-bonded base pairs with a similar sequence of nucleotides of another single-stranded region, such that A pairs with U or T and C pairs with G (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (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, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0298] By "preferential hybridization" or "specific hybridization" is meant that under stringent hybridization assay conditions, probes hybridize to their target sequences or copies thereof to form stable probe:target hybrids, while the formation of stable probe:non-target hybrid is minimized. Thus, the extent of hybridization of a probe to a target sequence or copy thereof is sufficiently greater than its extent of hybridization to a non-target sequence to enable one of ordinary skill in the art to accurately detect RNA copies or complementary DNA (cDNA) of the target sequence formed during amplification. Appropriate hybridization conditions are well known in the art and can be predicted based on sequence composition or can be determined by using routine testing methods (e.g., §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57 of Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0299] By "capture oligonucleotide" or "capture oligomer" or "capture probe" is meant a nucleic acid oligomer that specifically hybridizes to a target nucleic acid to be captured and provides a means for separating and / or concentrating the target from other sample components. An example of a capture oligomer includes two binding regions: a target binding region and an immobilization probe binding region, whereby the capture oligomer can form a hybridization complex in which the target binding region of the capture oligomer binds to a target sequence and the immobilization probe binding region binds to an oligomer immobilized on a solid support (see U.S. Patent Nos. 6,110,678 and 6,280,952 to Weisburg et al.). Although the target binding region and the immobilization probe binding region are typically on the same capture oligomer, the two functional regions can be present on two different oligomers that are joined together by one or more linkers. For example, the immobilization probe binding region can be present on a first oligomer, the target binding region can be present on a second oligomer, and the two oligomers are joined by hydrogen bonding to a third oligomer that is a sequence-specific hybrid to the first and second oligomers. The target binding region of a capture probe can also be referred to as a target-specific portion of a capture probe and the immobilization probe binding region can be referred to as a tail portion. Examples of tail portions include homopolymers (e.g., poly dT or poly dA) or non-homopolymers (e.g., T 0-3 A 15-30 ), preferably linked to the 3' end of the target-specific portion of the oligomer.

[0300] By "immobilized probe" or "immobilized oligomer" is meant a nucleic acid oligomer that is directly or indirectly attached to an immobilization support. Immobilized probes attached to a solid support facilitate the separation of bound target sequences from unbound material in a sample. Any known solid support can be used, such as matrices and particles in solution, for example, nitrocellulose, nylon, glass, polyacrylamide, mixed polymers, polystyrene, silane polypropylene, and metal particles, preferably magnetically attractable particles. A preferred support is a monodisperse paramagnetic sphere (e.g., uniform size ± 5%) to provide consistent results, to which the immobilized probe is attached directly (e.g., by direct covalent linkage, chelation, or ionic interaction) or indirectly (e.g., through one or more linkers), where the linkage or interaction is stable during nucleic acid hybridization conditions.

[0301] "Sample preparation" refers to any step or process of treating a sample for subsequent amplification and / or detection of human parvovirus nucleic acid present in the sample. A sample can be a complex mixture of components, of which the target nucleic acid is a minor component. Sample preparation can include any known method of concentrating components (e.g., microorganisms or nucleic acids) from a larger sample volume, such as by filtering airborne or waterborne particles from a larger volume sample or by isolating microorganisms from a sample using standard microbiological methods. Sample preparation can include physical disruption and / or chemical lysis of cellular components to release intracellular components into an essentially aqueous or organic phase and remove debris, such as by using filtration, centrifugation, or adsorption. Sample preparation can include the use of nucleic acid oligonucleotides that selectively or non-specifically capture target nucleic acids and separate them from other sample components (e.g., as described in U.S. Patent 6,110,678 and PCT Publication No. WO 2008 / 016988).

[0302] By "isolating" or "purifying" is meant removing one or more components of a biological sample from at least one other component of the sample. Sample components generally include aqueous solutions of nucleic acids, salts, proteins, carbohydrates, and lipids. Steps to isolate or purify nucleic acids can remove at least about 70%, preferably at least about 90%, and more preferably at least about 95% of other components in the sample.

[0303] By "label" is meant a molecular moiety or compound that can be detected or that can produce a detectable signal. The label is directly or indirectly attached to the nucleic acid probe. Direct labeling utilizes a linkage or interaction, including a covalent bond or a noncovalent interaction, such as a hydrogen bond, a hydrophobic interaction, and an ionic interaction, or by forming a chelate or coordination complex, that links the label to the probe. Indirect labeling utilizes a bridging moiety or "linker" (e.g., an oligonucleotide or an antibody) to link the label to the probe. The linker can be used to amplify the detectable signal. The label is any known detectable moiety, such as a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group, or a chromophore, such as a dye or a detectable particle (e.g., a latex bead or a metal particle), a luminescent compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label), and a fluorescent compound. Preferably, the label on the labeled probe is detectable in a homogeneous reaction (i.e., in a mixture, the bound labeled probe exhibits a detectable change, such as stability or differential degradation, compared to the unbound labeled probe). One example of a label for use in a homogeneous assay is a chemiluminescent compound (e.g., described in detail in U.S. Patent Nos. 5,656,207 to Woodhead et al., 5,658,737 to Nelson et al., and 5,639,604 to Arnold, Jr., et al.). A preferred chemiluminescent label is an acridinium ester (AE) compound, such as a standard AE or a derivative thereof (e.g., naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamoyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and 2-methyl-AE).Synthesis and methods for attaching labels to nucleic acids and detecting the labels are well known in the art (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; U.S. Patent Nos. 4,581,333 to Kourilsky et al., 5,658,737 to Nelson et al., 5,656,207 to Woodhead et al., 5,547,842 to Hogan et al., 5,283,174 to Arnold, Jr. et al., and European Patent Publication No. 0747706 to Becker et al.). Another example of a label for use in a homogeneous assay is a fluorescent compound attached to a probe, where a quencher compound is in functional proximity to the fluorescent label when the probe is not hybridized to its target (e.g., U.S. Patent Nos. 5,118,801 and 5,312,728 to Lizardi et al., and 6,361,945 Bl to Becker et al.).

[0304] A "homogeneous detectable label" is a label whose presence can be detected in a homogeneous manner based on whether the labeled probe is hybridized to a target sequence (i.e., can be detected without physically removing unhybridized label or labeled probe). Examples of homogeneous detectable labels and methods for detecting them have been described (U.S. Patent Nos. 5,283,174 to Arnold et al., 5,656,207 to Woodhead et al., 5,658,737 to Nelson et al., 5,118,801 and 5,312,728 to Lizardi et al., and 6,361,945 Bl to Becker et al.).

[0305] By "consisting essentially of is meant that additional components and method steps can be included which do not materially affect the basic and novel characteristic of the invention. Such features include salts, buffers, nucleic acid oligomers, and similar biochemical reagents which do not materially affect the properties of the claimed components or method steps described herein for detecting nucleic acid sequences of Hepatitis A virus and / or Parvovirus types 1, 2, and 3. Similarly, additional method steps can be included which do not materially affect the basic nature of the assay.

[0306] As used herein, an oligonucleotide having a "sequence comprising or consisting of or consisting essentially of a sequence selected from a particular group of sequences" means that as a basic and novel characteristic, the oligonucleotide is capable of stable hybridization under stringent hybridization conditions to a nucleic acid having the exact complement of one of the nucleic acid sequences of the listed group. The exact complement includes the corresponding DNA or RNA sequence.

[0307] As used herein, an oligonucleotide "corresponding to" a specified nucleic acid sequence means that the referenced oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence, and thus it will hybridize under stringent hybridization conditions to the same target nucleic acid sequence. It will be understood by those skilled in the art that the "corresponding oligonucleotide" can vary from the referenced sequence and still hybridize to the same target nucleic acid sequence. It will also be understood that a first nucleic acid corresponding to a second nucleic acid includes the complement thereof and includes both the RNA and DNA thereof. This variation from the nucleic acid can be expressed in terms of the percent of identical bases within the sequence or the percent of completely complementary bases between the probe or primer and its target sequence. Thus, an oligonucleotide "corresponds to" a reference nucleic acid sequence if the percent of these base identities or complementarities is from 100% to about 80%. In preferred embodiments, the percent is from 100% to about 85%. In more preferred embodiments, the percent can be from 100% to about 90%; in other preferred embodiments, the percent is from 100% to about 95%. Similarly, a region of a nucleic acid or amplified nucleic acid can be referred to herein as corresponding to a reference nucleic acid sequence. It will be understood by those skilled in the art that different modifications to the hybridization conditions can be required at different percent complementarity to allow hybridization to a particular target sequence without producing unacceptable levels of non-specific hybridization.

[0308] The term "amplicon" or the term "amplification product" as used herein refers to a nucleic acid molecule that is complementary or homologous to a sequence contained within a target sequence that is produced during an amplification procedure. This complementary or homologous sequence of an amplicon is sometimes referred to herein as a "target-specific sequence." An amplicon can be double-stranded or single-stranded and can comprise DNA, RNA, or both. For example, a DNA-dependent RNA polymerase transcribes a single-stranded amplicon from double-stranded DNA during a transcription-mediated amplification procedure. These single-stranded amplicons are RNA amplicons and can be either strand of a double-stranded complex; this depends on how the amplification oligomers are designed. Thus, an amplicon can be single-stranded RNA. A RNA-dependent DNA polymerase synthesizes a DNA strand that is complementary to the RNA template. Thus, an amplicon can be a double-stranded DNA and RNA hybrid. RNA-dependent DNA polymerases often include RNase activity or are used in conjunction with an RNase that degrades the RNA strand. Thus, an amplicon can be single-stranded DNA. RNA-dependent DNA polymerases and DNA-dependent DNA polymerases synthesize a complementary DNA strand from a DNA template. Thus, an amplicon can be double-stranded DNA. RNA-dependent RNA polymerases synthesize RNA from an RNA template. Thus, an amplicon can be double-stranded RNA. DNA-dependent RNA polymerases synthesize RNA from a double-stranded DNA template, also known as transcription. Thus, an amplicon can be single-stranded RNA. Amplicons and methods for producing amplicons are known to those skilled in the art. For convenience herein, a single strand of RNA or a single strand of DNA can represent an amplicon produced by the amplification oligomer combinations of the present invention. Such a statement is not intended to limit the amplicon to the statement shown. One of skill in the art having the benefit of the present disclosure will use the amplification oligomers and polymerases to produce any of a number of types of amplicons; all within the spirit of the present invention.

[0309] A "non-target-specific sequence" as used herein refers to a region of an oligomer sequence that does not stably hybridize to a target sequence under standard hybridization conditions. Oligomers having non-target-specific sequences include, but are not limited to, a primer and a molecular beacon. An amplification oligomer can contain sequences that are not complementary to a target or template sequence; for example, the 5' region of a primer can include a promoter sequence that is not complementary to the target nucleic acid (referred to as a "promoter primer"). One of skill in the art will appreciate 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 the promoter sequence or synthesized in the absence of a promoter sequence and still function as a primer. A 3' blocked amplification oligomer can provide a promoter sequence and function as a polymerization template (referred to as a "promoter provider"). Thus, an amplicon produced by an amplification oligomer member such as a promoter primer will contain a target-specific sequence and a non-target-specific sequence.

[0310] As used herein, the term "relative light unit" ("RLU") is an arbitrary unit of measurement indicative of the relative number of photons emitted by a sample at a given wavelength or band of wavelengths. RLU varies with the characteristics of the detection means used for the measurement.

[0311] The term "specificity" in the context of amplification and / or detection systems is used herein to refer to a system characteristic that describes the ability of the system to distinguish between target and non-target sequences depending on sequence and assay conditions. With respect to nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of by-products (e.g., signal to noise ratio). With respect to detection, specificity generally refers to the ratio of signal produced by target nucleic acids to signal produced by non-target nucleic acids.

[0312] The term "sensitivity" is used herein to refer to the precision 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 number of copies of target nucleic acid that can be reliably detected in an amplification system, and will depend on, for example, the detection assay used as well as the specificity of the amplification reaction, e.g., the ratio of specific amplicons to by-products.

[0313] The assay of the present invention detects the presence of human parvovirus in a biological sample (e.g., blood, serum, plasma, sputum, bronchial lavage). In one embodiment, the assay detects parvovirus and / or HAV target nucleic acids in a plasma sample from an individual donor or from a pooled collection of donor samples. To prepare a plasma specimen, a whole blood sample is centrifuged using standard methods, and the plasma is stored at 4°C or -20°C prior to testing. To lyse the virions in the specimen, a lysis reagent containing detergent is mixed with the specimen to release the target nucleic acids from the virions. Specimen processing can combine viral lysis with purification of the viral target nucleic acids by including a capture oligomer and an immobilization oligomer in the lysis reagent. The method thus includes a target capture step in which the target nucleic acid is specifically hybridized to the capture oligomer, and then hybridized to the immobilization oligomer, and each bound complex (i.e., immobilization oligomer, capture oligomer, and target nucleic acid) is substantially separated from other sample components. Washing the solid support with a parvovirus binding complex containing wash can wash residual sample components out. Thus, the target nucleic acid is separated from other sample components and concentrated in the bound complex without releasing the bound target nucleic acid from the solid support.

[0314] A typical sample processing involves the following steps (described in detail in U.S. Patent No. 6,110,678, International Application Publication No. WO 2008 / 016988, and U.S. Patent Application No. 2006 / 0068417). Virions in a body fluid (e.g., 0.5 ml of plasma) are lysed upon contact with a target capture reagent (790 mM HEPES, 680 mM LiOH, 10% lithium dodecyl sulfate (LLS), 230 mM succinate, at least one 7 pm / ml of a capture probe, and 100 μg / ml of poly-dT14bound to magnetic particles (SERADYN TM The capture oligomer comprises a 5' target binding region sequence. The capture oligomer also comprises a homopolymer or heteropolymer 3' tail sequence that hybridizes to a complementary oligomer linked to a solid support (e.g., oligo-dT linked to a solid support and the oligo-dA tail portion of the capture oligomer). Target capture hybridization occurs in this reaction mixture by incubating the mixture at a first temperature (60°C) such that the capture oligomer specifically binds to its complementary target sequence in the target nucleic acid. The mixture is then cooled to a temperature of 40°C or less (e.g., room temperature) to allow the 3' tail of the capture oligomer to hybridize to its complementary oligomer on the particle. After the second hybridization, the mixture is processed to separate the solid support and its bound nucleic acid complex from other sample components, e.g., by using gravity, centrifugation, or magnetic separation. Typically, the separation uses a rack containing magnets to attract the magnetic particles with bound nucleic acid complex to the side of the tube. The bound complex on the particles is then removed from the supernatant by suspending the magnetic particles in a wash buffer, separating the particles to the side of the tube, and removing the supernatant, and washing the particles with 1 ml of wash buffer (10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v / v) anhydrous ethanol, 0.02% (w / v) methyl paraben, 0.01% (w / v) propyl paraben, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), pH 7.5).

[0315] Following sample preparation, amplification of the hepatitis A virus and / or parvovirus target nucleic acids to produce amplicons is achieved by using amplification oligomers that define the 5' and 3' ends of the region amplified by in vitro enzyme-mediated nucleic acid synthesis. One embodiment uses a transcription-mediated amplification (TMA) method, essentially as described in U.S. Patent Nos. 5,399,491 and 5,554,516, which is a substantially isothermal system that produces large amounts of detectable amplification product (RNA transcripts). Preferred embodiments of this method use a mixture of amplification oligomers, in which at least one primer is combined with at least one primer.

[0316] One preferred embodiment of an amplification oligomer set comprises a primer oligomer member and a promoter-based oligomer member. Preferably, the promoter-based amplification oligomer is a promoter primer comprising a 5' RNA polymerase promoter sequence and a 3' target binding sequence. RNA polymerase promoter sequences known in the art include, but are not limited to, sp6 RNA polymerase promoter sequences, T3 RNA polymerase promoter sequences, and T7 RNA polymerase promoter sequences. In a preferred embodiment, the promoter primer comprises a 5' T7 RNA polymerase promoter sequence and a 3' target binding sequence. Most preferably, the 5' T7 RNA polymerase promoter sequence is SEQ ID NO: 196.

[0317] In one preferred embodiment, the 3' target binding sequence of the promoter-based amplification oligomer is from about 10 to about 40 nucleic acid bases in length and comprises a nucleic acid sequence configured to specifically hybridize to a region within a target sequence of a human parvovirus nucleic acid or a hepatitis A virus target nucleic acid. Other preferred promoter primers comprise an internal tag sequence flanked at its 5' end by a promoter sequence and flanked at its 3' end by a target binding sequence. The internal tag sequence is also referred to herein as an intervening sequence. The internal tag sequence is any nucleic acid sequence that is preferably not stably hybridized to the target nucleic acid or that does not interfere with hybridization of the target binding sequence to the target nucleic acid. In addition, the internal tag sequence preferably has sufficient length and composition such that once incorporated into an amplification product, the tag-specific amplification oligomer can be used to participate in subsequent rounds to generate amplification products. One preferred tag sequence is from about 10 nucleotides to about 50 nucleotides in length. In addition, it is recognized that the intervening sequence can comprise any of the promoter-based oligomer members of the present application.

[0318] In one preferred embodiment, the amplification oligomer set comprises at least one primer amplification oligomer member. Preferred primer amplification oligomers have a length of from about 10 nucleic acid bases to about 50 nucleic acid bases and have a nucleotide composition configured to specifically hybridize to a hepatitis A virus or human parvovirus types 1, 2, and 3 when used in an amplification reaction of the present application to generate a detectable amplification product. One preferred primer oligomer is from about 10 to about 50 nucleic acid bases in length. Primer oligomer members of the present application are described herein. These descriptions need not be repeated here. Other preferred primer oligomer members comprise a 5' tag sequence. The 5' tag sequence is any nucleic acid sequence that is preferably not stably hybridized to the target nucleic acid or that does not interfere with hybridization of the target binding sequence to the target nucleic acid. In addition, the 5' tag sequence preferably has sufficient length and composition such that once incorporated into an amplification product, the tag-specific amplification oligomer can be used to participate in subsequent rounds to generate amplification products. One preferred 5' tag sequence is from about 10 nucleotides to about 50 nucleotides in length. Another preferred tag sequence is about 12 nucleotides in length. In addition, it is recognized that the 5' tag sequence can comprise any of the primer oligomer members of the present application.

[0319] Amplification of a target nucleic acid by transcription-mediated amplification can produce many nucleic acid strands from a single copy of the target nucleic acid, thus allowing detection of the target by detecting a probe that hybridizes to the sequence of the amplification product. Typically, the reaction mixture includes the target nucleic acid and at least two amplification oligomers, which comprise at least one primer, at least one promoter primer, reverse transcriptase and RNA polymerase activity, nucleic acid synthesis substrates (deoxyribonucleotide triphosphates and ribonucleotide triphosphates), and an appropriate salt- and buffer-containing solution to produce multiple RNA transcripts from the nucleic acid template. Briefly, the promoter primer specifically hybridizes to a portion of the target sequence. Reverse transcriptase, which includes RNase activity, produces a first strand cDNA by 3' extension of the promoter primer. The cDNA hybridizes to a primer downstream of the promoter primer and uses reverse transcriptase to synthesize a new DNA strand from the 3' end of the primer to produce dsDNA with a functional promoter sequence at one end. RNA polymerase binds to the promoter sequence of the dsDNA and transcribes multiple transcripts or amplicons. These amplicons are further used in the amplification process, serving as templates for new rounds of replication, to ultimately produce a large number of single-stranded amplified nucleic acids (e.g., 100 to 3,000 copies of RNA synthesized from a single template) from the initial target sequence. The process uses substantially constant reaction conditions (i.e., essentially isothermal). A typical 100 μΐ amplification reaction uses 75 μΐ of an amplification reagent mixture (11.6 mM Tris base, 15.0 mM Tris-HCl, 22.7 mM MgCl2, 23.3 mM KCl, 3.33% glycerol, 0.05 mM zinc acetate (dihydrate), 0.665 mM each of dATP, dCTP, dGTP, and dTTP, 5.32 mM each of ATP, CTP, GTP, and UTP, pH 7) and 25 μΐ of an enzyme reagent mixture (700 U of T7 RNA polymerase, 1400 U of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT), 16 mM HEPES (free acid, dihydrate), 70 mM N-acetyl-L-cysteine, 3 mM EDTA, 0.05% (w / v) sodium azide, 20 mM Tris base, 50 mM KCl, 20% (v / v) anhydrous glycerol, 10% (v / v) X-102 and 150 mM trehalose (di-hydrate), pH 7), preferably mixed with the captured target's nucleic acid that remains on the solid particle. For enzymatic activity, 1 U of T7 RNA polymerase incorporates 1 nmol of ATP into RNA in 1 hour at 37 °C using a T7 promoter-containing DNA template, while 1 U of MMLV-RT incorporates 1 nmol of dTTP into DNA in 10 minutes at 37 °C using 200-400 μmol oligo dT primed poly(A) as a template. Amplification oligos are in the range of about 5-20 picomoles / reaction, or more typically about 7-15 picomoles / reaction, or more typically about 5-15 picomoles / reaction or more typically about 5-10 picomoles / reaction, these ranges including all integers and fractions therein.

[0320] In a preferred embodiment, a TMA reaction is performed using a combination of amplification oligomers, wherein the combination includes at least one promoter primer oligomer member and at least one primer oligomer member, and wherein the combination is configured to produce amplification products for detection of hepatitis A virus and / or human parvovirus types 1, 2, and 3. In a particular aspect, a TMA reaction is performed using at least one hepatitis A virus non-T7 amplification oligomer (SEQ ID NOs: 1-11) and at least one hepatitis A virus promoter-based amplification oligomer (SEQ ID NOs: 12-28). In a particular aspect, a TMA reaction is performed using at least one parvovirus non-T7 amplification oligomer (SEQ ID NOs: 75-87) and at least one parvovirus promoter-based amplification oligomer (SEQ ID NOs: 88-107). In a particular aspect, a multiplex TMA reaction is performed using at least one hepatitis A virus non-T7 amplification oligomer (SEQ ID NOs: 1-11) and at least one hepatitis A virus promoter-based amplification oligomer (SEQ ID NOs: 12-28). In a particular aspect, a multiplex TMA reaction is performed using at least one parvovirus non-T7 amplification oligomer (SEQ ID NOs: 75-87) and at least one parvovirus promoter-based amplification oligomer (SEQ ID NOs: 88-107). In a particular aspect, a multiplex TMA reaction is performed using at least one parvovirus non-T7 amplification oligomer (SEQ ID NOs: 75-87), at least one parvovirus promoter-based amplification oligomer (SEQ ID NOs: 88-107), at least one hepatitis A virus non-T7 amplification oligomer (SEQ ID NOs: 1-11), and at least one hepatitis A virus promoter-based amplification oligomer (SEQ ID NOs: 12-28). In an aspect of this embodiment, the combination of amplification oligomers includes at least one promoter primer oligomer member comprising a 5' promoter sequence, an internal tag sequence, and a 3' target binding sequence. In an aspect of this embodiment, the combination of amplification oligomers includes at least one promoter primer oligomer member comprising a 5' promoter sequence, an internal tag sequence, and a 3' target binding sequence, and further includes at least one promoter primer oligomer member comprising a 5' promoter sequence and a 3' target binding sequence. In an aspect of this embodiment, the combination of amplification oligomers includes at least one primer oligomer member comprising a 5' tag sequence and a 3' target binding sequence. In an aspect of this embodiment, the combination of amplification oligomers includes at least one primer oligomer member comprising a 5' tag sequence and a 3' target binding sequence, and further includes at least one primer oligomer member comprising a 3' target binding sequence.

[0321] In another preferred embodiment, the TMA reaction is performed using an amplification oligomer combination comprising at least one promoter primer oligomer member and at least one primer oligomer member, wherein the combination is configured to generate amplification products for the detection of human parvovirus types 1, 2, and 3, and / or using an amplification oligomer combination comprising at least one promoter primer oligomer member and at least one primer oligomer member, wherein the combination is configured to generate amplification products for the detection of hepatitis A virus. In one embodiment, the TMA reaction for parvovirus is a quantitative amplification and detection reaction. In one embodiment, the TMA reaction for hepatitis A virus is a quantitative amplification and detection reaction. In one embodiment, the TMA reaction for parvovirus and the TMA reaction for hepatitis A virus are quantitative amplification and detection reactions. In one aspect, the amplification reactions are multiplex amplification reactions, and detection of the amplification products is performed in a detection step using one or more detection probes of SEQ ID NOs: 58-74 and / or one or more detection probes of SEQ ID NOs: 137-169. In one aspect, the amplification reactions are singleplex amplification reactions, and detection of the amplification products is performed in a detection step using one or more detection probes of SEQ ID NOs: 58-74 and / or one or more detection probes of SEQ ID NOs: 137-169.

[0322] Following or during the amplification reaction, the amplification sequences generated from the hepatitis A virus target nucleic acid and / or from the parvovirus target nucleic acid are detected, preferably by hybridization with at least one labeled nucleic acid probe that specifically hybridizes to a portion of the amplification sequence. Probe embodiments include probes having a Tm in the range of about 80°C to about 85°C mthose. Some preferred probe embodiments include oligomers having a nucleotide length of about 15 to about 40 nucleotides and a nucleic acid sequence that is DNA, RNA, or a combination thereof and configured for region-specific hybridization to all or a portion of a target sequence of a hepatitis A virus nucleic acid or a human parvovirus nucleic acid or amplified nucleic acid. Detection oligomers of the present application can also comprise one or more LNA residues. Detection of the probe is accomplished by detection of a label that can be detected in a homogeneous reaction. Thus, some preferred embodiments also comprise probes labeled with acridinium ester (AE) compounds using well-known methods that allow for homogeneous detection (e.g., labeling and detection methods are described in detail in U.S. Patent Nos. 5,283,174 to Arnold, Jr. et al., 5,656,207 to Woodhead et al., and 5,658,737 to Nelson et al.). Chemiluminescent AE compounds are attached to the probe sequence via a linker compound (substantially as described in U.S. Patent Nos. 5,585,481 and 5,639,604 to Arnold, Jr. et al., e.g., see column 10, line 6 through column 11, line 3, and Example 8). In one embodiment, the labeled probe oligomer has at least one 2'-0-methoxy linkage in the nucleic acid backbone. In one embodiment of a typical detection step, the probe reagent comprises 100 mM succinate, 2% (w / v) LLS, 230 mM LiOH (monohydrate), 15 mM 2,2'-dithiodipyridine (ALDRITHIOL-2), 1.2 M LiCl, 20 mM EDTA, 20 mM EGTA, 3% (v / v) absolute ethanol, with LiOH to about pH 4.7, and the selection reagent for hydrolysis of label on unbound probe comprises 600 mM boric acid, 182 mM NaOH, 1% (v / v) Triton® X-100. The detection probe is added to the detection reaction in the range of about 1E7-5E7 relative light units (RLU) per reaction, more typically about 1E7-3E7 RLU per reaction, more typically 2E7-5E7 RLU per reaction, or more typically 2E7-4E7 RLU per reaction; wherein said range includes all integers and fractions therein. The signal is detected in RLU format using a luminometer (e.g., LEADER 2000® luminometer (Tecan, Inc., Durham, NC) or Wallac Victor® 1420 Multilabel Counter (PerkinElmer, Inc., Waltham, MA) or Wallac Microbeta® Plus X-100. The detection probe is added to the detection reaction in the range of about 1E7-5E7 relative light units (RLU) per reaction, more typically about 1E7-3E7 RLU per reaction, more typically 2E7-5E7 RLU per reaction, or more typically 2E7-4E7 RLU per reaction; wherein said range includes all integers and fractions therein. The signal is detected in RLU format using a luminometer (e.g., LEADER TM 450HC+ (Gen-Probe Incorporated, San Diego, CA) in RLU format.

[0323] To select DNA sequences suitable for use as capture oligomers, amplification oligomers, and detection probes, DNA sequences (including portions or complements) available from public access databases (e.g., GenBank) are aligned by matching regions of identical or similar sequences and compared using well-known molecular biology techniques. Although sequence comparisons can be facilitated by the use of algorithms, such comparisons can readily be performed by the skilled artisan, manually and visually. Generally, portions of sequences that contain relatively few variants between the compared sequences are selected as the basis for designing synthetic oligomers used in the present invention. Other considerations for designing oligomers include the relative CG content (which affects T m ) and the relative absence of predicted secondary structures within the sequence (which can form intra-molecular hybrids), as determined by using well-known methods.

[0324] In one embodiment, the assay is performed using 0.5 to 1 ml of a sample of a bodily fluid (e.g., plasma) in a single tube to detect target nucleic acids at a sensitivity of about 100 to 500 copies / ml of target DNA per reaction. In other embodiments, the assay detects a higher number of target nucleic acids in a sample, which can be a pooled sample of individual samples.

[0325] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. General definitions for many of the terms used herein are provided in Dictionary of Microbiology and Molecular Biology, 2nded. (Singleton et al., 1994, John Wiley & Sons, New York, NY); The Harper Collins Dictionary of Biology (Hale and Marham, 1991, Harper Perennial, New York, NY); and Taber's Cyclopedic Medical Dictionary, 17th ed. (F.A. Davis Co., Philadelphia, PA, 1993). Unless otherwise mentioned, techniques used or contemplated herein are standard methods well known to those of ordinary skill in the art. The following examples illustrate some preferred embodiments of the present application and are provided merely as illustration.

[0326] Example 1: Amplification of HAV targets using primer and promoter providers and without target capture .

[0327] The Hepatitis A Virus Amplification Assay was set up as in Example 1, except that the assay included an initial target capture step with different dilutions and except that detection of the amplification product was performed using a detection probe (SEQ ID NO: 62). The assay was set up by first capturing HAV targets from each individual dilution and adding the captured HAV target nucleic acids to individual wells on a 96-well plate. The target capture oligomers used were from an oligomer pool with a dT3 dA30 tail and a K18 target capture region. The target capture region of the target capture oligomers was synthesized to have a random combination of G and U residues, so the population of target capture oligomers used in this example was a mixed collection of sequences. The detection step was an end-point detection reaction and the results showed that the assay system had a 95% detection rate for at least 3.87 IU / mL of HAV target nucleic acid (Table 1).

[0328] Example 2: Amplification of HAV targets using primer and promoter providers and with target capture .

[0329] The Hepatitis A Virus Amplification Assay was set up as in Example 1, except that the assay included an initial target capture step with different dilutions and except that detection of the amplification product was performed using a detection probe (SEQ ID NO: 62). The assay was set up by first capturing HAV targets from each individual dilution and adding the captured HAV target nucleic acids to individual wells on a 96-well plate. The target capture oligomers used were from an oligomer pool with a dT3 dA30 tail and a K18 target capture region. The target capture region of the target capture oligomers was synthesized to have a random combination of G and U residues, so the population of target capture oligomers used in this example was a mixed collection of sequences. The detection step was an end-point detection reaction and the results showed that the assay system had a 95% detection rate for at least 3.87 IU / mL of HAV target nucleic acid (Table 1).

[0330] Table 1 .

[0331]

[0332] This experiment showed that using a non-specific target capture system in combination with amplification and detection conditions provided sensitive amplification and detection of HAV target nucleic acid, but the amplification and detection was not as sensitive as seen in Example 1 where known amounts of target were added directly to the amplification reaction.

[0333] Example 3: Amplification of parvovirus genotypes 1, 2, and 3 using primer and promoter providers and with target capture Example 4: Amplification and detection of hepatitis A virus in the presence of parvovirus .

[0334] An amplification reaction was set up to test multiple combinations of amplification oligomers for amplifying different concentrations of parvovirus genotype 1. An amplification assay was set up in which all combinations of non-T7 and T7 amplification oligomers could be derived from combining each of SEQ ID NOS: 75-87 with each of SEQ ID NOS: 88-99, where each combination was exactly one non-T7 and one T7. Each amplification oligomer was designed to amplify parvovirus genotypes 1, 2, and 3. The target nucleic acid was SEQ ID NO: 203. The stock nucleic acid was diluted and added to each well of a reaction plate at 0 copies per reaction, 10 copies per reaction, 100 copies per reaction, and 100,000 copies per reaction. Primerless amplification reagents were prepared as generally described above and added to each well of each reaction plate for 0, 10, 100, and 100,000 copy reactions. Various primer conditions from the above combinations of non-T7 and T7 amplification oligomers were added to separate wells on these plates. The amplification reaction was isothermal and included an initial incubation step at about 62 °C and about 42 °C for 10 minutes and 20 minutes, respectively, followed by amplification at 42 °C for 50 minutes in the presence of polymerase. Detection of amplification products was performed using SEQ ID NO: 145 and Leader HC luminometer. The RLU detected for the plate with 0 copies of parvovirus was 934 RLU to 14,698 RLU. The RLU detected for the plate with 10 copies of parvovirus per reaction was 2,015 RLU to 8,040,373 RLU, with more than half of the combinations providing a signal well above background. The RLU detected for the plate with 100 copies of parvovirus per reaction was 2,439 RLU to 8,114,133 RLU. The RLU detected for the plate with 100,000 copies of parvovirus per reaction was 495,680 RLU to 8,308,252 RLU. From the results of these assays, multiple combinations of amplification oligomers were identified that could be used for a parvovirus amplification reaction that could amplify as few as 10 copies of parvovirus.

[0335] Another assay was set up for amplification and detection of each of parvovirus genotypes 1, 2, and 3. These amplification reactions used the following amplification oligo conditions: SEQ ID NO: 80 and 92; SEQ ID NO: 80 and 91; and SEQ ID NO: 81 and 92, each of which was designed to amplify three parvovirus genotypes. The target nucleic acid was an in vitro transcript of a portion of each parvovirus genotype 1-3 (SEQ ID NO: 200, 201, and 202, respectively) and was provided in the assay as serial dilutions from a stock concentration. Target capture was performed using the non-specific target capture system discussed in Example 2. Detection was an end-point detection using SEQ ID NO: 146. The results of this amplification and detection assay showed that the three systems had consistent sensitivity down to at least 80 copies of target per milliliter, and had good sensitivity down to 5 copies per milliliter, but there was variation in detection between the three genotypes (e.g., at 5 copies per milliliter, 40% of the wells with genotype 1 were reactive, while only about 20% of the wells of genotype 3 were reactive).

[0336] Another amplification reaction was performed to amplify and detect parvovirus nucleic acid, where the amplification oligo combination contained a non-T7 amplification oligo and two T7 amplification oligos. The combinations of amplification oligos were as follows: SEQ ID NO: 80, 90, and 99; SEQ ID NO: 80, 91, and 99; SEQ ID NO: 80, 92, and 99; SEQ ID NO: 81, 90, and 99; SEQ ID NO: 81, 91, and 99; SEQ ID NO: 81, 92, and 99; SEQ ID NO: 82, 90, and 99; SEQ ID NO: 82, 91, and 99; and SEQ ID NO: 82, 92, and 99. The amplification and detection reactions were set up as generally described above, and each reaction was performed in 10 wells. The target nucleic acid was SEQ ID NO: 203. The results showed that SEQ ID NO: 80, 90, and 99; SEQ ID NO: 80, 91, and 99; SEQ ID NO: 80, 92, and 99; and SEQ ID NO: 81, 91, and 99 all detected down to 50 copies of target nucleic acid with 100% reactivity for each well, while the other combinations were about 80% reactive to non-reactive.

[0337] The following combinations were then tested against SEQ ID NO: 200-202: SEQ ID NO: 80, 91, and 99; SEQ ID NO: 80, 92, and 99; and SEQ ID NO: 82, 90, and 99, each target nucleic acid provided at 45 copies per reaction, 15 copies per reaction, and 5 copies per reaction. Each reaction condition was tested in 10 separate wells. Amplification and detection reactions were set up as generally described above. All three amplification oligo combinations were 100% reactive for detecting 45 copies of each parvovirus genotype. The three amplification oligo combinations were 100% reactive to 70% reactive for detecting 15 copies of the three parvovirus genotypes. The three amplification oligo combinations were 70% reactive to 40% reactive for detecting 5 copies of the three parvovirus genotypes. Thus, the combination of parvovirus amplification oligos was able to detect as few as 5 copies of each genotype with about 40-70% efficiency.

[0338] Example 5: Quantitative amplification and detection of parvovirus target nucleic acids .

[0339] An amplification assay was performed to amplify and detect Hepatitis A Virus target nucleic acid in the presence of parvovirus nucleic acid. The HAV target nucleic acid was the WHO standard described in Example 1. The HAV standard was serially diluted and each dilution was added to a separate well on a 96 well plate. 5E6 copies of SEQ ID NO: 200 was added to each well. The HAV amplification reaction mix was prepared to include 5-10 pM / rxn of SEQ ID NO: 2 and 18. The endpoint detection reaction mix was prepared to include 5E6 RLU per reaction of SEQ ID NO: 62. The amplification reaction was performed, then the reaction was terminated and the detection reaction was performed. The results of the assay showed that 100% of the wells containing as few as 40 copies of HAV and 90% of the wells containing as few as 20 copies of HAV were detectable in the presence of 5E6 copies of parvovirus.

[0340] Table 2 .

[0341] A quantitative assay for the amplification and detection of parvovirus genotypes 1, 2 and 3 was performed. The parvovirus target nucleic acid was nucleic acid from the WHO International Reference Panel for parvovirus B19 genotypes (NIBSC 99 / 800, which is 5.98 log10 IU / mL genotype 1, 5.94 log10 IU / mL genotype 2, 5.97 log10 IU / mL genotype 3, where one IU is approximately 0.12 copies of each genotype). The parvovirus target nucleic acid was diluted down to 10,000 IU / mL and 1,000 IU / mL and each dilution was added to separate wells of a 96 well plate. The parvovirus amplification oligomers were SEQ ID NO: 91 and 131. The detection probe oligomer was SEQ ID NO: 144. The parvovirus competitor sequence (SEQ ID NO: 197) was added to the amplification reaction mixture. The results are shown in Table 2.

[0342] Example 6: Quantitative amplification and detection of parvovirus target nucleic acids spiked into parvovirus negative human plasma samples

[0343]

[0344] These results show that the quantitative assay of the present application shows good linearity with the WHO standard target nucleic acid, but the genotype 2 assay quantitative is relatively higher variation compared to the standard.

[0345] Example 7: Amplification and detection of hepatitis A virus nucleic acids and parvovirus target nucleic acids using specific target capture .

[0346] The quantitative amplification and detection assay was set up according to Example 5 above, except that the 10,000 IU / lM of target nucleic acid was resuspended in human plasma from a donor that was determined to be parvovirus negative. The expected amount of parvovirus detected in each sample was 4 Log (copies / mL), and the average result observed was 3.92 (± 0.059) Log (copies / mL). Thus, the quantitative parvovirus assay provides accurate results for the amplification and detection of all parvovirus target nucleic acid in human plasma samples.

[0347] Table 3: Exemplary oligomers, reference sequences, and regions ​ .

[0348] An amplification and detection reaction is performed on target nucleic acids isolated from a sample using target specific target capture oligomers. Target capture oligomers for capturing Hepatitis A virus nucleic acids are SEQ ID NOs: 46-51, and target capture oligomers for capturing parvovirus nucleic acids are SEQ ID NOs: 128-131. The target capture oligomers are used individually and in combination (meaning two or more target capture oligomers targeting Hepatitis A virus or two or more target capture oligomers targeting parvovirus) in a target capture reaction. The sample and reaction conditions are as described above for the Hepatitis A virus singleplex reaction and for the parvovirus singleplex reaction. The specific target capture oligomers, both individually and in combination, show improved sensitivity and capture efficiency compared to non-specific target capture methods.

[0349] ​

[0350]

[0351]

[0352]

[0353]

[0354]

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[0356]

[0357]

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[0364]

[0365] The application has been described in the context of particular examples and preferred embodiments. Those skilled in the art will recognize other embodiments within the scope of the application as defined by the following claims. SEQUENCE LIST <110> GEN-PROBE INCORPORATED <120> COMPOSITIONS AND METHODS FOR DETECTING HUMAN PARVOVIRUS NUCLEIC ACIDS AND HEPATITIS A VIRUS NUCLEIC ACIDS <130> GP274-PCT <150> 61 / 508,597 <151> 15-JUL-2011 <160> 203 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> SYNTHETIC OLIGO <400> 1 cagagaatta tgaaagtgga 20 <210> 2 <211> 23 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> SYNTHETIC OLIGO <400> 2 agtcagagaa ttatgaaagt gga 23 <210> 3 <211> 27 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> SYNTHETIC OLIGO <400> 3 ugagagtcag agaattatga aagtgga 27 <210> 4 <211> 22 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> SYNTHETIC OLIGO <400> 4 agtcagagaa ttatgaaagt gg 22 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 5 cagagaatta tgaaagtgg 19 <210> 6 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 6 tgagagtcag agaattatga aagtgg 26 <210> 7 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 7 tcagtgttca atgaatgt 18 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 8 tttactcagt gttcaatgaa tgt 23 <210> 9 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 9 ggagtttact cagtgttcaa tgaatgt 27 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 10 atgaaagtgg agtttactca gtgttcaatg aatgt 35 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 11 gaaagtcaga gaataatgaa agt 23 <210> 12 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 12 aatttaatac gactcactat agggagagga aaattaatca tggttttatc aatgtg 56 <210> 13 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <220> <221> misc_feature <222> (48)..(48) <223> N is inosine <400> 13 aatttaatac gactcactat agggagagga aaattaatca tggttttntc aatgtg 56 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 14 aatttaatac gactcactat agggagagca ggaaaattaa tcatg 45 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 15 aatttaatac gactcactat agggagagca ggaaaattaa tcat 44 <210> 16 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 16 aatttaatac gactcactat agggagagca ggaaaattaa tca 43 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 17 aatttaatac gactcactat agggagagca ggaaaattaa tc 42 <210> 18 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 18 aatttaatac gactcactat agggagaggc atagctgcag gaaaattaat catg 54 <210> 19 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 19 aatttaatac gactcactat agggagaggc atagctgcag gaaaattaat cat 53 <210> 20 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 20 aatttaatac gactcactat agggagaggc atagctgcag gaaaattaat ca 52 <210> 21 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 21 aatttaatac gactcactat agggagaggc atagctgcag gaaaattaat c 51 <210> 22 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 22 aatttaatac gactcactat agggagaact ctttctaaaa agcgttttgg agac 54 <210> 23 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligomer <400> 23 aatttaatac gactcactat agggagatct ttctaaaaag cgttttggag ac 52 <210> 24 <211> 54 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 24 aatttaatac gactcactat agggagaact ctttctaaag agcgttttgg agac 54 <210> 25 <211> 52 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 25 aatttaatac gactcactat agggagatct ttctaaagag cgttttggag ac 52 <210> 26 <211> 52 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 26 aatttaatac gactcactat agggagaact ctttctaaaa agcgttttgg ag 52 <210> 27 <211> 52 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 27 aatttaatac gactcactat agggagaact ctttctaaag agcgtttggag 52 <210> 28 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 28 aatttaatac gactcactat agggagagga aaattaatca tggttttatc aatgt 55 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 29 ggaaaattaa tcatggtttt atcaatgtg 29 <210> 30 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature <222> (21)..(21) <223> N is inosine <400> 30 ggaaaattaa tcatggtttt ntcaatgtg 29 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 31 gcaggaaaat taatcatg 18 <210> 32 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 32 gcaggaaaat taatcat 17 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 33 gcaggaaaat taatca 16 <210> 34 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 34 gcaggaaaat taatc 15 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 35 ggcatagctg caggaaaatt aatcatg 27 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 36 ggcatagctg caggaaaatt aatcat 26 <210> 37 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 37 ggcatagctg caggaaaatt aatca 25 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 38 ggcatagctg caggaaaatt aatc 24 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 39 actctttcta aaaagcgttt tggagac 27 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 40 tctttctaaa aagcgttttg gagac 25 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 41 actctttcta aagagcgttt tggagac 27 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 42 tctttctaaa gagcgttttg gagac 25 <210> 43 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 43 actctttcta aaaagcgttt tggag 25 <210> 44 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 44 actctttcta aagagcgttt tggag 25 <210> 45 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 45 ggaaaattaa tcatggtttt atcaatgt 28 <210> 46 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 46 ggucccucug aaauuaacau ugguguucca aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59 <210> 47 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligo <400> 47 guuccaucau ucuuuuuaug aacatttaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 57 <210> 48 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligo <400> 48 ccuucgccuu uuccucucca ugccugauct ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aa 62 <210> 49 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligo <400> 49 ucgccuuuuc cucuccaugc cugaucttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59 <210> 50 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligo <400> 50 ccuucgccuu uuccucucca ugccugttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59 <210> 51 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligo <400> 51 ggccccacca cacauuccag gaagtttaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 57 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 52 ggucccucug aaauuaacau ugguguucc 29 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 53 guuccaucau ucuuuuuaug aaca 24 <210> 54 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 54 ccuucgccuu uuccucucca ugccugauc 29 <210> 55 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 55 ucgccuuuuc cucuccaugc cugauc 26 <210> 56 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligomer <400> 56 ccuucgccuu uuccucucca ugccug 26 <210> 57 <211> 24 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 57 ggccccacca cacauuccag gaag 24 <210> 58 <211> 18 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 58 ucaguguuca augaaugu 18 <210> 59 <211> 18 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 59 ucaguguuca augaaugu 18 <210> 60 <211> 18 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 60 ucaguguuca augaaugu 18 <210> 61 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 61 uguucaauga auguggucuc c 21 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 62 uguucaauga auguggucuc c 21 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 63 uguucaauga auguggucuc c 21 <210> 64 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 64 guucaaugaa uguggucucc aaaacgcu 28 <210> 65 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 65 guucaaugaa uguggucucc aaaacgcu 28 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 66 gaaugugguc uccaaaacgc u 21 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 67 gaaugugguc uccaaaacgc t 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 68 gaaugugguc uccaaaacgc t 21 <210> 69 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 69 guggucucca aaacgcuuuu uaga 24 <210> 70 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 70 guggucucca aaacgcuuuu uaga 24 <210> 71 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 71 guggucucca aaacgcuuuu uaga 24 <210> 72 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 72 guggucucca aaacgcuuuu uaga 24 <210> 73 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 73 guggucucca aaacgcuuuu uaga 24 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 74 gucuccaaaa cgcuuuuuag 20 <210> 75 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <220> <221> misc_feature <222> (7)..(7) <223> N is G or inosine <220> <221> misc_feature <222> (12)..(12) <223> N is G or inosine <400> 75 ccagtancag tna 13 <210> 76 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 76 tcatccagta gcagtg 16 <210> 77 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 77 tcacccagta acag 14 <210> 78 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 78 tctgaccacc cccatgcctt atca 24 <210> 79 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 79 ctgaccaccc ccatgcctta tca 23 <210> 80 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 80 ccacccccat gccttatca 19 <210> 81 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 81 ggacagttat ctgaccaccc ccat 24 <210> 82 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 82 catggacagt tatctgacca cc 22 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 83 catcattttc aaagtcatgg acag 24 <210> 84 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 84 catcattttc agagtcatgg acag 24 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 85 ctctccagac ttatatagtc at 22 <210> 86 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 86 cctctctgtt tgacttagtt gctc 24 <210> 87 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 87 cctctttgtt tgacttagtt gctc 24 <210> 88 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 88 aatttaatac gactcactat agggagagct aacttgccca ggcttgtgt 49 <210> 89 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 89 aatttaatac gactcactat agggagaacg ctaacttgcc caggcttgtg t 51 <210> 90 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 90 aatttaatac gactcactat agggagagtt gtacgctaac ttgcccaggc ttgtgt 56 <210> 91 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 91 aatttaatac gactcactat agggagagct aacttgccca ggcttgtg 48 <210> 92 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 92 aatttaatac gactcactat agggagaacg ctaacttgcc caggcttgtg 50 <210> 93 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 93 aatttaatac gactcactat agggagagtt gtacgctaac ttgcccaggc ttgtg 55 <210> 94 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 94 aatttaatac gactcactat agggagaaac atagttagta ccgggta 47 <210> 95 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 95 aatttaatac gactcactat agggagacaa catagttagt accgggta 48 <210> 96 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 96 aatttaatac gactcactat agggagacca acatagttag taccgggta 49 <210> 97 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 97 aatttaatac gactcactat agggagatgc gggggcccag cttgta 46 <210> 98 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 98 aatttaatac gactcactat agggagaggc tatacctaaa gtcatg 46 <210> 99 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 99 aatttaatac gactcactat agggagaggc tatacctaaa gtcatga 47 <210> 100 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 100 aatttaatac gactcactat agggagagct atacctaaag tcatga 46 <210> 101 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 101 aatttaatac gactcactat agggagaacc taaagtcatg aatccttgca g 51 <210> 102 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 102 aatttaatac gactcactat agggagacca acatagttag taccgggggt 50 <210> 103 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 103 aatttaatac gactcactat agggagacca acatagttag taccggggg 49 <210> 104 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 104 aatttaatac gactcactat agggagacca acatagttag taccgggg 48 <210> 105 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 105 aatttaatac gactcactat agggagacca acatagttag taccgg 47 <210> 106 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 106 aatttaatac gactcactat agggagacca acatagttag taccgg 46 <210> 107 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 107 aatttaatac gactcactat agggagacca acatagttag taccg 45 <210> 108 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 108 gctaacttgc ccaggcttgt gt 22 <210> 109 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 109 acgctaactt gcccaggctt gtgt 24 <210> 110 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 110 gttgtacgct aacttgccca ggcttgtgt 29 <210> 111 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 111 gctaacttgc ccaggcttgt g 21 <210> 112 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 112 acgctaactt gcccaggctt gtg 23 <210> 113 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 113 gttgtacgct aacttgccca ggcttgtg 28 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 114 AATAGTTAGT ACCGGGTAT 20 <210> 115 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 115 CAACATAGTT AGTACC GGGTA 21 <210> 116 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 116 CCAACATAGT TAGTACC GGGTA 22 <210> 117 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <220> <221> misc_feature <222> (7)..(7) <223> N is G or Inosine <400> 117 CCAGTANCAG 10 <210> 118 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 118 GGCTATACCT AAAGT CATG 19 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> ​​​​​<223> synthetic oligomer <400> 119 ggctatacct aaagtcatga 20 <210> 120 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 120 gctataccta aagtcatga 19 <210> 121 <211> 24 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 121 acctaaagtc atgaatcctt gcag 24 <210> 122 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 122 ccaacatagt tagtaccggg ggt 23 <210> 123 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 123 ccaacatagt tagtaccggg gg 22 <210> 124 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 124 ccaacatagt tagtaccggg g 21 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 125 ccaacatagt tagtaccggg 20 <210> 126 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 126 ccaacatagt tagtaccgg 19 <210> 127 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 127 ccaacatagt tagtaccg 18 <210> 128 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 128 atgcgagcaa ctaagtcaaa cagggagttt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 <210> 129 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 129 tgcgggggcc cagcttgtat ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 52 <210> 130 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 130 tggctatacc taaagtcatg aatcctttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59 <210> 131 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 131 agtactgaaa tccatatcgg ttgtttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 56 <210> 132 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 132 atgcgagcaa ctaagtcaaa cagggag 27 <210> 133 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 133 tgcgggggcc cagcttgta 19 <210> 134 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 134 tggctatacc taaagtcatg aatcct 26 <210> 135 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 135 agtactgaaa tccatatcgg ttg 23 <210> 136 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 136 gagtatatgg gtttattccc a 21 <210> 137 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 137 uacagaaccu agaggagaaa aug 23 <210> 138 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 138 uacagaaccu agaggagaa 19 <210> 139 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligomer <400> 139 gaaccuagag gagaaaaug 19 <210> 140 <211> 17 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 140 cagaaccuag aggagaa 17 <210> 141 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 141 cagaaccuag aggagaaaau g 21 <210> 142 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 142 cgugcagaac cuagaggaga a 21 <210> 143 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 143 cauacagaac cuagaggaga a 21 <210> 144 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic oligomer <400> 144 aguacagaac cuagaggaga a 21 <210> 145 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 145 uacagaaccu agaggagaa 19 <210> 146 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 146 ugcagaaccu agaggagaa 19 <210> 147 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 147 agugcagaac cuagaggaga a 21 <210> 148 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 148 cagaaccuag aggagaa 17 <210> 149 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 149 cagaaccuag aggagaa 17 <210> 150 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 150 cgugcagaac cuagaggaga a 21 <210> 151 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 151 cgugcagaac cuagaggaga a 21 <210> 152 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 152 cgugcagaac cuagaggaga a 21 <210> 153 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 153 cgugcagaac cuagaggaga a 21 <210> 154 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 154 cgugcagaac cuagaggaga a 21 <210> 155 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 155 cguacagaac cuagaggaga a 21 <210> 156 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 156 cgugcagaac cuagaggagg agau 24 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 157 gugcagaacc uagaggagaa 20 <210> 158 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 158 ugcagaaccu agaggagaa 19 <210> 159 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 159 gaaccuagag gagaagaug 19 <210> 160 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 160 caugcagaac cuagaggaga a 21 <210> 161 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 161 caugcagaac cuagaggaga a 21 <210> 162 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 162 gcagaaccua gaggagaaaa ug 22 <210> 163 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 163 gaaccuagag gagaaaaug 19 <210> 164 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 164 gaacuuagag gagaaaaug 19 <210> 165 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 165 cagaaccuag aggagaa 17 <210> 166 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 166 cagaaccuag aggagaa 17 <210> 167 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 167 cagaaccuag aggagaa 17 <210> 168 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 168 gaaccuagag gagaaaaug 19 <210> 169 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 169 guauuaucua gugaagacuu ac 22 <210> 170 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 170 cagagaatwa tgaaagt 17 <210> 171 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 171 cagagaatwat gaaagtgg 19 <210> 172 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 172 tgaragtcag agaatwatga aagtgga 27 <210> 173 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 173 gaatwatgaa ag 12 <210> 174 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 174 gaaaattgar agtcagagaa twatgaaagt ggarttyac 39 <210> 175 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 175 ggaaaattaa tc 12 <210> 176 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <220> <221> misc_feature <222> (33)..(33) <223> N is A or inosine <400> 176 ggcatagctg caggaaaatt aatcatggtt ttntcaatgt g 41 <210> 177 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <220> <221> misc_feature <222> (39)..(39) <223> N is A or inosine <400> 177 gaaaaaggca tagctgcagg aaaattaatc atggttttnt caatgtgatg atg 53 <210> 178 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 178 tcagtgttca atgaatgtgg tctccaaaac gctttttaga 40 <210> 179 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 179 ccacccccat 10 <210> 180 <400> 180 000 <210> 181 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 181 catcattttc aragtcatgg acagttatct gaccaccccc atgccttatc atccagtagc 60 agtca 65 <210> 182 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 182 tctgaccacc cccatgcctt atcatccagt agcagtca 38 <210> 183 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 183 ccacccccat gccttatca 19 <210> 184 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 184 catcattttc aragtcatgg acagttatct gaccaccccc at 42 <210> 185 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 185 catcattttc aragtcatgg acagttatct gaccacc 37 <210> 186 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 186 gctaacttgc ccaggcttgt g 21 <210> 187 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 187 gttgtacgct aacttgccca ggcttgtgt 29 <210> 188 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 188 cttgcccagg c 11 <210> 189 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 189 gggtagttgt acgctaactt gcccaggctt gtgtaagtc 39 <210> 190 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 190 acctaaagtc atg 13 <210> 191 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 191 ggctatacct aaagtcatga atccttgcag 30 <210> 192 <400> 192 000 <210> 193 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 193 cagttggcta tacctaaagt catgaatcct tgcagcactg 40 <210> 194 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 194 mrtrcagaac ytagaggaga aratgcagta ttatctagtg aagacttac 49 <210> 195 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 195 mrtrcagaac ytagaggaga aratg 25 <210> 196 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 196 aatttaatac gactcactat agggaga 27 <210> 197 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 197 cttatcacac aagcctgggc aagttagct 29 <210> 198 <211> 7477 <212> DNA <213> Hepatitis A Virus <300> <308> GenBank / AB020564.1 GI:4001732 <309> 04-Aug-2001 <313> (1)..(7477) <400> 198 ttcaagaggg gtctccggag ttttccggag cccctcttgg aagtccatgg tgaggggact 60 tgatacctca ccgccgtttg cctaggctat aggctaaatt tccctttccc tgtccttccc 120 ctattcccct ttgttttgtt tgtaaatatt aattcctgca ggttcagggt tctttaatct 180 gtttctctat aagaacactc aattttcacg ctttctgtct tctttcttcc agggctctcc 240 CCTTGGCCTA GGCTCTGGCC GTTGCGCCCG GC GGGGTCAAC TCCATGATTA GCA TGGAGC 300 TGTAAGAATC TAAATTGGGG AC GC AGATGTTTGGGACGTCACCTTGCAGT GTTAAC T TGG 360 CTTTCATGAA CCTCTTTGAT CTTTCACAAG AGGTAGGCTA CGG GTGAAACCTCTTAGGCT 420 AATACTTCTA TGAAGAGATG CCTTGGATAG GGTAACAGCG GC GGATATTGGTGAGTTGTT 480 AAGACAAAAA CCATTCAACG CC GAAGGACTG GTTCTCATCC AGTGGATGC ATTAGTGAA 540 TTGATTGTCA AGGCTGTCTC TAGGTTTAAT CCCAGACCTC TCTGTGCTTA GGGCAAACAC 600 TATTTGGCCT TAAATGGGAT CCTGTGAGAG GGGGTCCCTC CATTGACAGC TGGACTGTT 660 TTTGGGGCCT TATGTGGTGT TTGCCTCTGA GGTACTCAGG GGCA TTTAGGTTTTTCCTCA 720 TTCTTAAACA ATAA TGAATAGTCC AAACAAGGAA TT TTC CAGACTGTTGGAGTGGCCT 780 TGACCACATC CTGTCTCTGG CAGATATTGA GGAAGAACAA ATGATTCAGT CC GTTGATAG 840 GACTGCAGTG ACTG GTGCTTCTTATTTTACTTCTGTGGACCAATCGTCAGTTCACACTGC 900 TGAGGTTGGC TCACATCAAAT TGAACCTTTG AAAACCTCT GTT GATAAACCTGGTTC TAA 960 gaagactcag ggggagaagt ttttcttgat tcactctgct gattggctta ctacacatgc 1020 tctttttcat gaagttgcaa aattggatgt ggtgaaatta ctgtataatg agcaatttgc 1080 tgtccaaggc ttgttgagat atcacacata tgcaagattt ggcattgaga ttcaagttca 1140 gataaaccct acaccctttc agcaaggggg attaatttgt gccatggttc ctggtgacca 1200 aagttatggt tcaatagcat ccttgactgt ttatcctcat ggtctgttaa attgtaacat 1260 caacaatgtt gttagaataa aggttccatt catttatact agaggtgctt atcattttaa 1320 agatccacag tacccagttt gggaattaac aatcagagtt tggtcagagt tgaatattgg 1380 aacaggaact tcggcttaca cttcacttaa cgttttagct aggtttacag atttggagtt 1440 acatggttta actcctcttt ctacacagat gatgagaaat gaatttagag ttagtactac 1500 tgaaaatgtt gtaaacttgt caaattatga agatgcaagg gcaaaaatgt cttttgcttt 1560 ggatcaggaa gattggaagt ctgatccctc tcaaggtggt ggaattaaaa ttactcattt 1620 tactacttgg acatccattc caaccttagc tgcccagttt ccgtttaatg cttcagattc 1680 ggttgggcaa caaattaaag ttattccagt ggacccatat tttttccaga tgacaaacac taatcctgac caaaaatgta taactgccct ggcttctatt tgtcagatgt tttgttttg gaggggagat cttgtttttg attttcaggt ttttccaact aaatatcatt caggtagtt attgttttgt tttgttcctg ggaatgagtt aatagatgtt actggaatta cattaagca ggcaactact gctccttgtg cagtgatgga cattacagga gtgcaatcaa ctttgagatt tcgtgttcct tggatttctg atacacccta tcgagtaaat aggtacacga agtcggcaca tcaaaaaggt gagtacactg ccattggaaa gcttattgtg tactgttaca atagactgac ttctccttct aatgttgctt ctcatgttag agttaatgtt tatctttcag caattaatct ggaatgtttt gctcctcttt atcatgctat ggatgttacc acacaagttg gagatgattc aggaggtttt tcaacaacag tttctactga gcagaatgtt cctgatcccc aagttggcat aacaaccatg agggacctaa aagggaaagc caatagagga aagatggatg tttcaggtgt gcaagcacct gtgggagcta tcacaacat tgaggatcca gttttagcaa agaaagtgcc TGAGACATTT CCTGAATTGA AACCTGGAGA GTCTAGACAT ACATCAGATC ATATGTCTAT 2460 TTATAAATTT ATGGGAAGGT CTCATTTTCT GTGTACTTTT ACTTTTAATT CAAATAATAA 2520 AGAGTACACA TTTCCAATAA CTCTGTCTTC GACTTCTAAT CCTCCTCATG GTTTACCATC 2580 AACATTAAGG TG GTTCTTCA ATCTGTTTCA GTTGTATAGA GGACCATTGG ATTTGACAAT 2640 TATTATCACAGGAGCCACTGATGTGGATGGTATGGCCTGGTTCACTCCAGTAGGCCTTGC 2700 TGTCGACACCCCTTGGGTGG AAAAGGAGTC AGCTTTGTCT ATTGATTACAA ACTGCTCT 2760 TGGAGCTGTTAGATTCAATACAAGAAGAACAGGGAATATT CAGAT TAGATTGCCATGGTA 2820 TTCTTATTTGTATGCCGTGTCTGGAGC ACTGGATGGTCTGGGGGATAAAACAGATTCCAC 2880 ATTTGGATTG GTTTCCATTCA GATTGCAAATTACAATCATTCTGATGAA TATTGTCC TT 2940 TAGTTGCTATT TGTCTGTTC AGAACAATC AGAGTTCTATT TTCCTAGAGC TCCATTAAA 3000 TTCAAATGCT ATGTTGTCCAC TGAGTCCAT GATGAGCAGA ATTGCAGCTG GGGACTTGG A 3060 GT CATCGGTGG ATGATCCTAG ATCAGAGGAG GACAGAAGATTTGAGAGTC ATATAGAAAG 3120 taggaaacca tacaaagaat tgagattgga ggttggcaaa caaagactca agtatgctca 3180 ggaagaactg tcaaatgagg tgcttccacc tcctaggaaa ataaaggggc tattttcaca 3240 agctaaaatt tctctttttt atactgagga gcatgaaata atgaaattttt cttggagagg 3300 agtaactgct gacactaggg ctttgagaag atttggattc tctatggctg ctggtagaag 3360 tgtgtggact cttgagatgg atgctggagt tcttactgga agattggtca gattgaatga 3420 tgagaaatgg acagaaatga aagatgataa aattgtttca ttaatcgaaa aattcacaag 3480 caacaaatat tggtctaaag tgaattttcc acatggaatg ttagatcttg aagaaattgc 3540 tgccaactct aaagattttc caaatatgtc tgagacagat ttgtgtttcc tgttgcattg 3600 gctgaatcca aagaaaataa atttagcaga tagaatgctt ggattgtctg gagtgcagga 3660 attaaagaa cagggtgttg gattgatagc agagtgtaga actttcttgg attctattgc 3720 tgggactctg aaatccatga tgtttggatt tcatcattct gtgactgttg aaattataaa 3780 tactgtgctt tgttttgtta agagtggaat tctactctat gtcatacaac aattgaacca 3840 agatgagcac tcccacataa ttggtttgtt gagagtcatg aattatgcag atattggctg 3900 ctcagttatt tcatgtggca aagttttttc taaaatgtta gaaacagttt ttaattggca 3960 aatggactcc agaatgatgg agctgagaac tcagagcttt tccaattggc taagagacat 4020 ttgttcagga attactattt ttaaaagttt taaggatgcc atatattggt tatatacaaa 4080 attgaaggat ttttatgaag taaattatgg caagaagaag gatgttctta atattcttaa 4140 agataaccag caaaaaatag aaaaagctat tgaagaagca gacaattttt gcattttgca 4200 aattcaagat gtagaaaaat ttgatcagta tcagaaaggg gttgatttaa tacaaaagct 4260 gagaactgtt cattcaatgg ctcaagttga ccctagcctt ggggttcatt tgtcacctct 4320 tagagattgt atagccagag tccaccaaaa gctcaagaat cttggatcta taaatcaggc 4380 catggtgaca agtagtgagc cagttgtttg ctatttatat ggcaaaagag gaggagggaa 4440 aagcttgact tcaattgcat tggcaaccaa gatttgtaaa cactatggtg ttgaacctga 4500 gaaaaatatt tacactaaac ctgtggcttc agactattgg gatggttata gtggacaatt 4560 ggtttgcatt attgatgata ttggccaaaa tacaacagat gaagattggt cagatttttg 4620 tcaattagtg tcaggatgcc caatgagatt gaatatggct tctcttgagg agaagggcag 4680 acatttttcc tctcctttta taatagcaac ttcaaattgg tcaaatccaa gtccaaaaac 4740 agtttatgtt aaggaagcaa ttgatcgtag gcttcatttt aaggttgaag ttaaacctgc 4800 ttcatttttt aaaaatcctc ataatgatat gttaaatgtt aatttggcta aaacaaatga 4860 tgcaattaag gacatgtctt gtgttgatct agtaatggat ggacataaca tttcattgat 4920 ggatttactt agttctttag tgatgacagt tgaaattagg aagcaaaata tgagtgaatt 4980 catggagttg tggtcccagg gaatctcaga tgatgacaat gatagtgcag tagctgagtt 5040 tttccaatct tttccatctg gtgaaccatc aaattccaaa ttatctagtt ttttccaatc 5100 tgtcactaat cacaagtggg ttgctgtggg agctgcagtt ggcattcttg gagtgcttgt 5160 gggaggatgg ttcgtgtaca agcatttctc ccgcaaagag gaagaaccaa ttccagctga 5220 aggggtttat catggcgtga ctaagcccaa acaggtgatt aaattggatg cagatccagt 5280 AGGTGCTGAGCTGGGGGGAAAGTGGAGTTTCTGTGGAGCTGCTGAGAAGAAGAAGAGC 120 AGTTGGGGAGAAAAATGGATGTGTGAGATGGGTTATGAA TGCCTTGGGAGTGAAGGATGA 5400 TTGTTTATAGTACCTTCTCATGCTTACAAGTTTGAAAAGGATTATGAAATGATGGAGTT 5460 TTATTTCAATAGAGGTGGAACTTACTATTCAATTTCA GCTGGAAATGTTGTTATTCAATC 5520 TTTAGATGTGGGTTTCAAGATGTTGTTC TAA TGAAGGTT CCTACAATTCCCAAGTTTAG 5580 AGATATTACTCAACATTTATTAAGAAAGGAGATGTACCTAGAGCCTTGAATCGCTTGGC 5640 AACATTAGTGACAAC TGT T AATGGAAC T CTTATGT T AATTC TGA G G G AC C AT T AAAG AT 5700 GGAGGAAAAAGCCACTTATGTTCATAAGAAGAATGATGGTACCACAGTTGATTTGACTGT 5760 TGATCAGGCATGGAGAGGAAAGGTGAAGGTC TTC TGG AATGTGTGGTGGGGCTCTGGT 5820 GT C AT C AAT C AGTCC AT AC AGAATGC AATTTTGGGTATTC AT GTTGC TGGAGGAAATTC 5880 AATTCTTGTTGGCAAAGTTGGTTACTCAAGAAATGTTCCAAAATATTGATAAGAAAATGAG 5940 AAGTCAGAGAATAATGAAAGTGGAATTCAC T C AGT GTT C AAT G AATGT AGT C T C C AA A AC 6000 gctttttaga aagagtccca ttcatcatca cattgataaa accatgatta attttcctgc 6060 agctatgcct ttttctaaag ttgaaattga tccaatggct gtgatgttgt ctaaatattc 6120 attacctctt gtagaagaac cagaggatta caaagaagct tcagtttttt atcaaaacaa 6180 gatagtaggc aagactcagt tagttgatga ctttttagat cttgatatgg ccattacagg 6240 ggctccaggc attgatgcta ttaatatgga ttcatctcct gggtttcctt atgttcaaga 6300 aaaattgact aaaagagatt taatttggtt ggatgaaaat ggtttgctgc taggagttca 6360 tccaagattg gctcagagaa ttttatttaa cactgtcatg atggaaaatt gttctgacct 6420 agatgttgtt tttacaactt gtccaaaaga tgaattgaga cctttagaga aagttttgga 6480 atcaaaaaca agagcaattg atgcttgtcc tttggattat acaattttat gtcgaatgta 6540 ctggggtcca gctattagtt attttcattt gaatccaggg tttcacacag gtgttgctat 6600 tggcatagat cctgatagac agtgggatga attatttaaa ccaatgataa gatttggaga 6660 tgttggtctt gatttagatt tttctgcctt tgatgctagt cttagtccat ttatgatcag 6720 ggaggcgggt agaatcatga gtgaattatc tggaacacca tctcattttg gaacggctct 6780 tatcaatact atcatttatt ctaaacattt gctgtacaat tgttgttatc atgtctgtgg 6840 ttcaatgcct tctgggtccc cttgtacagc tttgttgaat tcaattatta acaacattaa 6900 tttgtattat gtgttttcta aaatatttgg aaagtctcca gttttctttt gtcaagctct 6960 gaggatcctt tgttatggag atgatgtttt gatagttttt tccagagatg ttcaaattga 7020 taatcttgat ttgattggac agaaaattgt ggatgaattc aaaaaacttg gcatgacagc 7080 cacttcagct gataaaaatg tgcctcaact gaagccagtt tcagaattga cctttcttaa 7140 aagatctttt aatttggtgg aggacagaat taggcctgca atttcagaaa agacaatttg 7200 gtctttgata gcttggcaga gaagcaacgc tgagtttgag cagaatttag aaaatgctca 7260 gtggtttgct tttatgcatg gctatgagtt ttatcagaaa ttttattatt ttgttcagtc 7320 ctgtttggag aaagagatga tagaatatag gcttaaatct tatgattggt ggagaatgag 7380 attttatgac cagtgtttca tttgtgacct ttcatgattt gtttaaacaa attttcttaa 7440 aatttctgag gtttgtttat ttcttttatc agtaaat 7477 <210> 199 <211> 5594 <212> DNA <213> Erythrovirus B19 <300> <308> GenBank / AF162273.1 GI:5670171 <309> 1999-08-02 <313> (1)..(5594) <400> 199 ccaaatcaga tgccgccggt cgccgccggt aggcgggact tccggtacaa gatggcggac 60 aattacgtca tttcctgtga cgtcatttcc tgtgacgtca cttccggtgg gcgggacttc 120 cggaattagg gttggctctg ggccagcttg cttggggttg ccttgacact aagacaagcg 180 gcgcgccgct tgtcttagtg gcacgtcaac cccaagcgct ggcccagagc caaccctaat 240 tccggaagtc ccgcccaccg gaagtgacgt cacaggaaat gacgtcacag gaaatgacgt 300 aattgtccgc catcttgtac cggaagtccc gcctaccggc ggcgaccggc ggcatctgat 360 ttggtgtctt cttttaaatt ttagcgggct tttttcccgc cttatgcaaa tgggcagcca 420 ttttaagtgt ttcactataa ttttattggt cagttttgta acggttaaaa tgggcggagc 480 ttttaagtgt ttcactataa ttttattggt cagttttgta acggttaaaa tgggcggagc 480GTAAGCTTGAAGCTTAATTCCTAGAAGAATAGGAAAGAATAG 60 CTTTTTCTTG GACTTTCTTG CTGTTTTTTT GTGAGCTAAC TAACAGGTAT TTATACTACT 600 GTAACTATAC TAACATGGAG CTATTTCGAG GGATGCTTCA AGTTTCTTCT AATGTTCTGG 660 ACTGTGCTAA CGATAACTGG TGATGCTCTT TACTGGATTA GACACTTCT GACTGGGAAC 720 CATACTCACT ACTAACAGAC TAATGGCAAT ATACTTAAGC AGTGTGGCT TCTAAGCTTG 780 ACTTTACCGG GGGGCCACTA GCAGGATGCT GTACTTTTTT CAAGTAGAAT GTAACAAAT 840 TTGAAGAAGG CTATCATATT CATGTGATTA TTGGGGGGCC AGGTTAAACC CCAGAAACC 900 TCACAGTGTG TGTAAGGGGT TATTAAATAA TGTACTTTAT CACCTTGTA ACTGAAAATG 960 TAAAGCTAAA ATTTTTGCCA GGAATGACTA CAAAAGGCAA ATACTTTAGA GATGGAGAGC 1020 AGTTATAGAA AACTATTTAA TGAAAAAAAT ACCTTAAATG TTGTATGGTG TGTTACTA 1080 ATATTGATGG ATATATAGAT ACCTGTATTT CTGCTACTTT TAGAAGGGGA GCTTGCCATG 1140 CCAAGAAACC CCACATTACC ACAGCCATAA ATGACACTAG TAGTGATGCT GGGGAGTCTA 1200 gcggcacagg ggcagaggtt gtgccaatta atgggaaggg aactaaggct agcataaagt ttcaaactat ggtaaactgg ttgtgtgaaa acagagtgtt acagaggat aagtggaaac tagttgactt taaccagtac actttactaa gcagtagtca cagtggaagt tttcaaattc aaagtgcact aaaactagca atttataaag caactaattt agtgcctaca agcacatttc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 tactttgtca aaactatgac cccctattag tggggcagca tgtgttaag tggattgata aaaaatgtgg caagaaaaat acactgtggt tttatgggcc gccaagtaca ggaaaaacaa acttggcaat ggccattgct aaaagtgttc cagtatatgg catggttaac tggaataatg aaaactttcc atttaatgat gtagcaggga aaagcttggt ggtctgggat gaggtatta 1800. 1800. 1800. 1800. 1800. 1800. atcaaaaaat gcgtggaagt gtagctgtgc ctggagtacc tgtggttata accagcaatg 1860. gtgacattac ttttgttgta agcgggaca ctacaacaac tgtacatgct aaagccttaa aagagcgaat ggtaaagtta aactttactg taagatgcag ccctgacatg gggttactaa 1980 cagaggctga tgtacaacag tggcttacat ggtgtaatgc acaaagctgg gaccactatg 2040 aaaactgggc aataaactac acttttgatt tccctggaat taatgcagat gccctccacc 2100 cagacctcca aaccacccca attgtcacag acaccagtat cagcagcagt ggtggtgaaa 2160 gctctgaaga actcagtgaa agcagctttt ttaacctcat caccccaggc gcctggaaca 2220 ctgaaacccc gcgctctagt acgcccatcc ccgggaccag ttcaggagaa tcatttgtcg 2280 gaagctcagt ttcctccgaa gttgtagctg catcgtggga agaagccttc tacacacctt 2340 tggcagacca gtttcgtgaa ctgttagttg gggttgatta tgtgtgggac ggtgtaaggg 2400 gtttacctgt gtgttgtgtg caacatatta acaatagtgg gggaggcttg ggactttgtc 2460 cccattgcat taatgtaggg gcttggtata atggatggaa atttcgagaa tttaccccag 2520 atttggtgcg gtgtagctgc catgtgggag cttctaatcc cttttctgtg ctaacctgca 2580 aaaaatgtgc ttacctgtct ggattgcaaa gctttgtaga ttatgagtaa agaaagtggc 2640 aaatggtggg aaagtgatga taaatttgct aaagctgtgt atcagcaatt tgtggaattt 2700 tatgaaaagg ttactggaac agacttagag cttattcaaa tattaaaaga tcactataat 2760 atttctttag ataatcccct agaaaaccca tcctctctgt ttgacttagt tgctcgtatt 2820 aaaaataacc ttaaaaactc tccagactta tatagtcatc attttcaaag tcatggacag 2880 ttatctgacc acccccatgc cttatcatcc agtagcagtc atgcagaacc tagaggagaa 2940 aatgcagtat tatctagtga agacttacac aagcctgggc aagttagcgt acaactaccc 3000 ggtactaact atgttgggcc tggcaatgag ctacaagctg ggcccccgca aagtgctgtt 3060 gacagtgctg caaggattca tgactttagg tatagccaac tggctaagtt gggaataaat 3120 ccatatactc attggactgt agcagatgaa gagcttttaa aaaatataaa aaatgaaact 3180 gggtttcaag cacaagtagt aaaagactac tttactttaa aaggtgcagc tgcccctgtg 3240 gcccattttc aaggaagttt gccggaagtt cccgcttaca acgcctcaga aaaataccca 3300 agcatgactt cagttaattc tgcagaagcc agcactggtg caggaggggg tggcagtaat 3360 cctgtcaaaa gcatgtggag tgagggggcc acttttagtg ccaactctgt aacttgtaca 3420 ttttccagac agtttttaat tccttatgac ccagagcacc attataaggt gttttctccc 3480 gcagcaagca gctgccacaa tgccagtgga aaggaggcaa aggtttgcac aattagtccc 3540 ataatgggat actcaacccc atggagatat ttagatttta atgctttaaa tttatttttt 3600 tcacctttag agtttcagca cttaattgaa aattatggaa gtatagctcc tgatgcttta 3660 actgtaacca tatcagaaat tgctgttaag gatgttacag acaaaactgg agggggggta 3720 caggttactg acagcactac agggcgccta tccatgttag tagaccatga atacaagtac 3780 ccatatgtgt taggacaagg tcaggatact ttagccccag aacttcctat ttgggtatac 3840 tttccccctc aatatgctta cttaacagta ggagatgtta acacacaagg aatctctgga 3900 gacagcaaaa aattagcaag tgaagaatca gcattttatg ttttggaaca cagttctttt 3960 cagcttttag gtacaggagg tacagcaact atgtcttata agtttcctcc agtgccccca 4020 gaaaatttag agggctgcag tcaacacttt tatgaaatgt acaatccctt atacggatcc 4080 CGCTTAGGGG TTCCTGACAC ATTAGGAGGT GACCCAAAAT TTAGATCTTT AACACATGAA 4140 GACCATGCAA TTCAGCCCCA AAACTTCATG CCAGGGCCAC TAGTAAACTC AGTGTC TACA 4200 AAGGAGGGAG ACAGCTCTAA TACTGGAGCT GGAAAAGCCT TAACAGGCCT TAGCACAGGC 4260 ACCTCTCAAA ACAC T AGAAT ATCCTTACGC CCTGGGCCAG TGT C AC AGCC ATACCACCAC 4320 TGGGACACAG ATAAATATGT TCCAGGAATA AATGCCATTT CT CATGGTCAG ACCACTTAT 4380 GGTAACGCTG AAGACAAGAG TATCAGCAAG GAGTGGGTAG ATTTCCAAAT GAAAAAGAA 4440 CAGCTAAAAC AGTTACAGGG TT T AAACATGC ACACCTATTT CCCCAATAA AGGAACCCAG 4500 CAATATACAG ATCAAATTGA GC G CCCCTAATGGTGGGTT CTGTATGGAA CAGAAGAGCC 4560 CTTC ACT ATG AAAGCCAGCT GTGGAGTAAA ATTCCAAATT TAGATGACAG TTT T AAAC T 4620 CAGTTTGCAG CCTTAGGAGG ATGGG GTTTGC ATCAGCCACC TCCTCAAAT ATTTTT AAA A 4680 ATATTACCAC AAAGTGGGCC AATTGGAGGT ATTAATCAAT GGGAATTACT ACCTTAGTT 4740 CAGTATGCCG TGGGAATTAT GACAGTAAC TATGACATTT AAATTGGGGC CCCTAAAGCT 4800 acgggacggt ggaatcctca acctggagta tatccccgc acgcagcagg tcatttacca 4860 tatgtactat atgaccccac agctacagat gcaaaacaac accacaggca tggatacgaa 4920 aagcctgaag aattgtggac agccaaaagc cgtgtgcacc cattgtaaac actccccacc 4980 gtgccctcag ccaggatgcg taactaaacg cccaccagta ccacccagac tgtacctgcc 5040 ccctcctgta cctataagac agcctaacac aaaagatata gacaatgtag aatttaagta 5100 cttaaccaga tatgaacaac atgttattag aatgttaaga ttgtgtaata tgtatcaaaa 5160 tttagaaaaa taaacatttg ttgtggttaa aaattatgt tgttgcgctt taaaaatttta 5220 aaagaagaca ccaaatcaga tgccgccggt cgccgccggt aggcgggact tccggtacaa 5280 gatggcggac aattacgtca tttcctgtga cgtcatttcc tgtgacgtca cttccggtgg 5340 gcgggacttc cggaattagg gttggctctg ggccagcgct tggggttgac gtgccactaa 5400 gacaagcggc gcgccgcttg tcttagtgtc aaggcaaccc caagcaagct ggcccagagc 5460 caaccctaat tccggaagtc ccgcccaccg gaagtgacgt cacaggaaat gacgtcacag 5520 GAAATGACGTAATTGTCCGCCATCTTGTA CCGGAAGTCCC GCCTACCGGCGGC GACCGGC 5580 GGCATCTGATTTGG 5594 <210> 200 <211> 472 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 200 AAATATTAAAAGATCATTATAATATTTCTTTAGATAATCCCCTAGAAAACCCATCCTCTC 60 TGTCTTGGTTAGTTGCTCGTATTAAAAATAACCTTAAAAACTCTCCAGACTTATATAGTC 120 ATCATTTTCAAAGTCAATGGACAGTTATCTGACCACCCCATGCCTTATCATCCAGTAGC A 180 GTCA TGC AGA ACCTAGAGGAGAAGATGCAGTATTATCTAGTGAAGACTTACACAAGCCTG 240 GGCAAGTTAGC GTACAAC TACCCG GTAC TAACTAT GTTGGGCCTGGCAATGAGCTACAAG 300 CTGGGCCCCC GCAAAGTGCT GTTGACAGTG CTGCAAGGAT TCA TGACTTT AGGTATAGCC 360 A ACTGGCTAAGTTGGGAATAAATCCATATACTCATTGGACTG TAGCAGATGAAGAGCTTT 420 TAAAAATAT AAAAAATGAAACCGGT TTC AAGCACAAGTAGTAAAGACTA 472 <210> 201 <211> 471 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligomer <400> 201 aatattaaaa gatcattaca atatttcttt agataatccc ctagaaaacc catcttccct 60 gtttgactta gttgctcgta ttaaaagtaa tcttaaagac tctccagacc tatatagtca 120 tcattttcaa agtcatggac agttatctga ccacccccat gccttatcac ccagtagcag 180 tcatacagaa cctagaggag aaaatgcagt attatctagt gaagacttac acaagcctgg 240 gcaagttagc atacaactac ccggtactaa ctatgttggg cctggcaatg agctacaagc 300 tgggcccccg caaagtgctg tggacagtgc tgcaaggatt catgacttta ggtatagcca 360 attggctaag ctgggaataa acccatatac ttattggact gtagcagatg aggaactgtt 420 aaaaaatata aaaaatgaaa ctgggtttca agcacaagca gtaaaagatt a 471 <210> 202 <211> 472 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomer <400> 202 aaattttaaa agatcattac aacatttctt tagacaatcc tttagaaaac ccctcttctt 60 tatttgactt agttgctcgc attaaaagca atcttaaaaa ctctccagac ctatatagtc 120 atcattttca gagccatgga cagttatctg accaccccca ttccttatca cccagtaaca 180 gtagtacaga acctagagga gaaaatgcag tattatctag tgaagactta cacaagcctg 240 ggcaagttag catacaatta cccggtacta actatgttgg gcctggcaat gagctacaag 300 ctgggcctcc gcagaatgct gtggacagtg ctgcaaggat tcatgacttt aggtatagcc 360 aattggctaa gttgggaata aatccttata ctcattggac ggtagcagat gaggaattgt 420 taaaaaatat aaaaaatgaa acagggtttc aagcacaagc agtaaaagac ta 472 <210> 203 <211> 1039 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 203 tttgtcggaa gcccagtttc ctccgaagtt gtagctgcat cgtgggaaga agccttctac 60 acacctttgg cagaccagtt tcgtgaactg ttagttgggg ttgattatgt gtgggacggt 120 gtaaggggct tacctgtgtg ttgtgtgcaa catattaaca atagtggggg aggcttggga 180 ctttgtcccc attgcattaa tgtaggggct tggtataatg gatggaatt tcgagaattt actccagatt tggtgcgatg tagctgccat gtgggagctt ctaatccctt ttctgtgcta 300 acctgtaaaa aatgtgctta cctgtctgga ttgcaaagct ttgtagatta tgagtaaaga aagtggcaaa tggtgggaaa gtaatgataa atttgctaaa gctgtgtatc agcaatttgt ggaattttat gaaaaagtta ctggaacaga cttagagctt attcaaatat taaagacca ttatatatt tctttagata atcccctaga aaacccatcc tctctgtttg acttagttgc tcgtattaaa aataacctta aaaactctcc agacttatat agtcatcatt ttcaaagtca tggacagtta tctgaccacc cccatgcctt atcatccagt agcagtcatg cagaacctag aggagaaaat gcagtattat ctagtgaaga cttacacaag cctgggcaag ttagcgtaca actacccggt actaactatg ttggggcctgg caatgagcta caagctgggc ccccgcaaag tgctgttgac agtgctgcaa ggattcatga ctttaggtat agccaactgg ctaagttggg 900. aataatcca tatactcatt ggactgtagc agatgaagag cttttaaaaa atataaaaaa TGAACCTGGGTTTCAAGCAC AAGTAGTA AAAGACTACTTT ACTTTAAAG GTGCAGCTGC 960 CCCCTGTGGCCCATTTC AAG GAAGTTTGCC GG AAGTTCCC GCTTACAACG CCTCAGAAAA 1020 ATACC C AAG CATGACTTCA 1039

Claims

1. An oligomer combination for detecting a Hepatitis A Virus (HAV) target nucleic acid in a sample, the oligomer combination comprising: (a) a first HAV amplification oligomer consisting of SEQ ID NO: 2; and (b) a second HAV amplification oligomer consisting of SEQ ID NO:

18.

2. The oligomer combination of claim 1, further comprising: at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57.

3. The oligomer combination of claim 2, wherein the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

4. The oligomer combination of claim 1, further comprising a HAV-specific detection probe oligomer configured for specific hybridization to the HAV amplification product under conditions whereby the presence or absence of the HAV amplification product is assayed, thereby indicating the presence or absence of HAV in the sample.

5. The oligomer combination of claim 4, wherein the HAV-specific detection probe oligomer comprises a target-hybridizing sequence 14 to 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from nucleotide position 5965 to nucleotide position 6028.

6. The oligomer combination of claim 5, wherein the HAV-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO:

178.

7. The oligomer combination of claim 6, wherein the HAV-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 58-74.

8. The oligomer combination of any one of claims 4-7, wherein the HAV-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

9. The oligomer combination of claim 8, wherein the HAV-specific detection probe comprises a fluorescent label, a quencher, or both.

10. The oligomer combination of claim 9, the HAV-specific detection probe is a TaqMan detection probe or a molecular beacon or a molecular torch.

11. The oligomer combination of any one of claims 1-7 and 9-10, wherein the oligomer combination is suitable for use in an isothermal amplification reaction.

12. The oligomer combination of any one of claims 1-7 and 9-10, wherein the sample is from an individual patient; or wherein the sample is pooled.

13. The oligomer combination of claim 12, wherein the pooled sample is a pooled plasma sample.

14. The oligomer combination of claim 1, further comprising an oligomer combination for amplifying a parvovirus nucleic acid target region, the oligomer combination for amplifying a parvovirus nucleic acid target region comprising: (a) a first parvovirus amplification oligomer comprising a first target hybridization sequence that is 14 to 27 consecutive nucleotides contained within the sequence of SEQ ID NO: 181 and includes at least the sequence of catggacag as set forth in SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) a second parvovirus amplification oligomer comprising a second target hybridization sequence selected from the group consisting of: (i) a sequence that is 14 to 30 consecutive nucleotides contained within the sequence of SEQ ID NO: 189 and includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is 14 to 30 consecutive nucleotides contained within the sequence of SEQ ID NO: 193 and includes at least the sequence of ctaaagtc as set forth in SEQ ID NO:

192.

15. The oligomer combination of claim 14, wherein the second parvovirus amplification oligomer is a promoter primer or a promoter provider further comprising a promoter sequence located 5' of the target hybridization sequence.

16. The oligomer combination of claim 15, wherein the promoter sequence is a T7 promoter sequence.

17. The oligomer combination of claim 16, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO:

196.

18. The oligomer combination of claim 17, wherein the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

19. The oligomer combination of any one of claims 14-18, further comprising at least one parvovirus-specific capture probe oligomer comprising a target hybridization sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein the target hybridization sequence is selected from the group consisting of SEQ ID NOs: 132-135.

20. The oligomer combination of claim 19, wherein the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

21. The oligomer combination of claim 14, further comprising a parvovirus-specific detection probe oligomer configured to specifically hybridize to a parvovirus amplification product under conditions whereby the presence or absence of the parvovirus amplification product is determined, thereby indicating the presence or absence of parvovirus in the sample.

22. The oligomer combination of claim 21, wherein, The parvovirus-specific detection probe oligomer comprises a target-hybridizing sequence 14 to 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 199 from nucleotide position 2921 to nucleotide position 2966, or from nucleotide position 2921 to nucleotide position 3067.

23. The oligomer combination of claim 22, wherein the parvovirus-specific detection probe target-hybridizing sequence is comprised in the sequence of SEQ ID NO: 194 or 195.

24. The oligomer combination of claim 23, wherein the parvovirus-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 137-169.

25. The oligomer combination of any one of claims 21-24, wherein the parvovirus-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

26. The oligomer combination of claim 25, wherein the parvovirus-specific detection probe comprises a fluorescent label, a quencher, or both.

27. The oligomer combination of claim 26, the parvovirus-specific detection probe is a TaqMan detection probe or a molecular beacon or a molecular torch.

28. The oligomer combination of claim 14, further comprising a parvovirus-specific detection probe oligomer and a HAV-specific detection probe oligomer configured for specific hybridization to a parvovirus amplification product and a HAV amplification product, respectively, under conditions whereby the presence or absence of the parvovirus amplification product and the HAV amplification product is determined, thereby indicating the presence or absence of parvovirus and HAV in the sample.

29. The oligomer combination of claim 28, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are differentially labeled.

30. The oligomer combination of claim 29, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a label independently selected from the group consisting of (a) a chemiluminescent label and (b) a fluorescent label.

31. The oligomer combination of claim 29, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a chemiluminescent label.

32. The oligomer combination of claim 31, wherein the chemiluminescent labels for the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are characterized by different luminescence kinetics sufficient to distinguish a parvovirus-specific chemiluminescent signal from a HAV-specific chemiluminescent signal.

33. The oligomer combination of claim 32, wherein the chemiluminescent label for each of the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer comprises acridinium ester (AE).

34. A kit comprising the oligomer combination of any one of claims 1-33.

35. A reaction mixture comprising the oligomer combination of any one of claims 1-33.

36. A method for detecting a target nucleic acid of hepatitis A virus (HAV) in a sample, the method comprising The method is not for disease diagnosis, the method comprising: (A) providing a sample suspected of containing HAV; (B) contacting the sample with an oligomer combination for amplifying a target region of HAV nucleic acid, the oligomer combination comprising: (a) a first HAV amplification oligomer consisting of SEQ ID NO: 2; and (b) a second HAV amplification oligomer consisting of SEQ ID NO: 18; (C) performing an in vitro nucleic acid amplification reaction in which any HAV target nucleic acid present in the sample is used as a template to generate a HAV amplification product; and (D) detecting the presence or absence of the HAV amplification product, thereby indicating the presence or absence of HAV in the sample.

37. The method of claim 36, further comprising purifying the HAV target nucleic acid from other components of the sample prior to step (B).

38. The method of claim 37, wherein the purifying comprises contacting the sample with at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57.

39. The method of claim 38, wherein the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

40. The method of claim 37, wherein the detecting step (D) comprises contacting the product of the in vitro nucleic acid amplification reaction with a HAV-specific detection probe oligomer configured for specific hybridization to the HAV amplification product under conditions whereby the presence or absence of the HAV amplification product is determined, thereby indicating the presence or absence of HAV in the sample.

41. The method of claim 40, wherein the HAV-specific detection probe oligomer comprises a target-hybridizing sequence 14 to 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from nucleotide position 5965 to nucleotide position 6028.

42. The method of claim 41, wherein the HAV-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO:

178.

43. The method of claim 42, wherein the HAV-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 58-74.

44. The method of claim 40, wherein the HAV-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

45. The method of any one of claims 40-44, wherein the detecting step (D) occurs during the amplifying step (C).

46. The method of claim 45, wherein the HAV-specific detection probe comprises a fluorescent label, a quencher, or both.

47. The method of claim 46, wherein the HAV-specific detection probe is a TaqMan detection probe or a molecular beacon or a molecular torch.

48. The method of any one of claims 36-44 and 46-47, wherein the amplification reaction of step (C) is an isothermal amplification reaction.

49. The method of any one of claims 36-44 and 46-47, wherein the sample is pooled.

50. The method of claim 49, wherein the pooled sample is a pooled plasma sample.

51. The method of claim 36, wherein the method is for additional detection of human parvovirus target nucleic acids, wherein the sample is also contacted with an oligomer combination for amplification of parvovirus nucleic acid target regions comprising: (a) a first parvovirus amplification oligomer comprising a first target-hybridizing sequence that is 14 to 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and includes at least the sequence catggacag set forth in SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) a second parvovirus amplification oligomer comprising a second target-hybridizing sequence selected from the group consisting of: (i) a sequence that is 14 to 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is 14 to 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and includes at least the sequence ctaaagtc set forth in SEQ ID NO:

192.

52. The method of claim 51, wherein the second parvovirus amplification oligomer is a promoter primer or a promoter provider that also comprises a promoter sequence located 5' of the target-hybridizing sequence.

53. The method of claim 52, wherein the promoter sequence is a T7 promoter sequence.

54. The method of claim 53, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO:

196.

55. The method of claim 54, wherein the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

56. The method of any one of claims 51-55, further comprising purifying the parvovirus target nucleic acid from other components of the sample prior to step (B).

57. The method of claim 56, wherein the purifying comprises contacting the sample with at least one parvovirus-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 132-135.

58. The method of claim 57, wherein the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

59. The method of claim 51, wherein the detecting step (D) further comprises contacting the product of the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer configured to specifically hybridize to the parvovirus amplification product under conditions whereby the presence or absence of parvovirus amplification product is determined, thereby indicating the presence or absence of parvovirus in the sample.

60. The method of claim 59, wherein the parvovirus-specific detection probe oligomer comprises a target-hybridizing sequence 14 to 40 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 199 from nucleotide position 2921 to nucleotide position 2966, or from nucleotide position 2921 to nucleotide position 3067.

61. The method of claim 60, wherein the parvovirus-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 194 or 195.

62. The method of claim 61, wherein the parvovirus-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 137-169.

63. The method of any one of claims 59-62, wherein the parvovirus-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

64. The method of claim 63, wherein the detecting step (D) occurs during the amplifying step (C).

65. The method of claim 64, wherein the parvovirus-specific detection probe comprises a fluorescent label, a quencher, or both.

66. The method of claim 65, wherein the parvovirus-specific detection probe is a TaqMan detection probe or a molecular beacon or a molecular torch.

67. The method of claim 51, wherein detection step (D) comprises contacting the product of the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer and a HAV-specific detection probe oligomer configured to specifically hybridize to parvovirus amplification product and HAV amplification product, respectively, under conditions whereby the presence or absence of parvovirus amplification product and HAV amplification product is determined, thereby indicating the presence or absence of parvovirus and HAV in the sample.

68. The method of claim 67, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are differentially labeled.

69. The method of claim 68, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a label independently selected from the group consisting of (a) a chemiluminescent label and (b) a fluorescent label.

70. The method of claim 68, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a chemiluminescent label.

71. The method of claim 70, wherein the chemiluminescent labels for the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are characterized by different luminescence kinetics sufficient to distinguish parvovirus-specific chemiluminescent signals from HAV-specific chemiluminescent signals.

72. The method of claim 71, wherein the chemiluminescent labels for the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise acridinium ester (AE).

73. Use of an oligomer combination for amplifying a HAV nucleic acid target region in the manufacture of a kit for a method of detecting a Hepatitis A Virus (HAV) target nucleic acid in a sample, the oligomer combination comprising: (a) a first HAV amplification oligomer consisting of SEQ ID NO: 2; and (b) a second HAV amplification oligomer consisting of SEQ ID NO: 18; the method comprising: (A) providing a sample suspected of containing HAV; (B) contacting the sample with the oligomer combination; (C) performing an in vitro nucleic acid amplification reaction in which any HAV target nucleic acid present in the sample is used as a template to generate a HAV amplification product; and (D) detecting the presence or absence of the HAV amplification product, thereby indicating the presence or absence of HAV in the sample.

74. The use of claim 73, wherein the method further comprises purifying the HAV target nucleic acid from other components of the sample prior to step (B).

75. The use of claim 74, wherein the purifying comprises contacting the sample with at least one HAV-specific capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 52-57.

76. The use of claim 75, wherein the HAV-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 46-51.

77. The use of claim 74, wherein detection step (D) comprises contacting the product of the in vitro nucleic acid amplification reaction with a HAV-specific detection probe oligomer configured for specific hybridization to a HAV amplification product under conditions whereby the presence or absence of the HAV amplification product is determined, thereby indicating the presence or absence of HAV in the sample.

78. The use of claim 77, wherein the HAV-specific detection probe oligomer comprises a target-hybridizing sequence 14 to 40 nucleotides in length and configured for specific hybridization to a target sequence contained within SEQ ID NO: 198 from nucleotide position 5965 to nucleotide position 6028.

79. The use of claim 78, wherein the HAV-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO:

178.

80. The use of claim 79, wherein the HAV-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 58-74.

81. The use of claim 77, wherein the HAV-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

82. The use of any one of claims 77-81, wherein detection step (D) occurs during the amplification step (C).

83. The use of claim 82, wherein the HAV-specific detection probe comprises a fluorescent label, a quencher, or both.

84. The use of claim 83, wherein the HAV-specific detection probe is a TaqMan detection probe or a molecular beacon or a molecular torch.

85. The use of any one of claims 73-81 and 83-84, wherein the amplification reaction of step (C) is an isothermal amplification reaction.

86. The use of any one of claims 73-81 and 83-84, wherein the sample is from an individual patient; or wherein the sample is pooled.

87. The use of claim 86, wherein the pooled sample is a pooled plasma sample.

88. The use of claim 73, wherein the method is for additional detection of a human parvovirus target nucleic acid, wherein the sample is also contacted with an oligomer combination for amplification of a parvovirus nucleic acid target region comprising: (a) a first parvovirus amplification oligomer comprising a first target hybridizing sequence that is 14 to 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 181 and includes at least the sequence of catggacag set forth in SEQ ID NO: 117, SEQ ID NO: 179, or SEQ ID NO: 180; and (b) a second parvovirus amplification oligomer comprising a second target hybridizing sequence selected from the group consisting of: (i) a sequence that is 14 to 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 189 and includes at least the sequence of SEQ ID NO: 188; and (ii) a sequence that is 14 to 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 193 and includes at least the sequence of ctaaagtc set forth in SEQ ID NO:

192.

89. The use of claim 88, wherein the second parvovirus amplification oligomer is a promoter primer or a promoter provider that also comprises a promoter sequence located 5' of the target hybridizing sequence.

90. The use of claim 89, wherein the promoter sequence is a T7 promoter sequence.

91. The use of claim 90, wherein the T7 promoter sequence has the sequence set forth in SEQ ID NO:

196.

92. The use of claim 91, wherein the second parvovirus amplification oligomer has a sequence selected from the group consisting of SEQ ID NOs: 88-93 and 98-101.

93. The use of any one of claims 88-92, wherein the method further comprises purifying the parvovirus target nucleic acid from other components of the sample prior to step (B).

94. The use of claim 93, wherein the purifying comprises contacting the sample with at least one parvovirus-specific capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, wherein the target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 132-135.

95. The use of claim 94, wherein the parvovirus-specific capture probe oligomer has a sequence selected from the group consisting of SEQ ID NOs: 128-131.

96. The use of claim 88, wherein detection step (D) further comprises contacting the product of the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer configured to specifically hybridize to a parvovirus amplification product under conditions whereby the presence or absence of parvovirus amplification product is determined, thereby indicating the presence or absence of parvovirus in the sample.

97. The use of claim 96, wherein the parvovirus-specific detection probe oligomer comprises a target-hybridizing sequence 14 to 40 nucleotides in length and configured to specifically hybridize to a target sequence contained within SEQ ID NO: 199 from nucleotide position 2921 to nucleotide position 2966, or from nucleotide position 2921 to nucleotide position 3067.

98. The use of claim 97, wherein the parvovirus-specific detection probe target-hybridizing sequence is contained within the sequence of SEQ ID NO: 194 or 195.

99. The use of claim 98, wherein the parvovirus-specific detection probe target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 137-169.

100. The use of any one of claims 96-99, wherein the parvovirus-specific detection probe comprises a label selected from the group consisting of: (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher; and (d) a combination of one or more of (a), (b), and (c).

101. The use of claim 100, wherein detection step (D) occurs during the amplification step (C).

102. The use of claim 101, wherein the parvovirus-specific detection probe comprises a fluorescent label, a quencher, or both.

103. The use of claim 102, wherein the parvovirus-specific detection probe is a TaqMan detection probe or a molecular beacon or a molecular torch.

104. The use of claim 88, wherein detection step (D) comprises contacting the product of the in vitro nucleic acid amplification reaction with a parvovirus-specific detection probe oligomer and a HAV-specific detection probe oligomer configured to specifically hybridize to a parvovirus amplification product and a HAV amplification product, respectively, under conditions whereby the presence or absence of parvovirus amplification product and HAV amplification product is determined, thereby indicating the presence or absence of parvovirus and HAV in the sample.

105. The use of claim 104, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer are differentially labeled.

106. The use of claim 105, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a label independently selected from the group consisting of (a) a chemiluminescent label and (b) a fluorescent label. ​ ​ ​ ​ 107. The use of claim 105, wherein the parvovirus-specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise a chemiluminescent label.

108. The use of claim 107, wherein the chemiluminescent labels for the parvovirus- specific detection probe oligomer and the HAV-specific detection probe oligomer are characterized by different luminescence kinetics sufficient to distinguish a parvovirus-specific chemiluminescent signal from a HAV-specific chemiluminescent signal.

109. The use of claim 108, wherein the chemiluminescent labels for the parvovirus- specific detection probe oligomer and the HAV-specific detection probe oligomer each comprise an acridinium ester (AE).

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